UNIVERSAL DNA ASSEMBLY

Abstract

The invention relates to a nucleic acid comprising at least one methylation-protectable restriction element, the methylation-protectable restriction element comprising: (i) a type IIS restriction enzyme recognition sequence, or a partial type IIS restriction enzyme recognition sequence, that is recognised by a type IIS restriction enzyme that cleaves outside of the recognition sequence; (ii) a DNA methylase recognition sequence that is recognised and methylated by a DNA methylase, wherein the DNA methylase recognition sequence is identical to, or is encompassed within, the type IIS restriction recognition sequence, such that methylation of the nucleic acid by the DNA methylase methylates the type IIS restriction enzyme recognition sequence and protects the nucleic acid from cleavage by the type IIS restriction enzyme; and (iii) a recognition sequence for a sequence-specific DNA-binding protein, wherein the recognition sequence is positioned such that the binding of the sequence-specific DNA-binding protein overlaps with the DNA methylase recognition sequence such that binding of the sequence-specific DNA-binding protein is capable of preventing methylation of the type IIS restriction enzyme recognition sequence by the DNA methylase such that it is not protected from cleavage by the type IIS restriction enzyme. The invention further relates to associated methods of nucleic acid assembly.

Claims

1. A nucleic acid comprising at least one methylation-protectable restriction element, the methylation-protectable restriction element comprising: (i) a type IIS restriction enzyme recognition sequence, or a partial type IIS restriction enzyme recognition sequence, that is recognised by a type IIS restriction enzyme that cleaves outside of the recognition sequence; (ii) a DNA methylase recognition sequence that is recognised and methylated by a DNA methylase, wherein the DNA methylase recognition sequence is identical to, or is encompassed within, the type IIS restriction recognition sequence, such that methylation of the nucleic acid by the DNA methylase methylates the type IIS restriction enzyme recognition sequence and protects the nucleic acid from cleavage by the type IIS restriction enzyme; and (iii) a recognition sequence for a sequence-specific DNA-binding protein, wherein the recognition sequence is positioned such that the binding of the sequence-specific DNA-binding protein overlaps with the DNA methylase recognition sequence such that binding of the sequence-specific DNA-binding protein is capable of preventing methylation of the type IIS restriction enzyme recognition sequence by the DNA methylase such that it is not protected from cleavage by the type IIS restriction enzyme.

2. The nucleic acid according to claim 1, wherein the type IIS restriction enzyme recognition sequence of the methylation-protectable restriction element comprises or consists of the sequence GGTCTC, or a partial sequence thereof, and the type IIS restriction enzyme is BsaI, or a variant thereof.

3. The nucleic acid according to claim 1, wherein the restriction enzyme that recognizes the restriction enzyme recognition sequence of the methylation-protectable restriction element is capable of cutting nucleic acid to leave at least a 2 bp overhang/sticky end.

4. The nucleic acid according to any preceding claim, wherein the sequence specific DNA binding protein is selected from: a nucleic acid-guided DNA binding protein; a second DNA methylase, such that the recognition sequence of the DNA binding protein is a second DNA methylase recognition sequence relative to the first DNA methylase recognition sequence of ii), and said sequences are different; a transcription activator-like effector; a deactivated endodeoxyribonuclease; and a sequence specific zinc finger protein.

5. The nucleic acid according to any preceding claim, wherein the sequence specific DNA binding protein is a deactivated RNA-guided DNA endonuclease enzyme.

6. The nucleic acid according to any preceding claim, wherein the methylation-protectable restriction element further comprises a methylase-switch element, wherein the methylase-switch element comprises a recognition sequence for a switch DNA methylase, wherein the methylase-switch element comprises the said type IIS restriction enzyme recognition sequence (i) of the methylation-protectable restriction element, and the switch DNA methylase recognition sequence is different to the DNA methylase recognition sequence (ii) of the methylation-protectable restriction element.

7. The nucleic acid according to claim 6, wherein the type IIS restriction enzyme recognition sequence (i) and the switch DNA methylase recognition sequence of the methylase-switch element overlap such that the base modified by the switch DNA methylase of the methylase-switch element lies within the type IIS restriction enzyme recognition sequence (i) such that methylation by the switch DNA methylase blocks the overlapping type IIS restriction enzyme recognition sequence (i).

8. The nucleic acid according to claim 6 or 7, wherein the switch DNA methylase for the methylase-switch element comprises or consists of M.Osp807II or M.Sen0738I.

9. The nucleic acid according to any preceding claim, wherein the nucleic acid further comprises a non-switchable type IIS restriction enzyme recognition sequence opposing the type IIS restriction enzyme recognition sequence (i) of the methylation-protectable restriction element.

10. The nucleic acid according to claim 9, wherein the opposing non-switchable restriction enzyme recognition sequence comprises the same sequence as the type IIS restriction enzyme recognition sequence (i) of the methylation-protectable restriction element, and is recognised by the same type IIS restriction enzyme that recognises the type IIS restriction enzyme recognition sequence (i) of the methylation-protectable restriction element.

11. The nucleic acid according to any one of claims 1 to 8, wherein the nucleic acid comprises an opposing methylation-protectable restriction element.

12. The nucleic acid according to claim 11, wherein the type IIS restriction enzyme recognition sequences of the first methylation-protectable restriction element and opposing methylation-protectable restriction element are the same.

13. The nucleic acid according to any of claims 9 to 12, wherein the nucleic acid comprises a maintenance-type design element wherein the opposing type IIS restriction enzyme recognition sequence opposing the methylation-protectable restriction element is arranged to direct the restriction enzyme to cut the nucleic acid at the same site as the type IIS restriction enzyme directed by the type IIS restriction enzyme recognition sequence (i) of the methylation-protectable restriction element, such that the same overhangs are produced regardless of which type IIS restriction enzyme recognition sequence of the maintenance-type design element direct the cutting.

14. The nucleic acid according to any of claims 9 to 13, wherein the nucleic acid comprises an excision-type design element, wherein the type IIS restriction enzyme recognition sequence (i) of the methylation-protectable restriction element and the opposing type IIS restriction enzyme recognition sequence are positioned close enough together such that the cut site of the opposing type IIS restriction enzyme recognition sequence is at least partially within the sequence of the type IIS restriction enzyme recognition sequence (i) of the methylation-protectable restriction element or the cut site is within the sequence that is in between the sequence of the type IIS restriction enzyme recognition sequence (i) of the methylation-protectable restriction element and the start of its cut site; or wherein the nucleic acid comprises an excision-type design element where the opposing non-switchable type IIS restriction enzyme cuts x bases from the opposing type IIS restriction enzyme recognition sequence and generates y bases adhesive end, the distance (d) for the number of bases between the type IIS restriction enzyme recognition sequence (i) of the methylation-protectable restriction element and the opposing restriction enzyme recognition sequence is provided by the following equation: d≤2*x.

15. The nucleic acid according to any of claims 9 to 14, wherein the nucleic acid comprises an insertional-type design element, wherein a sequence-insert, comprising a comprise a functional sequence, is provided in between the methylation-protectable restriction element and the opposing type IIS restriction enzyme recognition sequence.

16. The nucleic acid according to any preceding claim, wherein the nucleic acid is a vector.

17. The nucleic acid according to any preceding claim, wherein the nucleic acid comprises at least two methylation-protectable restriction elements.

18. The nucleic acid according to claim 17, wherein the nucleic acid sequence between the cut sites of the two methylation-protectable restriction elements comprises two type IIS restriction enzyme recognition sequences, which respectively oppose the two methylation-protectable restriction elements.

19. The nucleic acid according to claim 17 or 18, wherein the nucleic acid sequence between the cut sites of the two methylation-protectable restriction elements is a discard sequence, optionally wherein the discard sequence comprises a selectable marker.

20. The nucleic acid according to claim 17, wherein the nucleic acid is a pre-cut linearized vector comprising a methylation-protectable restriction element at each end.

21. The nucleic acid according to any preceding claim, wherein the nucleic acid is: (a) isolated or derived from a bacterial strain that expresses a DNA methylase that recognises (and methylates) the DNA methylase recognition sequence (ii) of the methylation-protectable restriction element; or (b) methylated by a DNA methylase that recognises (and methylates) the DNA methylase recognition sequence.

22. The nucleic acid according to any preceding claim, wherein the nucleic acid is: (a) isolated or derived from a bacterial strain that expresses a switch DNA methylase that recognises (and methylates) the switch DNA methylase recognition sequence of the methylation-protectable restriction element; or (b) methylated by a switch DNA methylase that recognises (and methylates) the switch DNA methylase recognition sequence of the methylation-protectable restriction element.

23. The nucleic acid according to any preceding claim, wherein the nucleic acid is: (a) isolated or derived from a bacterial strain that expresses the sequence specific DNA binding protein, and associated guide nucleic acid where necessary; or (b) bound by (i.e. complexed with) the sequence specific DNA binding protein, and associated guide nucleic acid where necessary.

24. A method of assembling DNA comprising: providing a linearised methylated nucleic acid according to any preceding claim, wherein the methylation-protectable restriction element comprises a methylation-switch element that is switched OFF by methylation of the type IIS restriction enzyme recognition sequence with the switch DNA methylase; providing two or more DNA/insert fragments of interest for assembly with the linearised methylated nucleic acid, wherein a first DNA fragment comprises a complementary overhang for ligation with a first end of the linearised methylated nucleic acid, and a second DNA fragment comprises a complementary overhang for ligation with the other/second end of the linearised methylated nucleic acid; and (i) the first and second DNA/insert fragments further comprise complementary overhangs for ligation with each other; or (ii) the first and second DNA/insert fragments further comprise complementary overhangs for ligation with one or more further DNA/insert fragments having complementary overhangs, such that ligating the DNA fragments and the linearised methylated nucleic acid with a DNA ligase would result in a single assembled DNA molecule; and ligating the DNA fragments and linearised methylated nucleic acid with a ligase to form a single assembled DNA molecule comprising the sequence of the assembled DNA fragments flanked by the restriction enzyme recognition sequences of the methylation-protectable restriction elements.

25. A method of assembling DNA comprising: providing a linearised methylated nucleic acid according to any of claims 1 to 23, wherein the methylation-protectable restriction element comprises a methylation-switch element that is switched OFF by methylation of the type IIS restriction enzyme recognition sequence with the switch DNA methylase; providing a DNA/insert fragment of interest for assembly with the linearised methylated nucleic acid, wherein the DNA/insert fragment of interest comprises complementary overhangs for ligation with the linearised methylated nucleic acid; and ligating the DNA fragments and linearised methylated nucleic acid with a ligase to form a single assembled DNA molecule comprising the sequence of the assembled DNA fragments flanked by the restriction enzyme recognition sequences of the methylation-protectable restriction elements.

26. The method according to any of claim 24 or 25 wherein, the linearised nucleic acid is provided by providing a nucleic acid according to any of claims 1 to 23 in the form of a circular destination vector comprising two methylated methylation-protectable restriction elements and a discard sequence therebetween, wherein each methylated methylation-protectable restriction element is opposed by an opposing non-switchable restriction enzyme recognition sequence in the discard sequence, and further comprise the step of cutting the circular destination vector with restriction enzymes that recognise the opposing non-switchable restriction enzyme recognition sequences in the discard sequence, thereby leaving a linearised nucleic acid having overhangs defined by the restriction enzymes.

27. A method of assembling DNA comprising: providing a linearised methylated nucleic acid according to any of claims 1 to 23, wherein the type IIS restriction enzyme recognition sequence of the methylation-protectable restriction element is protected from cutting by methylation with the DNA methylase that recognises the DNA methylase recognition sequence (ii) of the methylation-protectable restriction element; providing two or more DNA/insert fragments of interest for assembly with the linearised methylated nucleic acid, wherein a first DNA fragment comprises a complementary overhang for ligation with a first end of the linearised methylated nucleic acid, and a second DNA fragment comprises a complementary overhang for ligation with the other/second end of the linearised methylated nucleic acid; and (i) the first and second DNA/insert fragments further comprise complementary overhangs for ligation with each other; or (ii) the first and second DNA/insert fragments further comprise complementary overhangs for ligation with one or more further DNA/insert fragments having complementary overhangs, such that ligating the DNA fragments and the linearised methylated nucleic acid with a DNA ligase would result in a single assembled DNA molecule; and ligating the DNA fragments and linearised methylated nucleic acid with a ligase to form a single assembled DNA molecule comprising the sequence of the assembled DNA fragments flanked by the restriction enzyme recognition sequences of the methylation-protectable restriction elements.

28. A method of assembling DNA comprising: providing a linearised methylated nucleic acid according to any of claims 1 to 23, wherein the type IIS restriction enzyme recognition sequence of the methylation-protectable restriction element is protected from cutting by methylation with the DNA methylase that recognises the DNA methylase recognition sequence (ii) of the methylation-protectable restriction element; providing a DNA/insert fragment of interest for assembly with the linearised methylated nucleic acid, wherein the DNA/insert fragment of interest comprises complementary overhangs for ligation with the linearised methylated nucleic acid; and ligating the DNA fragments and linearised methylated nucleic acid with a ligase to form a single assembled DNA molecule comprising the sequence of the assembled DNA fragments flanked by the restriction enzyme recognition sequences of the methylation-protectable restriction elements.

29. The method according to any of claim 27 or 28 wherein, the linearised nucleic acid is provided by providing a nucleic acid according to any one of claims 1 to 23 in the form of a circular destination vector comprising two pairs of opposing methylation-protectable restriction elements and a discard sequence therebetween, wherein one pair of opposing methylation-protectable restriction elements comprises an outside methylation-protectable restriction element that will remain in the vector after linearization and an opposing inside methylation-protectable restriction element that is in the discard sequence, and wherein the second pair of opposing methylation-protectable restriction elements also comprise an outside methylation-protectable restriction element that will remain in the vector after linearization and an opposing inside methylation-protectable restriction element that is in the discard sequence, wherein the opposing methylation-protectable restriction elements of a pair comprise different sequence specific DNA binding recognition sequences, wherein the outside methylation-protectable restriction elements are methylated and thereby protected from cutting by the type IIS restriction enzyme that recognises the type IIS recognition sequences of the outside methylation-protectable restriction elements, and the inside methylation-protectable restriction elements are not methylated and thereby not protected from cutting by the type IIS restriction enzyme that recognises the type IIS recognition sequences of the inside methylation-protectable restriction elements; and cutting the circular destination vector with the type IIS restriction enzyme that recognises the type IIS recognition sequences of the inside methylation-protectable restriction elements, thereby producing the linearised nucleic acid.

30. The method according to claim 29, wherein the outside methylation-protectable restriction elements are methylated and thereby protected from cutting by preparing/isolating the vector in a strain that expresses the DNA methylase that recognises the DNA methylase recognition sequence (ii) of the methylation-protectable restriction elements, but the strain does not express a functional sequence specific DNA binding protein that recognises the sequence specific DNA binding protein recognition sequence of the outside methylation-protectable restriction elements, and the inside methylation-protectable restriction elements are not methylated and thereby not protected from cutting as the strain expresses the DNA methylase that recognises the DNA methylase recognition sequence (ii) of the methylation-protectable restriction elements, and expresses a functional sequence specific DNA binding protein that recognises the sequence specific DNA binding protein recognition sequence of the inside methylation-protectable restriction elements.

31. The method according to any of claims 24 to 30, wherein the DNA fragment(s) of interest for assembly with the nucleic acid are provided in a circular donation vector, wherein the method comprises the step of cutting the circular donation vector to release the DNA fragment(s) of interest.

32. The method according to claim 31, wherein the circular donation vector comprises two methylation-protectable restriction elements with a DNA fragment of interest therebetween.

33. The method according to claim 32, wherein the circular donation vector is methylated or at least exposed to methylation by the DNA methylase that recognises the DNA methylase recognition sequence (ii) in the presence of the sequence specific DNA binding protein.

34. The method according to any one of claims 31 to 33, wherein the circular donor vector(s) is purified/isolated from a bacterial strain that expresses the sequence-specific DNA-binding protein that recognises the sequence-specific DNA-binding protein recognition sequence of the methylation-protectable restriction element and the DNA methylase that recognises the DNA methylase recognition sequence (ii) of the methylation-protectable restriction element; or wherein the circular donor vector(s) is methylated in vitro in the presence of the sequence-specific DNA-binding protein that recognises the sequence-specific DNA-binding protein recognition sequence of the methylation-protectable restriction element and the DNA methylase that recognises the DNA methylase recognition sequence (ii) of the methylation-protectable restriction element.

35. The method according to any one of claims 31 to 34, wherein the steps of restricting and ligating are combined, such that the circular destination vector, the donor vector(s), the restriction enzyme and the ligase are provided in the same composition.

36. A method of scarless DNA assembly of DNA fragments comprising the steps of: (A) providing a first intermediate vector, comprising the steps of: providing a first linearised methylated nucleic acid by providing an assembly vector comprising a nucleic acid according to any of claims 1 to 23, wherein the assembly vector comprises a maintenance-type design element and an excision-type design element flanking a discard sequence, wherein the type IIS restriction enzyme recognition sequences (i) of the maintenance-type design element and the excision-type design element are selectively methylated, such that the outside type IIS restriction enzyme recognition sequences (i) of the maintenance-type design element and excision-type design element in the vector backbone are methylated, and the inside type IIS restriction enzyme recognition sequences of the maintenance-type design element and excision-type design element in the discard sequence are not methylated, and cutting the assembly vector with the type IIS restriction enzyme that recognises the opposing type IIS restriction enzyme recognition sequences of the maintenance-type design element and excision-type design element that are in the discard sequence, and which are not methylated, further providing a first DNA/insert fragment for assembly with the first linearised methylated nucleic acid, the first DNA/insert fragment having overhang ends that are adapted to ligate to the overhang ends of the first linearised methylated nucleic acid, wherein any DNA methylase recognition sequences in the DNA fragment have been methylated with the DNA methylase that recognises the DNA methylase recognition sequence (i); ligating the first DNA/insert fragment for assembly and first linearised methylated nucleic acid with a ligase to form a first methylated intermediate vector comprising a first DNA/insert fragment for assembly flanked by methylation-protectable restriction elements; transforming the first methylated intermediate vector into a bacterial strain that expresses the DNA methylase that recognises the DNA methylase recognition sequence (i) and the sequence-specific DNA binding protein that recognises the sequence-specific DNA binding protein recognition sequence of the methylation-protectable restriction elements in the first methylated intermediate vector, such that any type IIS restriction enzyme recognition sequences in the first DNA/insert fragment are methylated and the type IIS restriction enzyme recognition sequence of the methylation-protectable restriction elements are not protected; isolating the first intermediate vector; (B) providing a second intermediate vector, comprising the steps of: providing a second linearised methylated nucleic acid by providing an assembly vector comprising a nucleic acid according to any of claims 1 to 23, wherein the assembly vector comprises a maintenance-type design element and an excision-type design element flanking a discard sequence, wherein the type IIS restriction enzyme recognition sequences (i) of the maintenance-type design element and the excision-type design element are selectively methylated, such that the outside type IIS restriction enzyme recognition sequences (i) of the maintenance-type design element and excision-type design element in the vector backbone are methylated, and the inside type IIS restriction enzyme recognition sequences of the maintenance-type design element and excision-type design element in the discard sequence are not methylated, and cutting the assembly vector with the type IIS restriction enzyme that recognises the opposing type IIS restriction enzyme recognition sequences of the maintenance-type design element and excision-type design element that are in the discard sequence, and which are not methylated, further providing a second DNA/insert fragment for assembly with the second linearised methylated nucleic acid, the second DNA/insert fragment having overhang ends that are adapted to ligate to the overhang ends of the second linearised methylated nucleic acid, wherein any DNA methylase recognition sequences in the second DNA/insert fragment have been methylated with the DNA methylase with the DNA methylase that recognises the DNA methylase recognition sequence (i); ligating the second DNA/insert fragment for assembly and second linearised methylated nucleic acid with a ligase to form a second methylated intermediate vector comprising a second DNA/insert fragment for assembly flanked by methylation-protectable restriction elements; transforming the second methylated intermediate vector into a bacterial strain that expresses the DNA methylase that recognises the DNA methylase recognition sequence (i), and the sequence-specific DNA binding protein that recognises the sequence-specific DNA binding protein recognition sequence of the methylation-protectable restriction elements in the second methylated intermediate vector, such that any type IIS restriction enzyme recognition sequences in the second DNA/insert fragment are methylated and the type IIS restriction enzyme recognition sequence of the methylation-protectable restriction elements are not protected; isolating the second intermediate vector; (C) cutting the first intermediate vector with a type IIS restriction enzyme that recognises the type IIS restriction enzyme recognition sequences of the methylation-protectable restriction elements, thereby forming a first adapted DNA/insert fragment that comprises a maintained-overhang sequence that is determined by the maintenance-type design element and an opposing native-overhang sequence that is determined by the native sequence of the first DNA/insert fragment for assembly; (D) cutting the second intermediate vector with a type IIS restriction enzyme that recognises the type IIS restriction enzyme recognition sequences of the methylation-protectable restriction elements, thereby forming a second adapted DNA fragment insert that comprises a maintained-overhang sequence that is determined by the maintenance-type design element and an opposing native-overhang sequence that is determined by the native sequence of the second DNA fragment for assembly; wherein (i) the first and second adapted DNA/insert fragments are end fragments wherein their native-overhang sequences are complementary, such that they are arranged to ligate together; or (ii) one or more middle DNA/insert fragments for assembly are provided wherein the first and second adapted DNA/insert fragments are respective end fragments in the assembly, and the one or more middle DNA fragments are arranged to be ligated between the first and second adapted DNA/insert fragments via complementary native-overhang sequences; further comprising the step of ligating together, with a ligase, the first and second adapted DNA/insert fragments, or the first and second adapted DNA/insert fragments and one or more middle DNA/insert fragments, to form an assembled DNA fragment having maintained-overhangs at each end, and optionally ligating the assembled DNA fragment into a linearised destination vector.

37. The method according to claim 36, wherein a middle DNA/insert fragment for assembly is provided by providing a further intermediate vector, comprising the steps of: providing a further linearised methylated nucleic acid by providing an assembly vector comprising a nucleic acid according to any of claims 1 to 23, wherein the assembly vector comprises a pair of excision-type design elements flanking a discard sequence, wherein the type IIS restriction enzyme recognition sequences (i) of the excision-type design elements are selectively methylated, such that the outside type IIS restriction enzyme recognition sequences (i) of the excision-type design elements in the vector backbone are methylated, and the inside type IIS restriction enzyme recognition sequences of the excision-type design elements in the discard sequence are not methylated, and cutting the assembly vector with the type IIS restriction enzyme that recognises the opposing type IIS restriction enzyme recognition sequences of the excision-type design elements that are in the discard sequence, and which are not methylated, further providing a middle DNA/insert fragment for assembly with the further linearised methylated nucleic acid, the middle DNA/insert fragment having overhang ends that are adapted to ligate to the overhang ends of the further linearised methylated nucleic acid, wherein any DNA methylase recognition sequences in the second DNA/insert fragment have been methylated with a DNA methylase that recognises the DNA methylase recognition sequence (ii) of the methylation-protectable restriction element; ligating the middle DNA/insert fragment for assembly and further linearised methylated nucleic acid with a ligase to form a further methylated intermediate vector comprising a middle DNA/insert fragment for assembly flanked by methylation-protectable restriction elements; transforming the further methylated intermediate vector into a bacterial strain that expresses the DNA methylase that recognises the DNA methylase recognition sequence (ii) of the methylation-protectable restriction element, and expresses the sequence-specific DNA binding protein that recognises the sequence-specific DNA binding protein recognition sequences of the methylation-protectable restriction element, such that any type IIS restriction enzyme recognition sequences in the second DNA/insert fragment are methylated and the type IIS restriction enzyme recognition sequence of the methylation-protectable restriction elements are not protected; isolating the further intermediate vector; cutting the further intermediate vector with a type IIS restriction enzyme that recognises the type IIS restriction enzyme recognition sequences of the methylation-protectable restriction elements, thereby forming a middle adapted DNA/insert fragment that comprises opposing native-overhang sequences that are determined by the native sequences of the middle DNA/insert fragment for assembly.

38. The method according to any of claim 36 or 37, wherein the first, second and/or middle DNA/insert fragment(s) for assembly are provided in one or more circular donation vectors, wherein the method comprises the step of cutting the circular donation vectors to release the DNA/insert fragment(s) for assembly.

39. The method according to claim 38, wherein the circular donation vectors comprise two methylation-protectable restriction elements with the DNA/insert fragment(s) for assembly therebetween.

40. The method according to claim 39, wherein the circular donation vectors are methylated or at least exposed to methylation by the DNA methylase that recognises the DNA methylase recognition sequence (ii) in the presence of the sequence specific DNA binding protein.

41. The method according to any one of claims 38 to 40, wherein the circular donor vectors are purified/isolated from a bacterial strain that expresses the sequence-specific DNA-binding protein that recognises the sequence-specific DNA-binding protein recognition sequence of the methylation-protectable restriction element and the DNA methylase that recognises the DNA methylase recognition sequence (ii) of the methylation-protectable restriction element; or wherein the circular donor vectors are methylated in vitro in the presence of the sequence-specific DNA-binding protein that recognises the sequence-specific DNA-binding protein recognition sequence of the methylation-protectable restriction element and the DNA methylase that recognises the DNA methylase recognition sequence (ii) of the methylation-protectable restriction element.

42. Use of a sequence-specific DNA binding protein for controlling the methylation and/or restriction of the methylation-protectable restriction element of the nucleic acid according to any of claims 1 to 23.

43. The use according to claim 42, wherein the sequence-specific DNA binding protein is used to sterically prevent the binding of the DNA methylase that recognises the DNA methylase recognition sequence (ii) of the methylation-protectable restriction element.

44. Use of a nucleic acid comprising opposing BsaI/M2.Eco31I recognition sequences in combination with a sequence-specific DNA binding protein.

45. A method of methylation protecting BsaI recognition sequences in a vector comprising the nucleic acid according to any of claims 1 to 23, wherein the vector comprises at least one BsaI recognition sequence that is not part of the methylation-protectable restriction element of the nucleic acid, wherein the methylation comprises methylating the vector with M2.Eco31I in the presence of the sequence-specific DNA binding protein which recognises and binds to the sequence-specific DNA binding protein recognition sequence of the methylation-protectable restriction element.

46. A modified bacterial strain that is modified to express the DNA methylase that recognises the DNA methylase recognition sequence (ii) of the methylation-protectable restriction element of the nucleic acid according to any of claims 1 to 23, and the sequence-specific DNA binding protein.

47. Use of a modified bacterial strain that is modified to express a DNA methylase that recognises the DNA methylase recognition sequence (ii) of the methylation-protectable restriction element of the nucleic acid according to any of claims 1 to 23, for manufacturing a nucleic acid molecule according to any of claims 1 to 23, and wherein the modified bacterial strain is further be modified to express a sequence-specific DNA-binding protein.

48. A composition comprising the nucleic acid according to any of claims 1 to 23, wherein the composition further comprises one or more of: a) a DNA methylase that recognises the DNA methylase recognition sequence (ii) of the methylation-protectable restriction element; b) a sequence-specific DNA-binding protein; and c) a switch DNA methylase; and optionally a DNA ligase.

49. A kit comprising the nucleic acid according to any of claims 1 to 23, wherein the composition further comprises one or more of: a) a DNA methylase that recognises the DNA methylase recognition sequence (ii) of the methylation-protectable restriction element; b) a sequence-specific DNA-binding protein; and c) a switch DNA methylase; and optionally a DNA ligase.

50. The kit according to claim 49, further comprising a modified bacterial strain that is modified to express one or more DNA methylases and/or a sequence-specific DNA binding protein, optionally with any guide nucleic acid as necessary.

51. A host cell comprising nucleic acid according to any of claims 1 to 23, wherein the host cell further comprises nucleic acid for the expression of one or more of: a) a DNA methylase that recognises the DNA methylase recognition sequence (ii) of the methylation-protectable restriction element; b) a sequence-specific DNA-binding protein; and c) a switch DNA methylase.

52. Use of the nucleic acid according to any of claims 1 to 23 for assembling DNA fragments of interest, wherein the use of the nucleic acid is with one or more of: a) a DNA methylase that recognises the DNA methylase recognition sequence (ii) of the methylation-protectable restriction element; b) a sequence-specific DNA-binding protein described herein; and c) a switch DNA methylase described herein.

53. Use of a sequence-specific DNA binding protein to protect a type IIS restriction enzyme recognition site from methylation by a DNA methylase that is capable of recognition and methylation of the type IIS restriction enzyme recognition site.

54. Use of a sequence-specific DNA binding protein for steric hindrance of methylation and/or restriction of a first type IIS restriction enzyme recognition sequence in a nucleic acid, wherein the sterically blocking is effected by binding to or near the first type IIS restriction enzyme recognition sequence in the nucleic acid, wherein the nucleic acid comprises a second type IIS restriction enzyme recognition sequence that is the same sequence as the first type IIS restriction enzyme recognition sequence and wherein the second type IIS restriction enzyme recognition sequence is not arranged to be bound by or sterically hindered by the sequence-specific DNA binding protein

55. A method of producing a nucleic acid in the form of a vector according to any of claims 16 to 23, the method comprising transforming a nucleic acid in the form of a vector according to claims 16 to 23 into a bacterial strain that is capable of replicating the vector, and wherein the bacterial strain is modified to express: a) a DNA methylase that recognises the DNA methylase recognition sequence (ii) of the methylation-protectable restriction element, and b) a sequence-specific DNA-binding protein described herein, for example dCas9 (optionally with the guide nucleic acid); and optionally growing the bacteria, such that the nucleic acid is replicated and/or isolating the nucleic acid from the bacteria.

56. A nucleic acid-protein complex comprising the nucleic acid according to any one of claims 1-23 and a sequence specific DNA binding protein that is arranged to bind to the associated sequence specific DNA binding protein recognition sequence in the nucleic acid.

Description

[0370] Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings.

[0371] FIG. 1. The methylation switching approach in the original MetClo system

[0372] A. A standard type IIS restriction site such as BsaI site (boxed) can be combined with a ‘switch’ methylase (M.Osp807II) recognition sequence (highlighted in grey, with the methylated base in bold) that partially overlaps with the restriction site to create a combined type IIS restriction site. The combined site is switchable by the ‘switch’ methylase in that methylation of the site by the M.Osp807II methylase blocks the restriction of the site by BsaI. The methylation can be removed by producing the plasmid in a normal E. coli strain that lacks the M.Osp807II switch methylase activity. An overlapping site that can be switched on or off by the M.Osp807II switch methylase is referred to as an M.Osp807II methylase-switchable site.

[0373] B. In contrast, a typical standard BsaI site does not have an overlap with an M.Osp807II switch methylase recognition site and so will not be methylated by the M.Osp807II switch methylase. Such sites can be restricted by BsaI and this cleavage is unaffected by the presence of absence of the of M.Osp807II switch methylase. The switching process can be represented using the symbols to the right, where the open triangles depict an unmethylated BsaI site, and the black triangles depict a methylated BsaI sites.

[0374] FIG. 2. Methylation switching using M.Sen0738I

[0375] A. A standard type IIS restriction site such as BsaI site (boxed) can be combined with a ‘switch’ methylase (M.Sen0738I) recognition sequence (highlighted in grey, with the methylated base in bold) that partially overlaps with the restriction site to create a ‘switchable’ type IIS restriction site. The combined site is switchable because methylation of the site by the M.Sen0738I switch methylase blocks the restriction of the site by BsaI. The methylation can be removed by producing the plasmid in a normal E. coli strain that does not express the M.Sen0738I switch methylase. An overlapping site that can be switched on or off in this way by the M.Sen0738I switch methylase is referred to as an M.Sen0738I methylase-switchable site.

[0376] B. In contrast, a typical standard BsaI site does not have an overlap with an M.Sen0738I switch methylase recognition site and so will not be methylated by the M.Sen0738I switch methylase. Such sites can be restricted by BsaI and this cleavage is unaffected by the presence of absence of the of M.Sen0738I switch methylase. The switching process can be represented using the symbols to the right, where the open triangles depict an unmethylated BsaI site, and the black triangles depict a methylated BsaI sites.

[0377] FIG. 3. Methylation switching in vivo using M.Osp807II and M.Sen0738I

[0378] A. Diagram depicting the strains for methylation switching in vivo. The DH10B-M.Osp807II expresses M.Osp807II from the arsB locus under the J23100 promoter, and a zeocin selection marker under EM2KC promoter. The strain DH10B-2W148R for M.Sen0738I-based methylation switching expresses M.Sen0738I and S.Sen0738I under J23100 promoter in tandem along with a zeocin selection marker under EM2KC promoter.

[0379] B. Experimental designs to test methylation switching in vivo. The test plasmids (pMOP_BsaNC for DH10B-M.Osp807II, and pMOP_testN10 for DH10B-2W148R) contain a head-to-head methylation-switchable BsaI site ˜220 bp away from an internal BamHI site, and a non-switchable BsaI site ˜370 bp away from the BamHI site. Restriction digestion by BamHI and BsaI of test plasmid prepared from a normal E. coli strain that does not express the switch methylase would result in cutting at both BsaI sites and the internal BamHI site resulting in release of both the ˜220 bp and ˜370 bp fragments from the vector backbone. Restriction digestion of test plasmid prepared from a strain expressing the switch methylase would not release the ˜220 bp fragment due to blocking of the methylation-switchable BsaI restriction sites by in vivo methylation. The head-to-head arrangement of overlapping methylation/restriction site allows the same assay to be used to detect any residual single strand nicking activity of the restriction enzyme towards the methylated restriction site.

[0380] C. Agarose gel electrophoresis analysis of the test plasmids prepared in E. coli strains DH10B-M.Osp807II (MOsp) or DH10B-2W148R (2W148R) that express the switch methylases M.Osp807II and M.Sen0738I respectively and digested with BamHI and BsaI-HFv2. The results show that in vivo methylation by each of the methylases successfully blocked the switchable BsaI restriction site.

[0381] FIG. 4. The methylation protection approach

[0382] A. A standard type IIS restriction enzyme site such as BsaI (boxed) can be methylated by the M2.Eco31I ‘protection’ methylase at the 4th base of the top strand (in bold) when the plasmid is prepared in an E. coli strain that expresses the M2.Eco31I methylase, dCas9 and a synthetic guide RNA (sgRNA) targeting a specific sequence (the standard BsaI site must not overlap with the sgRNA-guided dCas9 binding sequence).

[0383] B. Combining a BsaI site (boxed) with the sgRNA-guided dCas9 binding site (the seed sequence and PAM sequence critical for dCas9 specificity are highlighted in grey) creates a BsaI site that overlaps with the dCas9-binding site. Preparation of the plasmid in an E. coli strain that expresses the M2.Eco31I protection methylase, dCas9 and the sgRNA that recognizes this combined site results in dCas9 binding to this site, which blocks the site from methylation by the M2.Eco31I protection methylase. Such site is referred to as a dCas9-protectable site. The methylation protection process can be represented by the symbols to the right; open triangles depicts an unmethylated BsaI site, and black triangles depict a methylated BsaI site.

[0384] FIG. 5. Methylation protection using dCas9

[0385] A. Diagram depicting the E. coli strains for methylation protection. The strains express a synthetic guide RNA (guide #360 for strain DH10B-2W213R or guide #401 for strain DH10B-2W214R, driven by a J23119 promoter and with L3S2P21 terminator), dCas9 (driven by a J23119 promoter with a B0034m* ribosomal binding sequence (RBS) and L3S1P51 terminator), the M2.Eco31I methylase (driven by a J23112 promoter with a B0034m* RBS and L3S1P32 terminator) and a zeocin-resistance gene (ZeoR, driven by an EM2KC promoter) from the arsB locus of the E. coli chromosome.

[0386] B. Diagram of the plasmids used to test the methylation protection approach. The test plasmids (pMOP_testN8 for DH10B-2W213R and pMOP_testN20 for DH10B-2W214R) carry a dCas9-protectable BsaI site for the corresponding guide RNA sequence, a BamHI site, and a normal (non-switchable) BsaI site. If this test plasmid is prepared in a normal strain, such as DH10B, then both BsaI sites should be cut by BsaI. Therefore, digestion with BamHI and BsaI should generate two small fragments of ˜370 bp and ˜220 bp. If this test plasmid is prepared in the methylation protection strain that expresses the protection methylase, dCas9 and the corresponding guide RNA, then the normal BsaI site will be methylated and so will not be cut, but the methylation-protectable BsaI site will bound by the dCas9 and so will not be methylated. When the plasmid is exposed to BamHI and BsaI, of the two BsaI sites, only the unmethylated methylation-protectable BsaI site can be cut and this results in only one small fragment of ˜220 bp.

[0387] C. Gel electrophoresis of the test plasmids prepared from the DH10B or methylation protection strains following BamHI and BsaI-HFv2 digestion. The digested samples demonstrate the expected pattern as predicted in B, confirming successful protection from methylation of the dCas9-protectable BsaI site by the guided dCas9 in the DH10B-2W213R and DH10B-2W214R strains.

[0388] FIG. 6. Combined BsaI sites invoking both methylation-protection and methylation-switching. (compatible with M.Osp807II-based methylation switching)

[0389] A. Combining a dCas9-protectable BsaI site (the nucleotides required for dCas9 binding specificity are highlighted in grey and the BsaI site is boxed) and an M.Osp807II protection methylase site (highlighted in grey with the methylated bases in bold) generates a combined BsaI site that is both dCas9-protectable and M.Osp807II-switchable.

[0390] B. Systems deploying both methylation-protection and methylation-switching may contain four different types of BsaI sites. BsaI sites that are dCas9-protectable can be cut if the plasmid was produced in a strain that coexpresses the M2.Eco31I protection methylase, dCas9 and the appropriate sgRNA. However, the other standard BsaI sites in a plasmid produced in this strain are methylated by the M2.Eco31I protection methylase. BsaI sites that are methylation-switchable are not cut by BsaI if the plasmid has been produced in a strain that expresses the M.Osp807II switch methylase as these sites are then methylated and so protected from digestion by BsaI. Any non-switchable BsaI sites will not be methylated and can be cut by the enzyme if the plasmid has been produced in such a strain that expresses the M.Osp807II switch methylase.

[0391] FIG. 7. Combined BsaI sites invoking both methylation-protection and methylation-switching. (compatible with M.Sen0738I-based methylation switching)

[0392] A. Combining a dCas9-protectable BsaI site (the nucleotides required for dCas9 binding specificity are highlighted in grey and the BsaI site is boxed) and an M.Sen0738I protection methylase site (highlighted in grey with the methylated bases in bold) generates a combined BsaI site that is both dCas9-protectable and M.Sen0738I-switchable.

[0393] B. Systems deploying both methylation-protection and methylation-switching may contain four different types of BsaI sites. BsaI sites that are dCas9-protectable can be cut if the plasmid was produced in a strain that coexpresses the M2.Eco31I protection methylase, dCas9 and the appropriate sgRNA. However, the other standard BsaI sites in a plasmid produced in this strain are methylated by the M2.Eco31I protection methylase. BsaI sites that are methylation-switchable are not cut by BsaI if the plasmid has been produced in a strain that expresses the M.Sen0738I switch methylase as these sites are then methylated and so protected from digestion by BsaI. Any non-switchable BsaI sites will not be methylated and can be cut by the enzyme if the plasmid has been produced in such a strain that expresses the M.Sen0738I switch methylase.

[0394] FIG. 8. Universal Assembly system based on both methylation-switching and methylation-protection (M.Osp807II-based methylation switching and dCas9/guide #360-based methylation protection as an example)

[0395] The diagram depicts the design of the implemented Universal Assembly system using methylation-protection and methylation-switching. The donor plasmids contain inserts flanked by dCas9/guide #360-protectable and M.Osp807II-switchable BsaI sites (the BsaI site is boxed and the nucleotides critical for dCas9 binding specificity are highlighted in grey) that would generate compatible adhesive ends following BsaI restriction. The internal standard BsaI sites in the insert do not overlap with guided-dCas9 binding sequence and therefore are not protected from methylation by the M2.Eco31I protection methylase. Transformation of the insert plasmids into an E. coli strain (DH10B-2W213R) that expresses the M2.Eco31I protection methylase, dCas9 and a sgRNA guide #360 targeting the dCas9-protectable BsaI site results in selective methylation of internal BsaI sites within the insert (the methylated base is shown in bold). The assembly vector contains a negative selection marker LacZalpha flanked by head-to-head BsaI sites that generate the same adhesive end as that produced by BsaI-based excision of the insert from the donor plasmid. The outer pair of BsaI sites are dCas9/guide #360-protectable and M.Osp807II methylation-switchable, whereas the inner pair of BsaI sites are nonswitchable. Preparation of the assembly recipient vector in an E. coli strain (DH10B-M.Osp807II) expressing the M.Osp807II switch methylase results in specific methylation of the outer pair of BsaI sites. The methylated insert donor plasmids and assembly recipient vector plasmids can be cut with BsaI. Ligation of the cut fragments in a one-pot reaction that contains BsaI favours the generation of correctly assembled DNA comprising the inserts ligated together with the assembly vector backbone, in which all the BsaI sites are methylated. Transformation into a normal E. coli (DH10B) that lacks dCas9-protection and methylation-switching activity removes methylation of all the BsaI sites in the assembled plasmid.

[0396] FIG. 9. Universal Assembly system based on both methylation-switching and methylation-protection (M.Sen0738I-based methylation switching and dCas9/guide #360-based methylation protection as an example)

[0397] The diagram depicts the design of the implemented Universal Assembly system using methylation-protection and methylation-switching. The donor plasmids contain inserts flanked by dCas9/guide #401-protectable and M.Sen0738I-switchable BsaI sites (the BsaI site is boxed and the nucleotides critical for dCas9 binding specificity are highlighted in grey) that would generate compatible adhesive ends following BsaI restriction. The internal standard BsaI sites in the insert do not overlap with guided-dCas9 binding sequence and therefore are not protected from methylation by the M2.Eco31I protection methylase. Transformation of the insert plasmids into an E. coli strain (DH10B-2W214R) that expresses the M2.Eco31I protection methylase, dCas9 and a sgRNA guide #401 targeting the dCas9-protectable BsaI site results in selective methylation of internal BsaI sites within the insert (the methylated base is shown in bold). The assembly vector contains a negative selection marker LacZalpha flanked by head-to-head BsaI sites that generate the same adhesive end as that produced by BsaI-based excision of the insert from the donor plasmid. The outer pair of BsaI sites are dCas9/guide #401-protectable and M.Sen0738I methylation-switchable, whereas the inner pair of BsaI sites are nonswitchable. Preparation of the assembly recipient vector in an E. coli strain (DH10B-2W148R) expressing the M.Sen0738I switch methylase results in specific methylation of the outer pair of BsaI sites. The methylated insert donor plasmids and assembly recipient vector plasmids can be cut with BsaI. Ligation of the cut fragments in a one-pot reaction that contains BsaI favours the generation of correctly assembled DNA comprising the inserts ligated together with the assembly vector backbone, in which all the BsaI sites are methylated. Transformation into a normal E. coli (DH10B) that lacks dCas9-protection and methylation-switching activity removes methylation of all the BsaI sites in the assembled plasmid.

[0398] FIG. 10. Universal Assembly system based on both methylation-switching and methylation-protection (M.Osp807II-based methylation switching and dCas9/guide #360-based methylation protection as an example, direct transformation into DH10B-2W213R).

[0399] The diagram depicts the basic design of Universal Assembly with the assembled DNA directly transformed into an E. coli strain that deploys methylation-protection. The insert donor plasmids contain inserts flanked by dCas9/guide #360 methylation-protectable and M.Osp807II methylase-switchable BsaI sites (the BsaI site is boxed, the nucleotides critical for dCas9 specificity are highlighted in grey) that would generate mutually compatible adhesive ends following BsaI restriction. The internal standard BsaI sites in the insert do not overlap with the sequence that is the target of the guide RNA for the dCas9 and therefore are not bound by dCas9 and so are methylated by the M2.Eco31I protection methylase. Preparation of the insert plasmids in an E. coli strain (DH10B-2W213R) that expresses the M2.Eco31I protection methylase, dCas9 and an sgRNA guide #360 targeting the dCas9/guide #360 methylation-protectable BsaI results in specific methylation of standard internal BsaI sites within the insert (the methylated base is shown in bold). The assembly vector contains a negative selection marker LacZalpha flanked by head-to-head BsaI sites that generate the same adhesive end. The outer pair of BsaI sites are dCas9/guide #360-protectable and M.Osp807II methylase-switchable, whereas the inner pair are non-switchable. Preparation of the assembly vector in an E. coli strain expressing M.Osp807II (DH10B-M.Osp807II) results in specific methylation of the outer pair of BsaI sites. The methylated insert plasmids and assembly vector can be cut with BsaI. Ligation of the cut fragments in a one-pot reaction that contains BsaI favours the generation of correctly assembled DNA comprising the inserts ligated together with the assembly vector backbone, in which all the BsaI sites are methylated. Transformation into in the E. coli strain (DH10B-2W213R) that expresses the M2.Eco31I protection methylase, dCas9 and an sgRNA guide #360 targeting the dCas9/guide #360-protectable BsaI site, results in a plasmid that has methylation at all the BsaI sites in the insert, but no methylation at the dCas9/guide #360-protectable insert-flanking BsaI sites. The assembled DNA carries a similar methylation pattern on its BsaI sites to the original insert plasmids and so, therefore can be used directly for the next round of Universal Assembly.

[0400] FIG. 11. Universal Assembly system based on both methylation-switching and methylation-protection (M.Sen0738I-based methylation switching and dCas9/guide #401-based methylation protection as an example, direct transformation into DH10B-2W214R).

[0401] The diagram depicts the basic design of Universal Assembly with the assembled DNA directly transformed into an E. coli strain that deploys the methylation-protection principle. The insert donor plasmids contain inserts flanked by dCas9/guide #401-protectable and M.Sen0738I methylase-switchable BsaI sites (the BsaI site is boxed, the nucleotides critical for dCas9 specificity are highlighted in grey) that would generate mutually compatible adhesive ends following BsaI restriction. The internal standard BsaI sites in the insert do not overlap with the sequence that is the target of the guide RNA for the dCas9 and therefore are not bound by dCas9 and so are methylated by the M2.Eco31I protection methylase. Preparation of the insert plasmids in an E. coli strain (DH10B-2W214R) that expresses the M2.Eco31I protection methylase, dCas9 and an sgRNA guide #401 targeting the dCas9/guide #401-protectable BsaI results in specific methylation of standard internal BsaI sites within the insert (the methylated base is shown in bold). The assembly vector contains a negative selection marker LacZalpha flanked by head-to-head BsaI sites that generate the same adhesive end. The outer pair of BsaI sites are dCas9/guide #401-protectable and M.Sen0738I methylase-switchable, whereas the inner pair are non-switchable. Preparation of the assembly vector in an E. coli strain expressing M.Sen0738I (DH10B-2W148R) results in specific methylation of the outer pair of BsaI sites. The methylated insert plasmids and assembly vector can be cut with BsaI. Ligation of the cut fragments in a one-pot reaction that contains BsaI favours the generation of correctly assembled DNA comprising the inserts ligated together with the assembly vector backbone, in which all the BsaI sites are methylated. Transformation into in the E. coli strain (DH10B-2W214R) that expresses the M2.Eco31I protection methylase, dCas9 and an sgRNA guide #401 targeting the dCas9/guide #401-protectable BsaI site, results in a plasmid that has methylation at all the BsaI sites in the insert, but no methylation at the dCas9/guide #360-protectable insert-flanking BsaI sites. The assembled DNA carries a similar methylation pattern on its BsaI sites to the original insert plasmids and so, therefore can be used directly for the next round of Universal Assembly.

[0402] FIG. 12. Practical DNA assembly using Universal Assembly

[0403] A. Diagram of the DNA fragment to assemble. Four fragments of human DNA (FragA, FragB, FragC and FragD), each containing an internal BsaI site were assembled together to produce a 3.6 kb fragment as the final product.

[0404] B. Gel electrophoresis of BsaI digestion of insert plasmids with pMOK360 backbone prepared in the normal E. coli strain, DH10B, (−) or the E. coli strain DH10B-2W213R (+) that expresses dCas9, a guide RNA guide #360 and the M2.Eco31I protection methylase following BsaI-HFv2 digestion. dCas9-protection prevents methylation of the insert-flanking dCas9-protectable BsaI sites, but the internal BsaI sites are methylated and so cannot be cut by BsaI, resulting in larger complete insert fragments that are suitable for assembly. For insert plasmids prepared in DH10B-2W213R, BsaI-HFv2 digestion of insert plasmids with both flanking BsaI sites fully protected from methylation generates a ˜4.3 kb vector backbone, whereas BsaI-HFv2 digestion of insert plasmids with one of the two flanking BsaI sites protected from methylation generates a single ˜5.3 kb fragment.

[0405] C. Gel electrophoresis of BsaI digestion of insert plasmids with pMOK401 backbone prepared in the normal E. coli strain, DH10B, (−) or the E. coli strain DH10B-2W214R (+) that expresses dCas9, a guide RNA guide #401 and the M2.Eco31I protection methylase following BsaI-HFv2 digestion. The sequence-specific methylation-protection prevents methylation of the insert-flanking dCas9-protectable BsaI sites, but the internal BsaI sites are methylated and so cannot be cut by BsaI, resulting in larger complete insert fragments that are suitable for assembly. For insert plasmids prepared in DH10B-2W214R, BsaI-HFv2 digestion of insert plasmids with both flanking BsaI sites fully protected from methylation generates a ˜4.3 kb vector backbone, whereas BsaI-HFv2 digestion of insert plasmids with one of the two flanking BsaI sites protected from methylation generates a single ˜5.3 kb fragment.

[0406] D. Gel electrophoresis of DNA clones assembled into DH10B-2W213R cells by Universal Assembly from insert plasmids prepared in DH10B-2W213R following digestion by DraIII, which releases the assembled fragment through DraIII sites in the vector backbones, but does not cut inside the assembled 3.6 kb fragment as the assembled 3.6 kb DNA lacks DraIII sites. 7 out 8 of the assembled 8 clones were verified by DNA sequencing (+).

[0407] E. Gel electrophoresis of DNA clones assembled into DH10B-2W214R cells by Universal Assembly from insert plasmids prepared in DH10B-2W214R following digestion by DraIII, which releases the assembled fragment through DraIII sites in the vector backbones, but does not cut inside the assembled 3.6 kb fragment as the assembled 3.6 kb DNA lacks DraIII sites. 7 out 8 of the assembled 8 clones were verified by DNA sequencing (+).

[0408] F. Gel electrophoresis of DNA clones assembled into DH10B-2W213R or DH10B-2W214R by Universal Assembly following digestion by BsaI-HFv2. For correctly assembled DNA with internal BsaI sites completely blocked by M2.Eco31I methylation and both flanking BsaI sites fully protected from methylation, BsaI-HFv2 digestion generates a 4.4 kb vector backbone and a 3.6 kb insert DNA, whereas for assembled DNA with one of the two flanking BsaI sites protected from methylation, BsaI-HFv2 digestion generates a single 8.0 kb fragment.

[0409] FIG. 13. Basic concepts of hierarchical assembly vector design

[0410] In a hierarchical assembly process using a single type IIS restriction enzyme, the insert fragments are flanked by type IIS restriction sites (boxed) in insert plasmids. One pot assembly with assembly vector generates assembled fragment flanked by the same restriction site in the assembled plasmids. The overhang sequence flanking the insert fragment and involved in ligation with the assembly vector backbone is referred to as “pre-assembly overhang” (‘aaaa’ and ‘cccc’), and the overhang sequence flanking the assembled fragment after DNA assembly is referred to as “post-assembly overhang” (‘dddd’ and ‘eeee’). The pre-assembly overhang and post-assembly overhang may or may not be the same depending on the vector design.

[0411] FIG. 14. Typical designs of hierarchical assembly vector using head-to-head restriction sites

[0412] Head-to-head arrangement of type IIS restriction sites can be used for design of assembly vector for hierarchical DNA assembly using a single type IIS restriction site. In the assembly vector, the negative selection marker LacZalpha is flanked by head-to-head type IIS restriction sites (BsaI sites as an example). The inside sites (boxed in solid line) close to LacZalpha are functional and once cut with BsaI generates adhesive ends compatible with the insert fragments (‘aaaa’ and ‘cccc’) for ligation. The outside sites (boxed in dotted line) close to the assembly vector backbone are blocked by DNA methylation by the switch methylase (methylated base in bold). Different designs of assembly vector can generate assembled fragments with post-assembly overhang sequence identical to (A), or completely different from (B) the pre-assembly overhang sequence.

[0413] FIG. 15. Maintenance type design

[0414] A. DNA assembly process using vectors with maintenance type design. The maintenance type design contains a functional inside restriction site (boxed in solid line) and a methylated outside restriction site (boxed in dotted line, methylated base in bold) blocked by methylation switching using M.Osp807II or M.Sen0738I in a head-to-head arrangement. The distance between the outside restriction site and the pre-assembly overhang sequence ‘aaaa’ (11 bp) is the same as the distance between the inside restriction site and the pre-assembly overhang sequence (11 bp). Restriction with BsaI generates vector backbone containing the methylated restriction site, which can be ligated with the cut insert fragment flanked by pre-assembly overhang sequence (‘aaaa’). Following transformation into an E. coli strain that lacks methylation switching activity, methylation in the outside restriction site is lost and the assembled fragment can be cut with BsaI and generates assembled fragment flanked by post-assembly overhang sequence identical to the pre-assembly overhang sequence (‘aaaa’).

[0415] B. Abbreviation of the assembly process.

[0416] FIG. 16. Excision-type design of BsaI site with 4 bp excision

[0417] A. DNA assembly process using vectors with an excision-type design. The excision-type design contains a functional inside restriction site (boxed in solid line) and a methylated outside restriction site (boxed in dotted line, methylated base in bold) blocked by methylation switching using M.Osp807II or M.Sen0738I in a head-to-head arrangement. The distance between the outside restriction site and the pre-assembly overhang sequence ‘CTCN’ (7 bp) is 4 bp less than the distance between the inside restriction site and the pre-assembly overhang sequence (11 bp). Restriction with BsaI generates vector backbone containing a partial methylated restriction site, which can be ligated with the cut insert fragment flanked by pre-assembly overhang sequence (‘CTCN’) to reconstitute a functional outside restriction site. Following transformation into an E. coli strain that lacks methylation switching activity, methylation in the outside restriction site is lost and the assembled fragment can be cut with BsaI and generates assembled fragment flanked by post-assembly overhang sequence (‘xxxx’). Compared with the insert fragment, the assembled fragment has had 4 bp excised from the end of the sequence.

[0418] B. Abbreviation of the assembly process.

[0419] FIG. 17. Problem with 7 bp excision-type design of methylation-switching BsaI system

[0420] With a design of assembly vector that potentially leads to excision of 7 bp at the end of the insert DNA during the assembly process, the insert fragment must contain a functional BsaI site inside the BsaI site flanking the insert fragment, in order to reconstitute the outside BsaI site in the assembly vector backbone. Because the insert plasmid contains two functional BsaI sites, digestion with BsaI generates a mixture of two insert fragments with different flanking overhang sequences, which will interfere with the assembly process.

[0421] FIG. 18. Excision-type design of BsaI site with 6 bp excision

[0422] A. DNA assembly process using vectors with excision-type design. The excision-type design contains a functional inside restriction site (boxed in solid line) and a methylated outside restriction site (boxed in dotted line, methylated base in bold) blocked by methylation switching using M.Osp807II or M.Sen0738I in a head-to-head arrangement. The distance between the outside restriction site and the pre-assembly overhang sequence ‘GTCT’ (5 bp) is 6 bp less than the distance between the inside restriction site and the pre-assembly overhang sequence (11 bp). Restriction with BsaI generates vector backbone containing a partial methylated restriction site, which can be ligated with the cut insert fragment flanked by pre-assembly overhang sequence (‘GTCT’) to reconstitute a functional outside restriction site. Following transformation into an E. coli strain that lacks methylation switching activity, methylation in the outside restriction site is lost and the assembled fragment can be cut with BsaI and generates assembled fragment flanked by post-assembly overhang sequence (‘xxxx’). Compared with the insert fragment, the assembled fragment has 6 bp excised from the end of the sequence. ‘N’ represents A, T, C, or G. ‘H’ represents A, T or C, and ‘h’ represents the nucleotide complementary to ‘H’.

[0423] B. Abbreviation of the assembly process.

[0424] FIG. 19. Problem with excision-type design of M2.Nme-based methylation switching of BpiI system

[0425] The 4 bp excision-type design of BpiI assembly system based on M2.NmeMC58II methylation switching is unfeasible because digestion of the assembly vector generates a partial outside BpiI site that lacks DNA methylation due to the position of base methylated by switch methylase M2.NmeMC58II. Ligation of the vector backbone with the insert fragment generates assembled plasmid with functional BpiI site (highlighted in grey), which will be repeatedly cut during the assembly process.

[0426] FIG. 20. Excision-type design of BsaI site with 6 bp excision using partial BsaI site as outside site

[0427] A. DNA assembly process using vectors with excision-type design. The excision-type design contains a functional inside restriction site (boxed in solid line) and a methylated partial restriction site (boxed in dotted line, methylated base in bold) methylated by M.Osp807II or M.Sen0738I in a head-to-head arrangement. The distance between the outside restriction site and the pre-assembly overhang sequence ‘GTCT’ (5 bp) is 6 bp less than the distance between the inside restriction site and the pre-assembly overhang sequence (11 bp). Restriction with BsaI generates vector backbone containing a methylated partial restriction site, which can be ligated with the cut insert fragment flanked by pre-assembly overhang sequence (‘GTCT’) to reconstitute a functional outside restriction site. Following transformation into an E. coli strain that lacks methylation switching activity, methylation in the outside restriction site is lost and the assembled fragment can be cut with BsaI and generates assembled fragment flanked by post-assembly overhang sequence (‘xxxx’). Compared with the insert fragment, the assembled fragment has 6 bp excised from the end of the sequence. ‘N’ represents A, T, C, or G. ‘H’ represents A, T or C, and ‘h’ represents the nucleotide complementary to ‘H’.

[0428] B. Abbreviation of the assembly process.

[0429] FIG. 21. Excision-type design with methylation protection mechanism

[0430] A. DNA assembly process using vectors with excision-type design based on methylation protection mechanism. The excision-type assembly vector design contains a functional inside restriction site (boxed in solid line) but lacks outside restriction site. The insert DNA contains 9 bp sequence to be excised (‘abGGTCTCN’) which itself provides an intact BsaI site (boxed in dotted line) to be used as the reconstituted outside BsaI site in the assembled plasmid. This site is blocked in the insert plasmid by methylation due to the methylation protection mechanism (boxed in dotted line, methylated base in bold), whereas the flanking BsaI site to release the insert fragment is functional due to methylation protection (boxed in solid line). The 5 bp seed sequence that defines the specificity of the methylation protection mechanism (‘SSSSa’) is highlighted in grey. Restriction with BsaI generates vector backbone containing overhang sequence compatible with the pre-assembly overhang sequence (‘abGG’) of the insert fragment for ligation. Following transformation into strains that deploys methylation protection mechanism, the reconstituted BsaI site in the assembled plasmid lost methylation due to methylation protection mechanism. As a result, compared with the insert fragment, the assembled fragment has 9 bp excised from the end of the sequence. The assembly vector sequence was designed so that following DNA assembly, the reconstituted outside restriction site is protected from methylation protection mechanism using dCas9-based methylation protection with the same specificity as the insert plasmid (dCas9 seed sequence for methylation protection ‘SSSSa’ remains the same).

[0431] B. Abbreviation of the assembly process.

[0432] FIG. 22. Three types of scarless assembly vectors

[0433] Diagram for the three types of scarless assembly vectors. Bases methylated by switch methylase M.Sen0738I are in bold, blocked BsaI sites boxed in dotted line and functional BsaI sites boxed in solid line. Vector VLeft carries maintenance type design at the left arm with overhang sequence ‘CTCC’ (‘u’), and 6 bp excision-type design at the right arm with overhang sequence ‘AGAC’ (‘v’). Vector VMiddle carries 4 bp excision-type design at the left arm with overhang sequence ‘CTCC’ (‘u’), and 6 bp excision-type design at the right arm with overhang sequence ‘AGAC’ (‘v’). Vector VRight carries 4 bp excision-type design at the left arm with overhang sequence ‘CTCC’ (‘u’), and maintenance type design at the right arm with overhang sequence ‘AGAC’ (‘v’).

[0434] FIG. 23. Directional insert DNA excision using the three types of scarless assembly vectors

[0435] A. Cloning of a single insert DNA fragment FragA with compatible adhesive ends (‘CTCC’ and ‘AGAC’) into scarless assembly vector VLeft. Internal BsaI sites in the middle of the insert fragments are methylated by methylation protection (boxed in dotted line, methylated base in bold). Cloning into vector VLeft excises 6 bp adaptor sequence (‘TGAGAC’) at the right end of the sequence, generating insert fragment flanked by post-assembly overhang sequence ‘CTCC’ at left end and ‘bbbb’ at right end.

[0436] B. Symbolic representation of the assembly reaction. [FragA](u,v) represents FragA flanked by overhang sequence ‘u’ (‘CTTC’) and ‘v’ (‘AGAC’) in the insert plasmid. Assembling into VLeft generates assembled plasmid [FragABC](u,d) with assembled fragment flanked by overhang sequences ‘u’ (‘CTCC’) and ‘bbbb’.

[0437] C and E. Cloning of FragA into assembly vector VMiddle (C) and VRight (E).

[0438] D and F. Symbolic representation of the cloning process using vector VMiddle (D) and VRight (F).

[0439] FIG. 24. DNA assembly and excision using the three types of scarless assembly vectors

[0440] A. Assembly of three fragments (FragA, FragB and FragC) with compatible adhesive ends (‘CTCC’, ‘bbbb, ‘cccc’, ‘AGAC’) into scarless assembly vector VLeft. Internal BsaI sites in the middle of the insert fragments are methylated by methylation protection (boxed in dotted line, methylated base in bold). Assembly into vector VLeft excises 6 bp at the right end of the sequence, generating assembled fragment flanked by post-assembly overhang sequence ‘CTCC’ at left end and ‘dddd’ at right end.

[0441] B. Symbolic representation of the assembly reaction. [FragA](u,a) represents Fragment A flanked by overhang sequence ‘u’ (‘CTCC’) and ‘aaaa’ in the insert plasmid. Assembling into VLeft generates assembled plasmid [FragABC](u,d) with assembled fragment flanked by overhang sequences ‘u’ (‘CTCC’) and ‘dddd’.

[0442] C and E. Assembly of three fragments into assembly vector VMiddle (C) and VRight (E).

[0443] D and F. Symbolic representation of the assembly process using vector VMiddle (D) and VRight (F).

[0444] FIG. 25. Hierarchical scarless assembly scheme

[0445] A and B. The starting fragment is first of all digitally broken into 9 fragments with 4 bp overlapping sequence.

[0446] C. For each starting fragment, the 4 bp adaptor sequence ‘CTCC’ and 6 bp adaptor sequence ‘TGAGAC’ is added to the left and right end of the sequence respectively, and cloned into plasmids to generate insert plasmids with insert DNA flanked by overhang sequences ‘u’ (‘CTCC’) and ‘v’ (‘AGAC’). The insert plasmids are prepared in methylation protection strain, so that internal BsaI sites inside the insert are methylated (boxed in dotted line, methylated base in bold), whereas the flanking BsaI sites are protected from methylation (boxed in solid line).

[0447] D and E. Each insert fragment is then cloned as single fragment into appropriate scarless assembly vectors depending on the position of the fragment in the next round of assembly.

[0448] F and G. In the first round of multi-fragment assembly, the fragments were assembled three per group into appropriate scarless assembly vectors depending on the position of the assembled fragment in the next round of assembly to generate assembled fragment [ABC], [DEF] and [GHI].

[0449] H and I. These three intermediate fragments can then be assembled into vector VMiddle to generate the fully assembled fragment [ABCDEFGHI] corresponding to the starting fragment with flanking overhang sequence ‘xxxx’ and ‘yyyy’ defined by the input sequence.

[0450] FIG. 26. Hierarchical scarless assembly scheme with simplified preparative cloning process

[0451] A and B. The starting fragment is first of all digitally broken into 9 fragments with 4 bp overlapping sequence.

[0452] C. Each fragment is cloned into insert plasmid using methylation protection strain, with adaptor sequence added depending on the position of the fragment in the first round of assembly. The left most fragments (Fragments A, D and G) has 4 bp adaptor sequence ‘CTCC’ added to the left end of the fragment. The rightmost fragments (Fragments C, F and I) has 6 bp adaptor sequence ‘TGAGAC’ added to the right end of the fragment. The middle fragments (Fragments B, E and H) have no adaptor sequences added at either end.

[0453] D and E. In the first round assembly, the fragments were assembled three per group into appropriate scarless assembly vectors depending on the position of the assembled fragment in the next round of assembly to generate assembled fragment [ABC], [DEF] and [GHI].

[0454] F and G. These three intermediate fragments can then be assembled into vector VMiddle to generate the fully assembled fragment [ABCDEFGHI] corresponding to the starting fragment with flanking overhang sequence ‘xxxx’ and ‘yyyy’ defined by the input sequence.

[0455] FIG. 27. Practical scarless assembly using Universal Assembly

[0456] A and B. A ˜90 kb sequence corresponding to the MICA locus was first digitally broken into 14 fragments with 4 bp overlapping sequence (F1-F14). The fragments were cloned by PCR as insert plasmids to be used for first round DNA assembly, and assembled 2-4 fragments per group using scarless Universal Assembly into intermediate fragments G1-G4. The four intermediate fragments were further assembled into a single ˜90 kb fragment H1.

[0457] C. Pulsed field electrophoresis of assembled plasmids carrying intermediate fragments G1-G4 (lane 1-4), or fully assembled fragments H1 (lane 5-12) following restriction using NotI, along with MidRange I PFG Marker (NEB, M1) or 1 kb DNA ladder (NEB, M2). 12 independent clones of fully assembled plasmids were screened and the assembled clones are 100% correct (12 out of 12 correct).

[0458] FIG. 28. Methylation protection using DNA methylase as the specific DNA binding protein

[0459] A. A standard type IIS restriction enzyme site such as BsaI (boxed) can be methylated by the M1.Eco31I ‘protection’ methylase at the 3rd base of the bottom strand (in bold) when the plasmid is prepared in an E. coli strain that expresses the M1.Eco31I methylase, M.Csp205I and S.Csp205I (the standard BsaI site must not overlap with M.Csp205I recognition sequence).

[0460] B. A standard type IIS restriction enzyme site such as BsaI (boxed) can be methylated by the M2.Eco31I ‘protection’ methylase at the 4th base of the top strand (in bold) when the plasmid is prepared in an E. coli strain that expresses the M2.Eco31I methylase, M.Csp205I and S.Csp205I (the standard BsaI site must not overlap with M.Csp205I recognition sequence).

[0461] C. Combining a BsaI site (boxed) with the M.Csp205I recognition sequence (highlighted in grey, methylated base in bold) creates a BsaI site that overlaps with the M.Csp205I recognition sequence. Preparation of the plasmid in an E. coli strain that expresses the M1.Eco31I or M2.Eco31I methylase, M.Csp205I and S.Csp205I that recognizes this combined site results in M.Csp205I binding to the site, which may block the site from methylation by the M1.Eco31I or M2.Eco31I protection methylase. Such site is referred to as a M.Csp205I-protectable site. M.Csp205I methylates the 5.sup.th base at bottom strand of the BsaI site within the M.Csp205I-protectable site. This methylation itself does not affect BsaI activity towards the BsaI site. The methylation protection process can be represented by the symbols to the right; open triangles depicts an unmethylated BsaI site, and black triangles depict a methylated BsaI site.

[0462] FIG. 29. In vivo methylation protection using M.Csp205I

[0463] A. Diagram depicting the E. coli strains tested for methylation protection using M.Csp205I. The strains express either M1.Eco31I or M2.Eco31I with either J23114 or J23112 promoters, B0034m* ribosomal binding sequence (RBS) and L3S2P21 terminator, M.Csp205I and S.Csp205I (both driven by a J23100 promoter with a B0034m* RBS, and with L3S1P51 terminator and L3S1P32 terminator respectively), and a zeocin-resistance gene (ZeoR, driven by an EM2KC promoter) from the arsB locus of the E. coli chromosome.

[0464] B. Diagram of the plasmids used to test the methylation protection approach. The test plasmid pMOP_testN7 carries a M.Csp205I-protectable BsaI site, a BamHI site, and a normal (non-switchable) BsaI site. If this test plasmid is prepared in a normal strain, such as DH10B, then both BsaI sites should be cut by BsaI. Therefore, digestion with BamHI and BsaI should generate two small fragments of ˜370 bp and ˜220 bp. If this test plasmid is prepared in the methylation protection strain that expresses the protection methylase, and the second methylase, then the normal BsaI site will be methylated and so will not be cut, but the methylation-protectable BsaI site will bound by the second methylase. With successful methylation protection, when the plasmid is exposed to BamHI and BsaI, of the two BsaI sites, only the unmethylated methylation-protectable BsaI site can be cut and this results in only one small fragment of ˜220 bp. C. Gel electrophoresis of the test plasmids prepared from the DH10B or methylation protection strains following BamHI and BsaI-HFv2 digestion. The digested samples demonstrate the expected pattern as predicted in B only in the strain that expresses M2.Eco31I under J23112 promoter (2W94R), confirming successful protection from methylation of the dCas9-protectable BsaI site by M.Csp205I in this strain.

[0465] FIG. 30. Universal Assembly system based on methylation-protection approach alone

[0466] The diagram depicts the design of the Universal Assembly system using methylation-protection approach alone. The donor plasmids contain inserts flanked by dCas9/guideX-protectable BsaI sites (the BsaI site is boxed and the nucleotides critical for dCas9 binding specificity guided by guide RNA guide X are highlighted in grey) that would generate compatible adhesive ends following BsaI restriction. The internal standard BsaI sites in the insert do not overlap with guideX-guided-dCas9 binding sequence and therefore are not protected from methylation by the M2.Eco31I protection methylase. Transformation of the insert plasmids into an E. coli strain (DH10B-guideX) that expresses the M2.Eco31I protection methylase, dCas9 and a sgRNA guideX targeting the seed sequence XXXXX of dCas9-protectable BsaI site results in selective methylation of internal BsaI sites within the insert (the methylated base is shown in bold). The assembly vector contains a negative selection marker LacZalpha flanked by head-to-head BsaI sites that generate the same adhesive end as that produced by BsaI-based excision of the insert from the donor plasmid. The outer pair of BsaI sites are dCas9/guideX-protectable, whereas the inner pair of BsaI sites are dCas9/guideY-protectable (seed sequence XXXXX is critical for selective binding of dCas9 in the presence of guide RNA guideX to the outer pair BsaI sites, and seed sequence YYYYY is critical for selective binding of dCas9 in the presence of guideY to the inner pair BsaI sites). Preparation of the assembly recipient vector in an E. coli strain (DH10B-guideY) expressing the M2.Eco31I protection methylase, dCas9 and a sgRNA guideY targeting the seed sequence YYYYY results in specific methylation of the outer pair of BsaI sites. The inner pair of BsaI sites are protected from M2.Eco31I by the guideY-guided dCas9. The methylated insert donor plasmids and assembly recipient vector plasmids can be cut with BsaI. Ligation of the cut fragments in a one-pot reaction that contains BsaI favours the generation of correctly assembled DNA comprising the inserts ligated together with the assembly vector backbone, in which all the BsaI sites are methylated. Transformation into a normal E. coli (DH10B) that lacks dCas9-protection and methylation-switching activity removes methylation of all the BsaI sites in the assembled plasmid. Seed sequences XXXXX and YYYYY represents two different 5 bp sequence.

[0467] FIG. 31. Universal Assembly system based on methylation protection approach alone

[0468] The diagram depicts the basic design of Universal Assembly with the assembled DNA directly transformed into an E. coli strain that deploys methylation-protection approach alone.

[0469] The donor plasmids contain inserts flanked by dCas9/guideX-protectable BsaI sites (the BsaI site is boxed and the nucleotides critical for dCas9 binding specificity guided by guide RNA guide X are highlighted in grey) that would generate compatible adhesive ends following BsaI restriction. The internal standard BsaI sites in the insert do not overlap with guideX-guided-dCas9 binding sequence and therefore are not protected from methylation by the M2.Eco31I protection methylase. Preparation of the insert plasmids into an E. coli strain (DH10B-guideX) that expresses the M2.Eco31I protection methylase, dCas9 and a sgRNA guideX targeting the seed sequence XXXXX of dCas9-protectable BsaI site results in selective methylation of internal BsaI sites within the insert (the methylated base is shown in bold). The assembly vector contains a negative selection marker LacZalpha flanked by head-to-head BsaI sites that generate the same adhesive end as that produced by BsaI-based excision of the insert from the donor plasmid. The outer pair of BsaI sites are dCas9/guideX-protectable, whereas the inner pair of BsaI sites are dCas9/guideY-protectable (seed sequence XXXXX is critical for selective binding of dCas9 in the presence of guide RNA guideX to the outer pair BsaI sites, and seed sequence YYYYY is critical for selective binding of dCas9 in the presence of guideY to the inner pair BsaI sites). Preparation of the assembly recipient vector in an E. coli strain (DH10B-guideY) expressing the M2.Eco31I protection methylase, dCas9 and a sgRNA guideY targeting the seed sequence YYYYY results in specific methylation of the outer pair of BsaI sites. The inner pair of BsaI sites are protected from M2.Eco31I by the guideY-guided dCas9. The methylated insert donor plasmids and assembly recipient vector plasmids can be cut with BsaI. Ligation of the cut fragments in a one-pot reaction that contains BsaI favours the generation of correctly assembled DNA comprising the inserts ligated together with the assembly vector backbone, in which all the BsaI sites are methylated. Transformation into in the E. coli strain (DH10B-guideX) that expresses the M2.Eco31I protection methylase, dCas9 and an sgRNA guideX targeting the dCas9/guideX-protectable BsaI site, results in a plasmid that has methylation at all the BsaI sites in the insert, but no methylation at the dCas9/guideX-protectable insert-flanking BsaI sites. The assembled DNA carries a similar methylation pattern on its BsaI sites to the original insert plasmids and so, therefore can be used directly for the next round of Universal Assembly with the same system. Seed sequences XXXXX and YYYYY represents two different 5 bp sequence.

[0470] FIG. 32. Methylation protection using dST1Cas9

[0471] A. A standard type IIS restriction enzyme site such as BsaI (boxed in solid line) can be methylated by the M2.Eco31I ‘protection’ methylase at the 4th base of the top strand (in bold) when the plasmid is prepared in an E. coli strain that expresses the M2.Eco31I methylase, dST1Cas9 and a chimeric single guide RNA (sgRNA) targeting a specific sequence (the standard BsaI site must not overlap with the sgRNA-guided dST1Cas9 binding sequence).

[0472] B. Combining a BsaI site (boxed in solid line) with the sgRNA-guided dST1Cas9 binding site (the 7 bp PAM motif is boxed in dotted line, and the nucleotides that form Watson-Crick base pairing with the sgRNA are shown in italic) creates a BsaI site that overlaps with the dST1Cas9-binding site, in which the sgRNA targets the bottom strand of the combined BsaI site, and the BsaI site overlaps with the PAM motif of the dST1Cas9 binding site. Preparation of the plasmid in an E. coli strain that expresses the M2.Eco31I protection methylase, dST1Cas9 and the sgRNA that recognizes this combined site results in dST1Cas9 binding to this site, which blocks the site from methylation by the M2.Eco31I protection methylase. Such site is referred to as a dST1Cas9-protectable site. The methylation protection process can be represented by the symbols to the right; open triangles depicts an unmethylated BsaI site, and black triangles depict a methylated BsaI site.

[0473] C. dST1Cas9-protectable BsaI sites can also be formed by combining a BsaI site (boxed in solid line) with a sgRNA-guided dST1Cas9 binding site (the 7 bp PAM motif is boxed in dotted line, and the nucleotides that form Watson-Crick base pairing with the sgRNA are shown in italic), whereas the sgRNA targets the top strand of the combined BsaI site, and the BsaI site lies within the sgRNA targeted sequence. Preparation of the plasmid in an E. coli strain that expresses the M2.Eco31I protection methylase, dST1Cas9 and the sgRNA that recognizes this combined site results in dST1Cas9 binding to this site, which blocks the site from methylation by the M2.Eco31I protection methylase.

[0474] FIG. 33. Methylation protection using dST1Cas9

[0475] A. Diagram depicting the E. coli strains for methylation protection. The strains express a sgRNA (guide #498 for strain DH10B-2W276R or guide #500 for strain DH10B-2W278R, driven by a J23119 promoter and with L3S2P21 terminator), dST1Cas9 (driven by a J23119 promoter with a B0034m* ribosomal binding sequence (RBS) and L3S1P51 terminator), the M2.Eco31I methylase (driven by a J23112 promoter with a B0034m* RBS and L3S1P32 terminator) and a zeocin-resistance gene (ZeoR, driven by an EM2KC promoter) from the arsB locus of the E. coli chromosome.

[0476] B. Diagram of the plasmids used to test the methylation protection approach. The test plasmids (pMOP_testN24 for DH10B-2W276R and pMOP_testN32 for DH10B-2W278R) carry a dST1Cas9-protectable BsaI site (GTCTAGTATGCGGATGCCAGTGAGAAGGTCTC (SEQ ID NO: 112) in pMOP_testN24, CTTCTCAAGCCCCAGAATGGTGGTCTC (SEQ ID NO: 113) in pMOP_testN32, with the BsaI recognition sequence underlined) for the corresponding guide RNA sequence, a BamHI site, and a non-protectable BsaI site. If this test plasmid is prepared in a normal strain, such as DH10B, then both BsaI sites should be cut by BsaI. Therefore, digestion with BamHI and BsaI should generate two small fragments of ˜370 bp and ˜220 bp. If this test plasmid is prepared in the methylation protection strain that expresses the protection methylase, dST1Cas9 and the corresponding guide RNA, then the normal BsaI site will be methylated and so will not be cut, but the methylation-protectable BsaI site will bound by the dST1Cas9 and so will not be methylated. When the plasmid is exposed to BamHI and BsaI, of the two BsaI sites, only the unmethylated methylation-protectable BsaI site can be cut and this results in only one small fragment of ˜220 bp.

[0477] C. Gel electrophoresis of the test plasmids prepared from the DH10B or methylation protection strains following BamHI and BsaI-HFv2 digestion. The digested samples demonstrate the expected pattern as predicted in B, confirming successful protection from methylation of the dST1Cas9-protectable BsaI site by the guided dST1Cas9 in the DH10B-2W276R and DH10B-2W278R strains.

[0478] FIG. 34. Combined BsaI sites invoking both dST1Cas9-based methylation-protection and M.Sen0738I-based methylation-switching.

[0479] A. Combining a dST1Cas9-protectable BsaI site (the 7 bp PAM motif boxed in dotted line, the nucleotides that form Watson-Crick base pairing with the sgRNA in italic, and the BsaI site boxed in solid line) in which the guide RNA targets the bottom strand of the combined BsaI site, and an M.Sen0738I switch methylase recognition sequence (methylase recognition sequence boxed in dotted line, and the methylated bases in bold) generates a combined BsaI site that is both dST1Cas9-protectable and M.Sen0738I-switchable.

[0480] B. A dST1Cas9-protectable and M.Sen0738I-switchable BsaI site can also be generated by combining a dST1Cas9-protectable BsaI site (the 7 bp PAM motif boxed in dotted line, the nucleotides that form Watson-Crick base pairing with the sgRNA in italic, and the BsaI site boxed in solid line) in which the guide RNA targets the top strand of the combined BsaI site, and an M.Sen0738I switch methylase recognition sequence (methylase recognition sequence boxed in dotted line, and the methylated bases in bold).

[0481] C. Systems deploying both methylation-protection and methylation-switching may contain four different types of BsaI sites. BsaI sites that are dST1Cas9-protectable can be cut if the plasmid was produced in a strain that coexpresses the M2.Eco31I protection methylase, dST1Cas9 and the appropriate sgRNA. However, the other standard BsaI sites in a plasmid produced in this strain are methylated by the M2.Eco31I protection methylase. BsaI sites that are methylation-switchable are not cut by BsaI if the plasmid has been produced in a strain that expresses the M.Sen0738I switch methylase as these sites are then methylated and so protected from digestion by BsaI. Any non-switchable BsaI sites will not be methylated and can be cut by the enzyme if the plasmid has been produced in such a strain that expresses the M.Sen0738I switch methylase.

[0482] FIG. 35. Universal Assembly system based on both methylation-switching and methylation-protection (M.Sen0738I-based methylation switching and dST1Cas9/guide #498-based methylation protection as an example)

[0483] The diagram depicts the design of the implemented Universal Assembly system using methylation-protection and methylation-switching. The donor plasmids contain inserts flanked by dST1Cas9/guide #498-protectable and M.Sen0738I-switchable BsaI sites (the 7 bp PAM motif boxed in dotted line, the nucleotides that form Watson-Crick base pairing with the sgRNA in italic, and the BsaI site boxed in solid line) that would generate compatible adhesive ends following BsaI restriction. The internal standard BsaI sites in the insert do not overlap with guided-dST1Cas9 binding sequence and therefore are not protected from methylation by the M2.Eco31I protection methylase. Transformation of the insert plasmids into an E. coli strain (DH10B-2W276R) that expresses the M2.Eco31I protection methylase, dST1Cas9 and a sgRNA guide #498 targeting the dST1Cas9-protectable BsaI site results in selective methylation of internal BsaI sites within the insert (the methylated base is shown in bold). The assembly vector contains a negative selection marker LacZalpha flanked by head-to-head BsaI sites that generate the same adhesive end as that produced by BsaI-based excision of the insert from the donor plasmid. The outer pair of BsaI sites are dST1Cas9/guide #498-protectable and M.Sen0738I methylation-switchable, whereas the inner pair of BsaI sites are nonswitchable. Preparation of the assembly recipient vector in an E. coli strain (DH10B-2W148R) expressing the M.Sen0738I switch methylase results in specific methylation of the outer pair of BsaI sites. The methylated insert donor plasmids and assembly recipient vector plasmids can be cut with BsaI. Ligation of the cut fragments in a one-pot reaction that contains BsaI favours the generation of correctly assembled DNA comprising the inserts ligated together with the assembly vector backbone, in which all the BsaI sites are methylated. Transformation into a normal E. coli (DH10B) that lacks dST1Cas9-protection and methylation-switching activity removes methylation of all the BsaI sites in the assembled plasmid.

[0484] FIG. 36. Universal Assembly system based on both methylation-switching and methylation-protection (M.Sen0738I-based methylation switching and dST1Cas9/guide #498-based methylation protection as an example, direct transformation into DH10B-2W276R).

[0485] The diagram depicts the basic design of Universal Assembly with the assembled DNA directly transformed into an E. coli strain that deploys the methylation-protection principle. The insert donor plasmids contain inserts flanked by dST1Cas9/guide #498-protectable and M.Sen0738I methylase-switchable BsaI sites (the 7 bp PAM motif boxed in dotted line, the nucleotides that form Watson-Crick base pairing with the sgRNA in italic, and the BsaI site boxed in solid line) that would generate mutually compatible adhesive ends following BsaI restriction. The internal standard BsaI sites in the insert do not overlap with the sequence that is the target of the guide RNA for the dST1Cas9 and therefore are not bound by dST1Cas9 and so are methylated by the M2.Eco31I protection methylase. Preparation of the insert plasmids in an E. coli strain (DH10B-2W276R) that expresses the M2.Eco31I protection methylase, dST1Cas9 and an sgRNA guide #498 targeting the dST1Cas9/guide #498-protectable BsaI results in specific methylation of standard internal BsaI sites within the insert (the methylated base is shown in bold). The assembly vector contains a negative selection marker LacZalpha flanked by head-to-head BsaI sites that generate the same adhesive end. The outer pair of BsaI sites are dST1Cas9/guide #498-protectable and M.Sen0738I methylase-switchable, whereas the inner pair are non-switchable. Preparation of the assembly vector in an E. coli strain expressing M.Sen0738I (DH10B-2W148R) results in specific methylation of the outer pair of BsaI sites. The methylated insert plasmids and assembly vector can be cut with BsaI. Ligation of the cut fragments in a one-pot reaction that contains BsaI favours the generation of correctly assembled DNA comprising the inserts ligated together with the assembly vector backbone, in which all the BsaI sites are methylated. Transformation into in the E. coli strain (DH10B-2W276R) that expresses the M2.Eco31I protection methylase, dST1Cas9 and an sgRNA guide #498 targeting the dST1Cas9/guide #498-protectable BsaI site, results in a plasmid that has methylation at all the BsaI sites in the insert, but no methylation at the dST1Cas9/guide #498-protectable insert-flanking BsaI sites. The assembled DNA carries a similar methylation pattern on its BsaI sites to the original insert plasmids and so, therefore can be used directly for the next round of Universal Assembly.

[0486] FIG. 37. Universal Assembly system based on both methylation-switching and methylation-protection (M.Sen0738I-based methylation switching and dST1Cas9/guide #500-based methylation protection as an example)

[0487] The diagram depicts the design of the implemented Universal Assembly system using methylation-protection and methylation-switching. The donor plasmids contain inserts flanked by dST1Cas9/guide #500-protectable and M.Sen0738I-switchable BsaI sites (the 7 bp PAM motif boxed in dotted line, the nucleotides that form Watson-Crick base pairing with the sgRNA in italic, and the BsaI site boxed in solid line) that would generate compatible adhesive ends following BsaI restriction. The internal standard BsaI sites in the insert do not overlap with guided-dST1Cas9 binding sequence and therefore are not protected from methylation by the M2.Eco31I protection methylase. Transformation of the insert plasmids into an E. coli strain (DH10B-2W278R) that expresses the M2.Eco31I protection methylase, dST1Cas9 and a sgRNA guide #500 targeting the dST1Cas9-protectable BsaI site results in selective methylation of internal BsaI sites within the insert (the methylated base is shown in bold). The assembly vector contains a negative selection marker LacZalpha flanked by head-to-head BsaI sites that generate the same adhesive end as that produced by BsaI-based excision of the insert from the donor plasmid. The outer pair of BsaI sites are dST1Cas9/guide #500-protectable and M.Sen0738I methylation-switchable, whereas the inner pair of BsaI sites are nonswitchable. Preparation of the assembly recipient vector in an E. coli strain (DH10B-2W148R) expressing the M.Sen0738I switch methylase results in specific methylation of the outer pair of BsaI sites. The methylated insert donor plasmids and assembly recipient vector plasmids can be cut with BsaI. Ligation of the cut fragments in a one-pot reaction that contains BsaI favours the generation of correctly assembled DNA comprising the inserts ligated together with the assembly vector backbone, in which all the BsaI sites are methylated. Transformation into a normal E. coli (DH10B) that lacks dST1Cas9-protection and methylation-switching activity removes methylation of all the BsaI sites in the assembled plasmid.

[0488] FIG. 38. Universal Assembly system based on both methylation-switching and methylation-protection (M.Sen0738I-based methylation switching and dST1Cas9/guide #500-based methylation protection as an example, direct transformation into DH10B-2W278R).

[0489] The diagram depicts the basic design of Universal Assembly with the assembled DNA directly transformed into an E. coli strain that deploys the methylation-protection principle. The insert donor plasmids contain inserts flanked by dST1Cas9/guide #500-protectable and M.Sen0738I methylase-switchable BsaI sites (the 7 bp PAM motif boxed in dotted line, the nucleotides that form Watson-Crick base pairing with the sgRNA in italic, and the BsaI site boxed in solid line) that would generate mutually compatible adhesive ends following BsaI restriction. The internal standard BsaI sites in the insert do not overlap with the sequence that is the target of the guide RNA for the dST1Cas9 and therefore are not bound by dST1Cas9 and so are methylated by the M2.Eco31I protection methylase. Preparation of the insert plasmids in an E. coli strain (DH10B-2W278R) that expresses the M2.Eco31I protection methylase, dST1Cas9 and an sgRNA guide #500 targeting the dST1Cas9/guide #500-protectable BsaI results in specific methylation of standard internal BsaI sites within the insert (the methylated base is shown in bold). The assembly vector contains a negative selection marker LacZalpha flanked by head-to-head BsaI sites that generate the same adhesive end. The outer pair of BsaI sites are dST1Cas9/guide #500-protectable and M.Sen0738I methylase-switchable, whereas the inner pair are non-switchable. Preparation of the assembly vector in an E. coli strain expressing M.Sen0738I (DH10B-2W148R) results in specific methylation of the outer pair of BsaI sites. The methylated insert plasmids and assembly vector can be cut with BsaI. Ligation of the cut fragments in a one-pot reaction that contains BsaI favours the generation of correctly assembled DNA comprising the inserts ligated together with the assembly vector backbone, in which all the BsaI sites are methylated. Transformation into in the E. coli strain (DH10B-2W278R) that expresses the M2.Eco31I protection methylase, dST1Cas9 and an sgRNA guide #500 targeting the dST1Cas9/guide #500-protectable BsaI site, results in a plasmid that has methylation at all the BsaI sites in the insert, but no methylation at the dST1Cas9/guide #500-protectable insert-flanking BsaI sites. The assembled DNA carries a similar methylation pattern on its BsaI sites to the original insert plasmids and so, therefore can be used directly for the next round of Universal Assembly.

[0490] FIG. 39. Practical DNA assembly using Universal Assembly with dST1Cas9-based methylation protection

[0491] A. Diagram of the DNA fragment to assemble. Four fragments of human DNA from the MICA locus (FragA, FragB, FragC and FragD), each containing an internal BsaI site were assembled together to produce a 3.6 kb fragment (Genbank KF724576.1:23-3633) as the final product. The coordinates of the DNA fragment to assemble are FragA (KF724576.1:23-1048), FragB (KF724576.1:1045-1894), FragC (KF724576.1:1891-2744), FragD (KF724576.1:2741-3633).

[0492] B. Gel electrophoresis of BsaI digestion of insert plasmids with pMOK498 backbone (pMOK498_F5A, pMOK498_F5B, pMOK498_F5C, pMOK498_F5D) prepared in the normal E. coli strain, DH10B, (−) or the E. coli strain DH10B-2W276R (+) that expresses dST1Cas9, a guide RNA guide #498 and the M2.Eco31I protection methylase following BsaI-HFv2 digestion. dST1Cas9-based methylation protection prevents methylation of the insert-flanking dST1Cas9-protectable BsaI sites, but the internal BsaI sites are methylated and so cannot be cut by BsaI, resulting in larger complete insert fragments that are suitable for assembly. For insert plasmids prepared in DH10B-2W276R, BsaI-HFv2 digestion of insert plasmids with both flanking BsaI sites fully protected from methylation generates a ˜4.3 kb vector backbone, whereas BsaI-HFv2 digestion of insert plasmids with one of the two flanking BsaI sites protected from methylation generates a single ˜5.3 kb fragment.

[0493] C. Gel electrophoresis of BsaI digestion of insert plasmids with pMOK500 backbone (pMOK500_F6A, pMOK500_F6B, pMOK500_F6C, pMOK500_F6D) prepared in the normal E. coli strain, DH10B, (−) or the E. coli strain DH10B-2W278R (+) that expresses dST1Cas9, a guide RNA guide #500 and the M2.Eco31I protection methylase following BsaI-HFv2 digestion. dST1Cas9-based methylation-protection prevents methylation of the insert-flanking dST1Cas9-protectable BsaI sites, but the internal BsaI sites are methylated and so cannot be cut by BsaI, resulting in larger complete insert fragments that are suitable for assembly. For insert plasmids prepared in DH10B-2W278R, BsaI-HFv2 digestion of insert plasmids with both flanking BsaI sites fully protected from methylation generates a ˜4.3 kb vector backbone, whereas BsaI-HFv2 digestion of insert plasmids with one of the two flanking BsaI sites protected from methylation generates a single ˜5.3 kb fragment.

[0494] D. Gel electrophoresis of DNA clones assembled into DH10B-2W276R or DH10B-2W278R cells by Universal Assembly from insert plasmids prepared in DH10B-2W276R or DH10B-2W278R following digestion by DraIII, which releases the assembled fragment through DraIII sites in the vector backbones, but does not cut inside the assembled 3.6 kb fragment as the assembled 3.6 kb DNA lacks DraIII sites. 6 out of 8 of clones assembled in DH10B-2W276R, and 8 out of 8 clones assembled in DH10B-2W278R were verified by DraIII digestion and by DNA sequencing.

[0495] E. Gel electrophoresis of DNA clones assembled into DH10B-2W276R or DH10B-2W278R by Universal Assembly following digestion by BsaI-HFv2. For correctly assembled DNA with internal BsaI sites completely blocked by M2.Eco31I methylation and both flanking BsaI sites fully protected from methylation, BsaI-HFv2 digestion generates a 4.4 kb vector backbone and a 3.6 kb insert DNA, whereas for assembled DNA with one of the two flanking BsaI sites protected from methylation, BsaI-HFv2 digestion generates a single 8.0 kb fragment.

[0496] FIG. 40. Practical scarless assembly of a ˜260 kb DNA using Universal Assembly

[0497] A and B. A ˜260 kb sequence corresponding to the CLEC16A locus was first digitally broken into 10 fragments with 4 bp overlapping sequence (L3G1-L3G10). The 10 starting fragments cloned in insert plasmids with flanking dCas9-guide #401 methylation protectable and M.Sen0738I methylation switchable BsaI sites carry suitable overhang sequences depending on position of the assembled fragment will take up in the next round of assembly. The fragments were assembled 2-4 fragments per group using scarless Universal Assembly into intermediate fragments L3H1, L3H2 and L3H3. The three intermediate fragments were further assembled into a single ˜260 kb fragment L3I1.

[0498] C. Pulsed field electrophoresis of assembled plasmids carrying intermediate fragments L3H1 (lane 1-6), L3H2 (lane 7-10) and L3H3 (lane 11-19), following restriction using NotI, along with MidRange I PFG Marker (NEB, ‘M’). Clones with correct restriction pattern are marked with an asterisk. The assembly vector backbone contains flanking NotI sites. Restriction of the assembled clones with NotI therefore generates the ˜6.3 kb assembly vector backbone and the insert fragment (˜51 kb for L3H1, ˜105 kb for L3H2 and ˜105 kb for L3H3). Fragment L3H1 contains an internal NotI site therefore restriction of L3H1 generates two fragments (˜33 kb and ˜19 kb) as predicted.

[0499] D. Pulsed field electrophoresis of assembled plasmids carrying fully assembled fragment L3I1 (lane 1-12), following restriction using NotI, along with MidRange I PFG Marker (NEB, ‘M’). Clones with correct restriction pattern are marked with an asterisk. The assembly vector backbone contains flanking NotI sites, and the fully assembled L3I1 (˜261 kb) contains an internal NotI site. Restriction of the assembled clones with NotI generates the ˜6.3 kb assembly vector backbone, and two fragments (˜242 kb and ˜19 kb) as predicted.

[0500] FIG. 41. Testing of methylation switching activity of recombinant M.Osp807II

[0501] A. Experimental designs to test methylation switching activity of M.Osp807II in vitro. The test plasmid (pMOP_BsaNC) contain a head-to-head M.Osp807II-methylation-switchable BsaI site (GACTTGGTCTCATGCCTGAGACCAAGTC) ˜600 bp away from a non-switchable BsaI site. Restriction digestion by BsaI of test plasmid would result in cutting at both BsaI sites resulting in generation of a 4.4 kb and a 600 bp fragment. Restriction digestion of test plasmid that has been methylated by M.Osp807II in vitro generate a single 5 kb fragment due to blocking of the methylation-switchable BsaI restriction sites by in vitro methylation. The head-to-head arrangement of overlapping methylation/restriction site allows the same assay to be used to detect any residual single strand nicking activity of the restriction enzyme towards the methylated restriction site.

[0502] B. Agarose gel electrophoresis analysis of the test plasmid (prepared in DH10B) methylated by M.Osp807II in vitro followed by digested with BsaI-HFv2. The results show that in vitro methylation by M.Osp807II successfully blocked the switchable BsaI restriction site.

[0503] FIG. 42. Testing of methylation activity of recombinant M2.Eco31I

[0504] A. Experimental designs to test methylation activity of M2.Eco31I in vitro. The test plasmid (pET-15b) contain an ApaI site and a BsaI site. Restriction digestion by ApaI/BsaI would generate a 3.3 kb and a 2.3 kb fragment. Restriction digestion of test plasmid that has been methylated by M2.Eco31I in vitro would generate a single 5.6 kb fragment due to blocking of the BsaI site by in vitro methylation.

[0505] B. Agarose gel electrophoresis analysis of the test plasmid (prepared in DH10B) methylated by M2.Eco31I in vitro followed by digested with BsaI-HFv2. The results show that in vitro methylation by M2.Eco31I successfully blocked BsaI restriction site.

[0506] FIG. 43. Testing of methylation activity of recombinant M2.BsaI

[0507] A. Experimental designs to test methylation activity of M2.BsaI in vitro. The test plasmid (pUC18) contain a BamHI site and a BsaI site. Restriction digestion by BamHI/BsaI would generate a 1355 bp and a 1331 bp fragment. Restriction digestion of test plasmid that has been methylated by M2.BsaI in vitro would generate a single 2686 bp fragment due to blocking of the BsaI site by in vitro methylation. B. Agarose gel electrophoresis analysis of the test plasmid (prepared in DH10B) methylated by M2.BsaI in vitro followed by digested with BsaI-HFv2. The results show that in vitro methylation by M2.BsaI successfully blocked BsaI restriction site.

[0508] FIG. 44. In vitro methylation protection process

[0509] The in vitro methylation protection consists of 3 steps. In step 1 DNA molecules were incubated with dCas9/sgRNA complex which selectively binds to the dCas9-protectable BsaI sites but not non-protectable standard BsaI sites. In step 2 methylases such as M2.Eco31I or M2.BsaI that blocks BsaI sites were added initiate the methylation reaction in vitro. This selectively methylates non-protectable standard BsaI sites, not the dCas9-protectable BsaI sites which were protected from methylation by the stably bound dCas9. In step 3 the sample was subject to heat-inactivation and cleaned up to remove the dCas9 and methylases from the reaction, generating purified DNA with selective methylation of non-protectable standard BsaI sites only.

[0510] FIG. 45. In vitro Universal Assembly based on in vitro methylation switching and in vitro methylation protection

[0511] The diagram depicts the design of the implemented Universal Assembly system using in vitro methylation-protection and in vitro methylation-switching. The donor plasmids contain inserts flanked by dCas9/guide #360-protectable and M.Osp807II-switchable BsaI sites (the BsaI site is boxed and the nucleotides critical for dCas9 binding specificity are highlighted in grey) that would generate compatible adhesive ends following BsaI restriction. The internal standard BsaI sites in the insert do not overlap with guided-dCas9 binding sequence and therefore are not protected from methylation by the M2.Eco31I protection methylase. In vitro methylation protection of the donor plasmids results in selective methylation of internal BsaI sites within the insert (the methylated base is shown in bold) but not the flanking dCas9-protectable BsaI sites. The assembly vector contains a negative selection marker LacZalpha flanked by head-to-head BsaI sites that generate the same adhesive end as that produced by BsaI-based excision of the insert from the donor plasmid. The outer pair of BsaI sites are dCas9/guide #360-protectable and M.Osp807II methylation-switchable, whereas the inner pair of BsaI sites are nonswitchable. In vitro methylation switching of the assembly vector results in specific methylation of the outer pair of BsaI sites only. The methylated insert donor plasmids and assembly recipient vector plasmids can be cut with BsaI. Ligation of the cut fragments in a one-pot reaction that contains BsaI favours the generation of correctly assembled DNA comprising the inserts ligated together with the assembly vector backbone, in which all the BsaI sites are methylated. Transformation into a normal E. coli (DH10B) that lacks dCas9-protection and methylation-switching activity removes methylation of all the BsaI sites in the assembled plasmid.

[0512] FIG. 46. Practical DNA assembly using in vitro Universal Assembly with in vitro methylation switching using M.Osp807II and in vitro methylation protection using dCas9/guide #360 with either M2.Eco31I or M2.BsaI

[0513] A. Diagram of the DNA fragment to assemble. Four fragments of human DNA from the MICA locus (FragA, FragB, FragC and FragD), each containing an internal BsaI site were assembled together to produce a 3.6 kb fragment (Genbank KF724576.1:23-3633) as the final product. The coordinates of the DNA fragment to assemble are FragA (KF724576.1:23-1048), FragB (KF724576.1:1045-1894), FragC (KF724576.1:1891-2744), FragD (KF724576.1:2741-3633).

[0514] B. Gel electrophoresis of DNA clones assembled by in vitro Universal Assembly following digestion by DraIII, which releases the assembled fragment through DraIII sites in the vector backbones, but does not cut inside the assembled 3.6 kb fragment as the assembled 3.6 kb DNA lacks DraIII sites. 4 out 5 clones screened were correctly assembled for using either M2.Eco31I or M2.BsaI-based system.

EXAMPLE 1—UNIVERSAL ASSEMBLY: SEQUENCE-INDEPENDENT DNA ASSEMBLY USING A TYPE IIS RESTRICTION ENZYME, WITH DCAS9 AS THE SPECIFIC DNA BINDING PROTEIN FOR METHYLATION PROTECTION OF THE METHYLATION-PROTECTABLE RESTRICTION ELEMENT, AND M.OSP807II OR M.SEN0738I AS THE FURTHER METHYLASE FOR METHYLATION SWITCHING

[0515] Summary

[0516] Efficient DNA assembly is of great value in biological research and biotechnology. Type IIS restriction enzyme-based assembly systems allow assembly of multiple DNA fragments in a one-pot reaction, but suffer from the limitation that the DNA fragments to assemble need to be free of restriction sites for the type IIS restriction enzyme used for the assembly. Here we developed a new system named Universal Assembly that overcomes this problem. The Universal Assembly system is built on the methylation protection approach, whereby a DNA methylase is used to methylate sites for the given type IIS restriction enzyme in the DNA, but a DNA binding protein is used to selectively protect type IIS restriction sites that overlap with a DNA binding protein recognition sequence from methylation. We have developed practical Universal Assembly systems for BsaI-based one-pot assembly using dCas9-based methylation protection. The Universal Assembly system effectively eliminates the need to remove internal restriction sites from DNA to be assembled. The versatile system has potential to become a standard for modular DNA assembly, and has wide applications given the ability of the system to assemble DNA with no sequence constraints.

[0517] Introduction

[0518] Here we describe a new method for type IIS restriction enzyme-based DNA assembly that overcomes the problem of sequence constraint and so eliminates the requirement to remove internal type IIS restriction sites from DNA parts to assemble. The method, termed Universal Assembly, is based on the methylation protection approach, whereby a DNA methylase is used to methylate and so block any internal restriction sites for the type IIS restriction enzyme in any DNA fragment to be assembled. In parallel, a programmable DNA binding protein, such as a deactivated CRISPR Cas9, is used to bind to and so protect from methylation, the particular type IIS restriction sites that are positioned on the flanks of the DNA fragment and are used for restriction digestion to release the insert DNA fragment during the assembly process. The use of several Universal Assembly systems with different guide RNA sequences effectively eliminates the need to remove internal forbidden sequences and so allows DNA assembly without sequence constraint. We have designed and constructed a practical Universal Assembly system using the type IIS enzyme BsaI and used it to assemble multiple fragments of DNA each containing an internal BsaI site.

[0519] Material and Methods

[0520] Reagents

[0521] Enzymes for molecular biology are from NEB unless otherwise stated. A high-fidelity version of BsaI restriction enzyme named BsaI-HFv2 is used in place of BsaI for all the experiments.

[0522] Plasmid Construction

[0523] Plasmids for testing specific strains and for proof-of-principle DNA assembly were constructed using standard restriction enzyme/ligation-based cloning techniques. DNA fragments for cloning were generated by gene synthesis (Integrated DNA Technologies or Invitrogen), or by PCR with Q5 polymerase (NEB). Plasmids for functional testing, and the assembly vector for proof-of-principle DNA assembly were based on the ampicillin resistant MoClo vector pICH47732. Insert plasmids for proof-of-principle assembly were based on the kanamycin-resistant vector backbone described previously for MetClo proof-of-principle assembly (Lin D et al. Nucleic Acids Res. 2018 Nov. 2; 46(19):e113. doi: 10.1093/nar/gky596. PMID 29986052, which is herein incorporated by reference).

[0524] Low copy number plasmid pMOBKZ-2W148 was constructed for coexpression of M.Sen0738I (under a J23100 promoter, a modified RBS based on B0034m named B0034m* and an L3S2P21 terminator) and S.Sen0738I (under a J23100 promoter, a B0034m* RBS and an L3S1P51 terminator), and plasmid pMOBKZ-2W213 or pMOBKZ-2W214 was constructed for coexpression of guide RNA guide #360 or guide #401 (under a J23119 promoter and an L3S2P21 terminator), dCas9 (under a J23119 promoter, a B0034m* RBS and an L3S1P51 terminator), and M2.Eco31I (under a J23112 promoter, a B0034m* RBS and an L3S1P32 terminator). Briefly, for the construction of these plasmids, the transcription units were each assembled from individual cloned DNA parts using BsaI-based MetClo with the MetClo vector set described previously (Lin D et al. Nucleic Acids Res. 2018 Nov. 2; 46(19):e113. doi: 10.1093/nar/gky596. PMID 29986052). The transcription units were then assembled using BsaI into a low copy number kanamycin-resistant vector with an F replication origin, insert-flanking arsB homologous sequences and a zeocin-selection marker. In each round of assembly, insert DNA fragments containing M2.Eco31I were generated by PCR to remove M2.Eco31I methylation in the DNA. The dCas9 transcription unit for DNA assembly was generated by PCR using a flanking primer containing the J23119 promoter to avoid toxicity of the dCas9 transcription unit in the p15A-based low copy plasmid.

[0525] Generation of E. coli Strain

[0526] The E. coli strain that constitutively expresses M.Osp807II (DH10B-M.Osp807II) has been described previously (Lin D et al. Nucleic Acids Res. 2018 Nov. 2; 46(19):e113. doi: 10.1093/nar/gky596. PMID 29986052). The E. coli strain that constitutively expresses M.Sen0738I and S.Sen0738I (DH10B-2W148R) was constructed by recombineering using a linear DNA fragment amplified by PCR from pMOBKZ-2W148 using primers TTCTGTTACCCATCCAATTGTTC and CAGGCGCTTACCCGCTTCAT. The E. coli strains that deploy dCas9-based methylation protection mechanisms (DH10B-2W213R and DH10B-2W214R) were generated by lambda-red recombineering into DH10B cells using a linear DNA fragment amplified by PCR from pMOBKZ-2W213 and pMOBKZ-2W214 respectively using primers TTCTGTTACCCATCCAATTGTTC and CAGGCGCTTACCCGCTTCAT.

[0527] Modular Assembly

[0528] For assembly of a ˜3.6 kb DNA element from 4 fragments, the standard assembly reaction was 30 fmol of assembly vector, 60 fmol of each insert plasmid, 15 U T4 DNA ligase HC (Thermo Fisher) and 10 U BsaI-HFv2 (NEB) in 20 ul 1×T4 ligase buffer (NEB). The reaction condition was: 3TC 15 min, followed by 45 cycles of 3TC 2 min plus 16° C. 5 min, then 3TC 20 min, and 80° C. 5 min. Assembly reactions were transformed into specific chemically competent cells, and plated on LB plates with AIX selection (ampicillin 100 μg/ml, IPTG 100 μM, X-gal 50 μg/ml) at 3TC overnight. White colonies were expanded and screened by restriction digestion using DraIII or BsaI and by DNA sequencing.

[0529] For modular assembly of plasmid pMOP360_F3W using M.Osp807II and a dCas9-guide #360-based system, insert plasmids pMOK360_F3A, pMOK360_F3B, pMOK360_F3C and pMOK360_F3D were prepared in DH10B-2W213R from overnight culture in LB medium supplemented with 1% glucose and 30 μg/ml kanamycin. Assembly vector pMOP360_F3V was prepared in DH10B-M.Osp807II from overnight culture in LB medium with 100 μg/ml ampicillin, and the assembly reaction was transformed into DH10B-2W213R. Assembled clones were cultured in LB medium supplemented with 1% glucose and 100 μg/ml ampicillin.

[0530] For modular assembly of plasmid pMOP401_F4W using M.Sen0738I and a dCas9-guide #401-based system, insert plasmids pMOK401_F4A, pMOK401_F4B, pMOK401_F4C and pMOK401_F4D were prepared in DH10B-2W214R from overnight culture in LB medium supplemented with 1% glucose and 30 μg/ml kanamycin. Assembly vector pMOP401_F4V was prepared in DH10B-2W148R from overnight culture in LB medium with 100 μg/ml ampicillin, and the assembly reaction was transformed into DH10B-2W214R. Assembled clones were cultured in LB medium supplemented with 1% glucose and 100 μg/ml ampicillin.

[0531] For scarless assembly of 90 kb DNA resulting in plasmid pMOBC401_L1H1 using M.Sen0738I and a dCas9-guide #401-based system, the DNA was assembled in two stages from 14 fragments. In stage one, the insert plasmids carrying the initial ˜7 kb fragments were prepared in DH10B-2W214R, and the recipient assembly vector in DH10B-2W148R. 30 fmol of each insert plasmid and 15 fmol of the recipient assembly vector were used in a 20 ul assembly reaction with BsaI-HFv2 and T4 ligase. 20 U BsaI-HFv2 was then added to the assembly reaction for incubation at 37° C. for 45 min followed by heat inactivation at 80° C. for 5 min. The assembly reaction was dialysed for 1 h with water, and transformed into NEB10b competent cells by electroporation. Plasmid DNA from positive clones were transformed into DH10B-2W214R, and used as insert plasmids for the stage two assembly. In stage two, the assembly reaction set up and transformation procedure was the same as stage one. Clones were screened by NotI digestion followed by pulsed-field electrophoresis.

[0532] Results

[0533] Design of Universal Assembly: Requirement for Selective Methylation Protection of Insert-Flanking Type IIS Sites

[0534] For the original MetClo approach that we described previously, we developed a method for switching the insert flanking type IIS restriction enzyme recognition sites on and off using methylation of these sites by a specific methylase (a ‘switch’ methylase) whose own recognition site sequence overlaps that of the restriction enzyme (FIG. 1 and FIG. 2). The methylation blocks the type IIS restriction enzyme activity towards type IIS restriction sites that overlap with the methylation sequence. In practice, other different methylases such as M.Osp807II and M.Sen0738I can also be used as switch methylases with appropriate design of switchable BsaI sites (FIGS. 1, 2 and 3).

[0535] The approach that we have developed and termed Universal Assembly uses a DNA methylase to methylate, and so block from restriction digestion, all the internal sites for the type IIS restriction enzyme used for the assembly. The insert-flanking restriction sites used for the assembly are protected from this methylation so that they can remain active when required during the assembly. To protect these flanking restriction sites from methylation, a DNA-binding protein such as an appropriately guided dCas9 is used to bind to and so block the flanking restriction sites from the action of the methylase. (FIG. 4). This protection from methylation must only happen at the insert-flanking type IIS restriction enzyme sites. In this way, all the internal type IIS sites are blocked by DNA methylation, whereas the flanking protected-type IIS restriction sites are free for the release of intact insert DNA fragment. The specificity of this protection is determined by the sequence that overlaps between the restriction site and the DNA-binding protein recognition sequence.

[0536] Site-Specific Protection from Methylation by Sequence-Programmable dCas9

[0537] To develop Universal Assembly using BsaI-based DNA assembly we chose the methylase M2.Eco31I from E. coli because it methylates the 4.sup.th position of the BsaI site (GGTCTC) and methylation at this position is known to block BsaI activity (Storch M et al. ACS Synth Biol. 2015 Jul. 17; 4(7):781-7. doi: 10.1021/sb500356d PMID 25746445). We then chose dCas9 as the DNA-binding protein because its DNA-binding specificity can be programmed by the sequence of the guide RNA used—dCas9 binding specificity is determined by the NGG PAM motif and an adjacent >=5 bp seed sequence (O'Geen H et al. Nucleic Acids Res. 2015 Mar. 31; 43(6):3389-404. doi: 10.1093/nar/gkv137 PMID 25712100).

[0538] To build this system, we first constructed a new E. coli strain (DH10B-2W213R) which coexpresses a CRISPR guide RNA guide #360, dCas9 and the M2.Eco31I methylase (FIG. 5A). We used the strong J23119 for both the guide RNA and dCas9 expression, and the weak J23112 promoter for the M2.Eco31I methylase expression. We constructed a test plasmid (pMOP_testN8) that contains a BsaI site that overlaps with the guide RNA sequence, and a standard BsaI site (FIG. 5B) and prepared this plasmid in the new strain (DH10B-2W213R). The test plasmid prepared in this strain was cut by BsaI at the BsaI site that overlaps with the guide RNA sequence, but not at the standard BsaI site (FIG. 5C). This demonstrates that when a plasmid is prepared in this strain, the dCas9 is able to selectively protect the site to which the guide RNA directs its DNA-binding activity from methylation by the M2.Eco31I methylase.

[0539] A similar system with a different guide RNA sequence (guide #401 comprising strain DH10B-2W214R and compatible test plasmid pMOP_testN20) also shows efficient sequence-specific methylation protection (FIGS. 5A, B and C), confirming that the principle of methylation protection is universal for the dCas9-M2.Eco31I system.

[0540] Design of Universal Assembly: One-Pot DNA Assembly Using the Methylation Protection

[0541] The methylation protection approach can be combined with the methylation switching approach used in the original MetClo method in a one-pot DNA assembly system, which effectively eliminates the need to remove internal type IIS restriction sites in the DNA fragments to be assembled. This can be done by constructing an insert-flanking type IIS restriction site that is both dCas9-protectable and methylation-switchable (FIG. 6 and FIG. 7). This site can be blocked through the methylation-switching mechanism, or be protected from methylation by the methylase that inactivates sites for the type IIS restriction enzyme that lie within the insert sequence through the dCas9-protection mechanism.

[0542] A one-pot assembly system can then be designed, with the donor plasmids containing inserts flanked by this methylation-switchable and dCas9-protectable type IIS restriction sites, and the assembly vector plasmid containing a negative selection marker flanked by head-to-head type IIS restriction sites, with the outer pair of the sites closer to the vector backbone being the methylation-switchable and dCas9-protectable type IIS restriction sites, and the inner pair being the non-switchable sites.

[0543] A one-pot assembly reaction can then be undertaken using donor plasmid(s) prepared in the strain that expresses the insert-blocking methylase, dCas9 and an appropriate guide RNA, and vector plasmid prepared in a strain that expresses the switch methylase. In this reaction, the type IIS enzyme does not cut any internal sites for the type IIS enzyme that lie within the insert in the donor plasmid as these sites have been methylated and so blocked by the insert-blocking methylase. In contrast, the type IIS enzyme does cut the insert-flanking type IIS sites in the donor plasmid because these sites were protected from methylation by the dCas9 which was bound to them. Thus, the type IIS restriction enzyme releases the insert from the donor plasmid, but does not cut the insert at any type IIS restriction sites within the insert itself. The recipient vector plasmid contains a negative selection marker which will be replaced with the assembled inserts in the correctly assembled plasmid. At each end of the marker there are two head-to-head sites type IIS sites, all for the same enzyme. The outer two of these four sites are the methylation-switchable and dCas9-protectable sites, which have been methylated by the switch methylase and so are not cut by the type IIS enzyme. However, the inner type IIS sites are not methylation-switchable therefore will be cut, and this releases the selectable marker to be replaced by the inserts. In the fully assembled plasmid, the assembled insert is now flanked by the two outer methylation-switchable type IIS restriction enzyme sites which cannot be cut by the type IIS enzyme because they are methylated. Thus the assembly is completed. The newly formed plasmid can then be transformed into a normal strain that does not express the switch methylase nor the dCas9-protection mechanism, which will remove all the specific methylation of the type IIS restriction sites for the given enzyme in the plasmid. The assembled plasmid can then be used for next stage assembly starting with transformation into the strain that expresses the insert-blocking methylation, dCas9 and appropriate guide RNA, because the assembled insert is flanked by the same methylation-switchable and dCas9-protectable type IIS restriction sites as the donor plasmids before the assembly (FIG. 8 and FIG. 9). Alternatively, the assembly reaction mixture can be transformed directly into the strain that expresses the insert-blocking methylase, dCas9 and appropriate guide RNA, which yields assembled plasmids with the internal type IIS restriction sites blocked and the flanking sites protected by dCas9 (FIG. 10 and FIG. 11). This assembled plasmid can be used directly as donor plasmid for next stage one-pot DNA assembly (FIG. 10 and FIG. 11).

[0544] It should be noted that the above systems described that utilize flanking BsaI sites that are both methylation-switchable and dCas9-protectable are designed for sequence-independent hierarchical DNA assembly whereas the assembled DNA can be used as insert plasmid for next stage of DNA assembly. If only one-round of DNA assembly is required such that the assembled DNA will not be used for next round DNA assembly, then the design requirements can be relaxed that in the donor plasmids the flanking BsaI sites must still be dCas9-protectable but not necessarily methylation-switchable, and in the assembly vector the outside pair of BsaI sites must be methylation-switchable but not necessarily dCas9-protectable.

[0545] DNA Assembly Using Universal Assembly

[0546] To demonstrate the utility of Universal Assembly for DNA assembly, we built a proof-of-principle Universal Assembly system using appropriately guided dCas9 binding to protect the insert-flanking BsaI type IIS restriction enzyme site from methylation by the M2.Eco31I methylase. This used in vivo methylation in the strain DH10B-2W213R. The guide RNA sequence (guide #360) was designed to protect a methylation-switchable insert-flanking BsaI site that can be switched on and off using the M.Osp807II methylase (FIG. 5A, FIG. 6, Table 1). This system was used to assemble a ˜3.6 kb fragment from four DNA fragments, each of which contained a single internal BsaI site (FIG. 12A, Table 1). The internal BsaI sites do not overlap with the guide RNA sequence used to protect the dCas9-protectable insert-flanking BsaI sites, and so are not protected from methylation when the plasmid is prepared in the DH10B-2W213R strain which expresses the guide RNA (guide #360) for the insert-flanking sites, dCas9 and the M2.Eco31I1 methylase (FIG. 5 and FIG. 10). The resulting methylation that occurs at any BsaI sites within the insert subsequently protects them from digestion by BsaI during the assembly process. However, the insert-flanking BsaI sites have not been methylated because of the bound dCas9 and so are cut during the assembly process (FIG. 6B). For insert plasmids prepared in DH10B-2W213R, the internal BsaI site is 100% methylated, and the percentage of insert plasmids with both flanking BsaI sites protected by dCas9 from M2.Eco31I methylation is around 50% (FIG. 12B). When the insert plasmids prepared in DH10B-2W213R were assembled into assembly vector prepared in a strain that expresses the M.Osp807II (DH10B-M.Osp807II), and the assembly reaction transformed into DH10B-2W213R, 50% of the colonies are white and >80% (7 out of 8) of the white colonies screened were correctly assembled as verified by restriction digest and DNA sequencing (FIG. 12D). The correctly assembled DNA was still flanked by the same guide-RNA (guide #360)-protectable BsaI sites. Test digestion shows that the internal BsaI sites in the assembled DNA prepared in DH10B-2W213R strain that deploys guide #360-based dCas9 methylation protection were completely blocked by M2.Eco31I methylation, whereas the flanking BsaI sites were protected from M2.Eco31I methylation. The percentage of the assembled plasmids with both flanking BsaI sites protected from M2.Eco31I methylation is over 50%. This prepares the assembled DNA for use as donor plasmid in the next stage one-pot assembly (FIG. 12F).

[0547] Similar results were obtained using a Universal Assembly system with a different guide RNA sequence (guide #401) and different switch methylase (M.Sen0738I). In this system, the guide RNA sequence (guide #401) is designed to be compatible with M.Sen0738I-based methylation switching (FIG. 5A and FIG. 7, Table 1). The M.Sen0738I switch methylase was chosen from a set of potentially suitable switch methylases for methylation-switching of BsaI sites, because unlike the M.Osp807II-compatible Universal Assembly system in which the 5 bp seed sequence of guide RNA is constrained by the M.Osp807II methylation motif (NGACN) (FIG. 6A), the seed sequence of guide RNA for methylation compatible with M.Sen0738I can be freely chosen (NNNNN) (FIG. 7A). This increases the design space of guide RNA seed sequence compatible with the same switch methylase considerably (FIG. 7). With the RNA guide #401-compatible insert plasmids prepared in strain DH10B-2W214R that deploys guide #401-based methylation protection (FIG. 12C), and an assembly vector prepared in strain DH10B-2W148R for M.Sen0738I-based methylation switching, a 3.7 kb fragment can be assembled from 4ט1 kb fragments with high efficiency (7/8 correct) (FIG. 12E). As the assembled DNA is flanked by the same guide #401-protectable BsaI sequence, when prepared in DH10B-2W214R, the internal BsaI sites of the assembled DNA were completely methylated, whereas the flanking BsaI sites in the plasmid were protected from methylation (FIG. 12F), therefore DNA assembled using the guide #401 based assembly system can be used as donor plasmid in next stage of one-pot Universal Assembly.

[0548] These data together demonstrate that the Universal Assembly system can efficiently assemble DNA with internal type IIS restriction sites using different guide RNA sequences that are compatible with a suitable switch methylase. The choice of guide RNA sequence therefore can be tailored to the DNA sequence to assemble, which enables the system to assemble DNA with no sequence constraints.

[0549] Hierarchical Scarless DNA Assembly Using a Fixed Set of Sequence-Independent Assembly Vectors

[0550] The Universal Assembly system enables hierarchical scarless DNA assembly with type IIS restriction enzymes using a fixed set of assembly vectors. Here we describe the scheme.

[0551] For hierarchical DNA assembly systems that use a single type IIS restriction enzyme for different stages of DNA assembly, the insert DNA fragments are cut to have compatible adhesive ends (‘overhangs’) with the assembly vector, are assembled into the assembly vector using the type IIS restriction enzyme, and the assembled DNA can then be released with a pair of flanking type IIS restriction sites for the same enzyme. We term the overhang sequence used to insert DNA into the vector plasmid backbone during the assembly process as the “pre-assembly overhang”, and the overhang sequence at the end of the assembled DNA when released from the vector backbone in the next stage DNA assembly as “post-assembly overhang” (FIG. 13). For clarity, the sequence of the top strand or ‘forward’ strand is used to define the overhang sequence, but it will be represented by the reverse complement of this on the opposite strand.

[0552] A simple design for a recipient assembly vector to implement scarless hierarchical assembly could have a negative selection marker flanked by head-to-head type IIS restriction sites, to enable the assembled DNA to be released as an insert for next stage DNA assembly. The inner pair of type IIS restriction sites closer to the interposing negative selection maker is used to cut the assembly vector open and so allow ligation of insert DNA into the vector. These inner sites are within the fragment containing the negative selection marker that is removed from the assembly vector. The outer pair of type IIS restriction sites closer to the vector backbone are left in place and after the assembly they will be flanking the assembled insert DNA. These outer sites can then be used to release the assembled DNA which can then be used as an insert for next stage of a hierarchical DNA assembly process. In this design, the pre-assembly overhang sequence depends on the inner type IIS restriction site closer to the negative selection marker, and the post-assembly overhang sequence depends on the outer type IIS restriction site closer to the vector backbone (FIGS. 14A and 14B).

[0553] The pre-assembly overhang and post-assembly overhang may or may not be the same depending on the distance between the head-to-head type IIS restriction sites. When the two restriction sites in the head-to-head arrangement are equidistant from the pre-assembly overhang, they will cut at the same position and so generate the same adhesive end; the pre-assembly overhang sequence and post-assembly overhang sequence are then the same. We term this arrangement of inner and outer sites as a ‘maintenance-type’ head-to-head arrangement (FIG. 15). In a maintenance-type design, the choice of pre-assembly overhang sequence is totally flexible.

[0554] If the head-to-head sites are arranged such that the distance between the outer site and the pre-assembly overhang is less than the distance between the inner site and the pre-assembly overhang then one or more bases will be excised from the end of the insert DNA following the DNA assembly process. If the distance between the outer site and the pre-assembly overhang is reduced enough the post-assembly overhang sequence can be from within the insert DNA sequence itself and totally independent of the pre-assembly overhang. This will arise when the number of excised bases is greater than the size of the pre-assembly overhang. We term this arrangement of inner and outer sites as an ‘excision-type’ head-to-head arrangement (FIG. 16). For example, BsaI leaves a 4 bp overhang, so for a BsaI-based MetClo recipient vector, the excision-type design requires the distance between the outer site and the pre-assembly overhang to be at least 4 bp less than the distance between the inner site and the pre-assembly overhang (FIG. 16).

[0555] With an excision-type arrangement the inner restriction site cuts open the recognition sequence of the outer restriction site when the negative selection marker is cut out during the assembly process. This outer restriction site is then reconstructed when the overhang of the insert DNA is ligated to the overhang of the assembly vector (FIG. 16). There are various implications of the design, which in part depend on whether the insert DNA preparation involves methylation protection.

[0556] If methylation protection is not used for insert DNA preparation, then for a hierarchical assembly system using only a single type IIS restriction enzyme, the insert DNA must not contain any sites for this type IIS restriction enzyme. This is because if the insert contains one or more sites for this enzyme, then the enzyme will generate multiple DNA fragments with different adhesive ends, which may interfere with the assembly process. This places a limit on the number of bases that can be excised in the excision-type design. For a BsaI-based assembly systems, the upper limit for excision is 6 bp (FIG. 17, FIG. 18).

[0557] Furthermore, the initial outer restriction site in the vector backbone and the reconstructed outer restriction site following assembly must be protected by methylation-switching. The feasibility of the excision-type design thus depends on the location and strand of the methylated base that is generated by the switch methylase.

[0558] This is because the methylated base must remain in the assembly vector backbone following digestion of the assembly vector by the type IIS restriction enzyme acting from the inner site. For example, both the M.Osp807II and M.Sen0738I switch methylases allows 4-6 basepairs excision-type design in a BsaI-based system (FIG. 16 and FIG. 18), whereas the M2.NmeMC58II switch methylase in a BpiI-based system does not allow excision-type design at all because BpiI cuts such that the methylated base is retained with the negative selection marker rather than in the assembly vector backbone (FIG. 19).

[0559] The outer restriction site does not need to be a full restriction site and a partial site can be reconstituted to a full site by the insert DNA, which increases the freedom of sequence design for the excision-type head-to-head restriction sites (FIG. 20). A partial site that deploys methylation-switching still needs to carry the switch methylase recognition motif for the excision-type design to be feasible. Both M.Osp807II and M.Sen0738I-based methylation switching in a BsaI-based system allows 4-6 basepair excision-type design using a partial outside restriction site (FIG. 20). Table 2 lists the allowable pre-assembly overhang sequences for all the excision-type designs that can be used for M.Osp807II and M.Sen0738I-based methylation switching in a BsaI-based assembly system.

[0560] If methylation protection is used for the insert DNA preparation, then all the internal type IIS restriction sites in the insert are methylated and so inactivated. This allows for excision of more base pairs at the end of the insert sequence. During assembly process, the outer restriction site is provided by an internal restriction site inside the insert DNA sequence, which is methylated by the methylation protection mechanism during insert DNA preparation (FIG. 21).

[0561] With the maintenance-type and excision-type design of head-to-head restriction sites, it is possible to design a scarless hierarchical DNA assembly scheme that requires only a fixed set of assembly vectors for assembly of any DNA. This set of assembly vectors consists of three special vector types. all carrying an negative selection marker flanked by head-to-head type IIS restriction sites with the same pre-assembly overhangs ‘u’ and ‘v’ on the ‘left’ and ‘right’ end respectively (FIG. 22). The head-to-head restriction sites flanking the negative selection marker are of the maintenance-type or excision-type design depending on the vector type. In vector type VLeft, the negative selection marker is flanked on the ‘left’ by a maintenance-type head-to-head arrangement and on the ‘right’ by an excision-type head-to-head arrangement. The ‘right’ excision-type arrangement leads to excision of 6 base pairs. In vector type VRight, the negative selection marker is flanked on the ‘left’ by an excision-type head-to-head arrangement and on the ‘right’ by a maintenance-type head-to-head arrangement. The ‘left’ excision-type arrangement leads to excision of 4 base pairs. In vector type VMiddle, the negative selection marker is flanked on both sides by an excision-type head-to-head arrangement. These two excision-type arrangements differ, such that the ‘left’ excision-type arrangement leads to excision of 4 base pairs, but the ‘right’ excision-type arrangement leads to excision of 6 base pairs. In these three vectors, the pre-assembly overhang sequences ‘u’ and ‘v’ and their reverse-complement sequences are designed to be different to minimize the probability of self-ligation of the assembly vector backbone, and to ensure the directionality of assembly relative to the vector backbone sequence, thus the directionality of the excision process. For scarless BsaI-based assembly using the M.Sen0738I switch methylase, we chose ‘CTCC’ as the 4 bp overhang sequence ‘u’ (the physical overhang associated with the vector backbone is the reverse complement GGAG), and ‘AGAC’ as the 6 bp overhang sequence ‘v’ (FIG. 22).

[0562] The design of these three types of assembly vectors allows any given insert DNA which has the appropriate overhangs ‘u’ (‘CTCC’) and ‘v’ (‘AGAC’) to be cloned into any one of the three vectors, but with different outcome depending on the type of vector (FIG. 23). For an insert DNA plasmid with insert DNA carrying overhang ‘u’ and ‘v’ at the left and right hand of the fragment, cloning into vector VLeft excises the 6 bp adaptor sequence ‘TGAGAC’ at the right end of the fragment only (FIGS. 23A and B). Cloning into vector VMiddle excises both the 4 bp adaptor sequence ‘CTCC’ at the left end of the fragment and the 6 bp adaptor sequence ‘TGAGAC’ at the right end of the fragment (FIGS. 23C and D). Cloning into vector VRight excises the 4 bp adaptor sequence ‘CTCC’ at the left end of the fragment only (FIGS. 23E and F).

[0563] The multi-fragment assembly process and adaptor excision process can be combined in a single reaction. FIG. 24 illustrates an example of the outcomes from assembling the same set of three inserts in each of the three vector types. The leftmost insert DNA fragment starts with overhang sequence ‘u’ (‘CTCC’) and the last insert DNA fragment ends with overhang sequence ‘v’ (‘AGAC’). Assembly into vector VLeft generates an assembled fragment with excision of 6 bp adaptor sequence (‘TGAGAC’) at the right end of the assembled sequence (FIGS. 24A and B). Assembly into vector VMiddle generates an assembled fragment with excision of 4 bp adaptor sequence (‘CTCC’) at the left end, and 6 bp adaptor sequence (‘TGAGAC’) at the right end of the assembled sequence (FIGS. 24C and D). Assembly into vector VRight generates assembled fragment with excision of 4 bp adaptor sequence (‘CTCC’) at the left end of the assembled sequence (FIGS. 24E and F).

[0564] With these three type of assembly vectors and the Universal Assembly system that deploys methylation protection and methylation switching, any DNA can be assembled scarlessly with no sequence constraints. FIG. 25 illustrates an example of DNA assembly of 9 fragments in two rounds with 3 fragments per group in each round using the above vector set design (‘CTCC’ as ‘u’ and ‘AGAC’ as ‘v’) with a BsaI-based Universal Assembly system. Vector DNA is prepared in a strain expressing the M.Sen0738I switch methylase and insert DNA in a strain expressing the guide #401-based methylation protection system. The antibiotic selection marker of the assembly vector differs between consecutive rounds of DNA assembly. The DNA sequence to be assembled (FIG. 25A) is divided into 9 fragments with 4 bp overlapping sequences that define the overhang sequences for ordered DNA assembly (FIG. 25B). These 8 ‘internal’ overhang sequences must differ from the pre-assembly overhang sequences ‘CTCC’ and ‘AGAC’ (and their reverse complements). In addition, each internal overhang sequence used in each round of the assembly must be different from each other (and their reverse-complement sequences). The starting fragments can then be synthesized by PCR or gene-synthesis with appropriate adaptor sequences added, and cloned into suitable vectors that encode methylation-protection/methylation-switching motifs. A sequence-independent cloning method can be used for this purpose such as blunt-end cloning. The adaptor sequences added are the adaptor sequences that will be excised when cloned into vectors with excision-type designs. For the current design, the 4 bp adaptor sequence ‘CTCC’ and 6 bp adaptor sequence ‘TGAGAC’ are added to the left and right end of each starting fragment respectively, generating insert plasmids carrying the insert fragments with pre-assembly overhang sequence ‘u’ (‘CTCC’) and ‘v’ (‘AGAC’) (FIG. 25C). The insert plasmids were then cloned as single fragment into a suitable type of vectors depending on the position the cloned fragment would take up in the next round assembly (FIGS. 25D and E). In this case, fragment ‘A’, ‘D’ and ‘G’ are cloned as single fragment into vector ‘VLeft’ because they are the left fragment in the next round assembly; fragments ‘B’, ‘E’ and ‘H’ are cloned into vector ‘VMiddle’ because they are the middle fragment in the next round; fragments ‘C’, ‘F’ and ‘I’ are cloned into vector ‘VRight’ because they are the right fragment in the next round. The cloned fragments are then assembled three fragments per group into a suitable vector which was chosen on the basis of the position that the assembled fragment would take up in the next round assembly (FIGS. 25F and G); that is, whether it would be the left, middle or right fragment in the next round. In this case, fragments ‘A’, ‘B’, ‘C’ are assembled as fragment ‘ABC’ into vector ‘VLeft’ because fragment ‘ABC’ is the left fragment in the next round assembly. Fragments ‘D’, ‘E’, ‘F’ are assembled into vector ‘VMiddle’ as ‘DEF’ is the middle fragment in the next round. Fragments ‘G’, ‘H’, ‘I’ are assembled into vector ‘VRight’ as ‘GHI’ is the right fragment in the next round. In the last round, the three intermediate fragments ‘ABC’, ‘DEF’ and ‘GHI’ are assembled into a single fragment in vector ‘VMiddle’, generating fragment ‘ABCDEFGHI’ as required (FIGS. 25H and I).

[0565] In practice, the cloning of linear double strand DNA into a vector and cloning of this insert into a suitable vector type can be combined into a single step (FIG. 26C), simplifying the scarless assembly scheme (FIG. 26).

[0566] In the scarless assembly scheme, the design of the assembly vector is independent of the DNA sequence to be assembled. Therefore, the scheme allows using a fixed set of assembly vectors to assemble any DNA with no sequence constraints. A small set of six assembly vectors representing the three different types and two antibiotic selection markers (e.g. pMOBK401_VL, pMOBK401_VM, pMOBK401_VR, pMOBC401_VL, pMOBC401_VM, pMOBC401_VR representing VLeft, VMiddle and VRight with kanamycin or chloramphenicol selection markers) is sufficient for hierarchical scarless assembly with arbitrary rounds of assemblies.

[0567] Hierarchical DNA Assembly Using the Scarless Scheme

[0568] To demonstrate the feasibility of this scarless assembly scheme, we assembled a ˜90 kb DNA fragment from the human MICA locus from 14 fragments in two stages (FIGS. 27A and B, Table 3). 14 starting fragments with suitable overhang sequences were generated by PCR and cloned by blunt-end ligation to generate insert plasmids that carry the starting fragments with suitable overhang sequences added depending on the position that the resulting fragment will take up in the next round of assembly. In round one of the assembly, groups of 2-4 fragments were assembled into suitable assembly vectors depending on the position that the assembled fragment would take up in the next round of assembly. In round two, the four intermediate insert plasmids prepared in DH10B-2W214R were assembled in a single reaction into the final product. The success rate of each round of assembly exceeds 80% (FIG. 27C and Table 3). This demonstrates that the scarless assembly scheme combined with Universal assembly can be used for efficient scarless assembly of an arbitrary DNA sequence.

[0569] Scarless Assembly Vector Examples

[0570] Vector with M.Osp807II Methylation Switching, 4 bp Excision on the Left, 6 bp on the Right.

[0571] M.Osp807II used for methylation switching for preparation of assembly vector. Insert prepared by methylation protection using M2.Eco31I and dCas9 with guide RNA sequence

TABLE-US-00017 (SEQ ID NO: 95)   NNNNNNNNNNNNNNNNGACN
(last 5 bp are the seed sequence that determines the specificity).

TABLE-US-00018 VL (vector left insert) (SEQ ID NO: 96) NNNNNNNNNNNNNNNNGACNNGGTCTCNCTCCNGAGACC(N.sup.X)GGTCTCN AGACCNNGTCNNNNNNNNNNNNNNNN VM (vector middle insert) (SEQ ID NO: 97) NNNNNNNNNNNNNNNNGACNNGGTCTCCNGAGACC(N.sup.X)GGTCTCNAGAC CNNGTCNNNNNNNNNNNNNNNN VR (vector right insert) (SEQ ID NO: 98) NNNNNNNNNNNNNNNNGACNNGGTCTCCNGAGACC(N.sup.X)GGTCTCNAGAC NGAGACCNNGTCNNNNNNNNNNNNNNNN

[0572] Vector with M.Sen0738I Methylation Switching, 4 bp Excision on the Left, 6 bp on the Right

[0573] M.Sen0738I used for methylation switching for preparation of assembly vector. Insert prepared by methylation protection using M2.Eco31I and dCas9 with guide RNA sequence

TABLE-US-00019 (SEQ ID NO: 99)   NNNNNNNNNNNCCAGNNNNN
(last 5 bp are the seed sequence that determines the specificity).

TABLE-US-00020 VL (vector left insert) (SEQ ID NO: 100) NNNNNNNNNNNCCAGNNNNNNGGTCTCNCTCCNGAGACC(N.sup.X)GGTCTCN AGACCNNNNNNCTGGNNNNNNNNNNN VM (vector middle insert) (SEQ ID NO: 101) NNNNNNNNNNNCCAGNNNNNNGGTCTCCNGAGACC(N.sup.X)GGTCTCNAGAC CNNNNNNCTGGNNNNNNNNNNN VR (vector right insert) (SEQ ID NO: 102) NNNNNNNNNNNCCAGNNNNNNGGTCTCCNGAGACC(N.sup.X)GGTCTCNAGAC NGAGACCNNNNNNCTGGNNNNNNNNNNN

[0574] (N.sup.x) may mean any number of A, C, T or G, or between 0 bp and 300 kbp of A, C, T, or G.

[0575] Discussions

[0576] Here we described a system for DNA assembly using type IIS restriction enzymes with no sequence constraints.

[0577] The design of the system is based on the methylation protection principle, whereas a sequence-specific DNA binding protein can protect specific type IIS restriction site that overlaps with the binding sequence from being methylated by a methylase that would otherwise methylate all the type IIS restriction sites for the given enzyme. This can be achieved by coexpressing the DNA binding protein and the methylase in the strain used for DNA propagation (in vivo), or carrying out the methylation reaction in the presence of the DNA binding protein (in vitro). In theory a number of different types of sequence specific DNA binding proteins can be used. This includes two types: one that has no enzymatic activities toward the DNA substrate, such as CRISPR based dCas9, TALEN, Zinc finger protein etc. (pure DNA binding protein). The other may have enzymatic activity toward the DNA substrate (for example sequence specific DNA methylase), however as long as the modification (for example methylation) does not affect restriction enzyme activity of the type IIS restriction enzyme towards the type IIS restriction site then it can be used (See Example 2, FIGS. 28 and 29).

[0578] With the methylation protection principle, double strand DNA can be generated where a linear insert fragment is flanked by “protected” unmethylated type IIS restriction sites, but all the internal type IIS restriction sites are methylated—the insert can therefore be cut out by the type IIS restriction enzyme intact from the vector backbone in the double stranded DNA when the DNA is prepared through the methylation protection mechanism. In this way we can release intact linear DNA without sequence constraints, with any adhesive ends (4 bp for BsaI with no sequence constraints), from a closed (circular) DNA molecule. This linear DNA can then be used for ligation in cloning. The linear DNA can also be used for other DNA assembly methods such as Gibson assembly or homologous recombination in yeast or B. subtilis etc.

[0579] This approach can be applied to one-pot DNA assembly using type IIS restriction enzyme. For a single stage DNA assembly, the design is very flexible. For example, all the fragments for DNA assembly including the vector backbone can be prepared using the methylation protection approach, as long as the assembled plasmid sequence does not contain methylation-protectable type IIS restriction sites protectable in the strain used to prepare the insert fragments.

[0580] For an idempotent hierarchical assembly system, whereas both the insert fragments and the assembled fragments at all the stages contain the same flanking protectable type IIS restriction sites, two different designs are feasible. One design combines the methylation protection principle with the methylation switching principle, whereas a combined methylation-protectable and switchable flanking type IIS restriction sites are used as outer pair of the head-to-head type IIS restriction sites in the assembly vector, and non-switchable type IIS restriction sites for the same enzyme as the inner pair. In this design the assembly vector is prepared in strains that deploy methylation switching to selectively block the outer pair type IIS restriction sites in the assembly vector backbone, the insert plasmids are prepared in, and assembled plasmid transformed into strains that deploys methylation protection to selectively block internal type IIS restriction sites within the insert while leaving flanking type IIS restriction sites active (FIG. 10 and FIG. 11).

[0581] Another idempotent design (Example 3) uses the methylation protection principle alone for both vector and insert DNA preparation. Here the assembly vector contains head-to-head type IIS restriction sites with the outer and inner pair of type IIS restriction sites protected with two different methylation-protection specificity. The insert plasmids are prepared in and the assembled plasmid transformed into strains that deploy methylation protection for one specificity to block internal type IIS restriction sites while leaving the flanking methylation-protectable restriction sites open. The assembly vector is prepared in strains that deploy methylation protection with a different specificity to selectively protect the inner pair of type IIS restriction sites from methylation (FIG. 30 and FIG. 31). Compared with the previous design, this design has the advantage that the assembly vector backbone sequence can also contain internal type IIS restriction sites. The potential drawbacks are potential high background uncut assembly vector in the transformation reaction due to inefficient methylation protection of inner type IIS restriction sites within the assembly vector. This may be eliminated by using suicide genes such as ccdB as negative selection marker.

[0582] The idempotent hierarchical assembly system that deploys both methylation protection and methylation switching can be further adapted into an assembly system for scarless DNA assembly of DNA without sequence constraints using a limited set assembly vectors. This unique design is based on the ‘maintenance type’ and ‘excision type’ head-to-head type IIS restriction sites for assembly vector design, which enables assembly of the same set of insert fragments into different types of assembly vectors, at the same time excising fixed number of nucleotides at specific ends of the assembled DNA depending on the choice of assembly vector (FIG. 24). With this design, hierarchical scarless assembly can be carried out using only a fixed set of assembly vectors for any DNA sequence with no sequence constraints (FIG. 25 and FIG. 26). The utility of such system is demonstrated by hierarchical assembly of a large 90 kb DNA.

EXAMPLE 2—DNA METHYLASE AS THE SPECIFIC DNA-BINDING PROTEIN FOR METHYLATION PROTECTION OF THE METHYLATION-PROTECTABLE RESTRICTION ELEMENT

[0583] Alternatively, a second DNA methylase can be used as the specific DNA-binding protein for methylation protection of type IIS restriction sites within methylation-protectable restriction sites, providing that DNA methylation by the second methylase itself does not interfere with the restriction activity of the type IIS restriction enzyme (FIG. 28).

[0584] Methods

[0585] Reagents

[0586] Enzymes for molecular biology are from NEB unless otherwise stated. A high-fidelity version of BsaI restriction enzyme named BsaI-HFv2 is used in place of BsaI for all the experiments.

[0587] Plasmid Construction

[0588] The plasmid for testing methylation protection of M.Csp205I (pMOP_testN7) was constructed using standard restriction enzyme/ligation-based cloning techniques. DNA fragments for cloning were generated by PCR with Q5 polymerase (NEB). The vector backbone was based on the ampicillin resistant MoClo vector pICH47732.

[0589] Low copy number plasmid pMOBKZ-2W89, pMOBKZ-2W91, pMOBKZ-2W92 and pMOBKZ-2W94 was constructed for coexpression of M1.Eco31I or M2.Eco31I (under a J23112 or J23114 promoter, a modified RBS based on B0034m named B0034m* and an L3S2P21 terminator), M.Csp205I (under a J23100 promoter, a B0034m* RBS and an L3S1P51 terminator), and S.Csp205I (under a J23100 promoter, a B0034m* RBS and an L3S1P32 terminator). Briefly, for the construction of these plasmids, the transcription units were each assembled from individual cloned DNA parts using BsaI-based MetClo with the MetClo vector set described previously (Lin D et al. Nucleic Acids Res. 2018 Nov. 2; 46(19):e113. doi: 10.1093/nar/gky596 PMID 29986052). The transcription units were then assembled using BsaI into a low copy number kanamycin-resistant vector with an F replication origin, insert-flanking arsB homologous sequences and a zeocin-selection marker. In each round of assembly, insert DNA fragments containing M1.Eco31I or M2.Eco31I were generated by PCR to remove M1.Eco31I or M2.Eco31I methylation in the DNA.

[0590] Generation of E. coli Strain

[0591] The E. coli strain that constitutively expresses M1.Eco31I or M2.Eco31I together with M.Csp205I and S.Csp205I (DH10B-2W89R, DH10B-2W91R, DH10B-2W92R and DH10B-2W94R) was constructed by recombineering using a linear DNA fragment amplified by PCR from the corresponding plasmids (pMOBKZ-2W89, pMOBKZ-2W91, pMOBKZ-2W92 and pMOBKZ-2W94 respectively) using primers TTCTGTTACCCATCCAATTGTTC (SEQ ID NO: 103) and CAGGCGCTTACCCGCTTCAT (SEQ ID NO: 104).

[0592] Results

[0593] To develop practical methylation protection system using a second DNA methylase, we designed systems using type I methylase M.Csp205I (with methylase subunit M.Csp205I and specificity subunit S.Csp205I) as the second DNA methylase for methylation protection of BsaI restriction sites from methylation by M1.Eco31I or M2.Eco31I. Methylation of BsaI sites by M1.Eco31I or M2.Eco31I blocks BsaI activity, whereas methylation of the 5.sup.th base adenine at the bottom strand of an overlapping BsaI site by M.Csp205I does not itself affect restriction of the BsaI site by BsaI. The aim is to set up a system to test whether M.Csp205I can be used for in vivo methylation protection in combination with either M1.Eco31I or M2.Eco31I. We first constructed new E. coli strains that coexpress M.Csp205I, S.Csp205I and M1.Eco31I (DH10B-2W89R and DH10B-2W91R) or M2.Eco31I methylases (DH10B-2W92R and DH10B-2W94R). We used the strong J23100 promoter for both M.Csp205I and S.Csp205I expression, and tested weaker promoter J23114 or J23112 for M1.Eco31I or M2.Eco31I expression (FIG. 29A). We then constructed a test plasmid (pMOP_testN7) that contains a BsaI site that overlaps with M.Csp205I recognition sequence, and a standard BsaI site (FIG. 29B) and prepared this plasmid in the new strains. Only in the strain that express M2.Eco31I under the weak J23112 promoter (DH10B-2W94R), the test plasmid prepared was cut by BsaI at the BsaI site that overlaps with the guide RNA sequence, but not at the standard BsaI site (FIG. 29C). This demonstrates that M2.Eco31I, not M1.Eco31I under the control of suitable promoter in combination M.Csp205I can be used for methylation protection of BsaI sites.

EXAMPLE 3 DESIGN OF DNA ASSEMBLY PROCESS BASED ON METHYLATION PROTECTION APPROACH ALONE

[0594] Alternatively, a one-pot DNA assembly system can be designed based on the methylation protection approach alone (FIG. 4), without using the methylation switching approach (FIGS. 1 and 2). The system requires a specific design of assembly vector, in which the negative selection marker is flanked by two pairs of type IIS restriction sites in head-to-head arrangement, all of which are methylation protectable. The outer pair of type IIS restriction sites closer to the vector backbone are methylation-protectable by dCas9-based methylation protection approach guided by guide RNA guideX, with 5 bp seed sequence specificity (‘XXXXX’), and the inside pair of BsaI sites closer to the negative selection marker LacZalpha are methylation-protectable by dCas9 through a different guide RNA guideY with a different specificity (‘YYYYY’) (FIG. 30). Preparation of the vector in strains that expression dCas9-methylation protection mechanism with guide RNA guideY will result in selective methylation of the outer pair of type IIS restriction sites, whereas preparation in strains with guideX will result in selective methylation of the inner pair of type IIS restriction sites.

[0595] With this unique assembly vector design, a one-pot assembly reaction can then be undertaken using donor plasmid(s) prepared in the strain that expresses the insert-blocking methylase, dCas9 and an appropriate guide RNA guideX, and vector plasmid prepared in the strain that expresses the same methylase, dCas9 and a different guide RNA guideY. The donor plasmid contains inserts flanked by methylation-protectable type IIS restriction sites protectable by dCas9 guided by guide RNA guideX. Preparation of the donor plasmid in methylation protection strain that coexpresses the insert-blocking methylase (M2.Eco31I), dCas9 and guide RNA guideX blocks the internal type IIS restriction sites that are not bound by dCas9 within the insert, whereas the flanking type IIS restriction sites are bound by dCas9/guideX therefore protected from methylation by M2.Eco31I. This enables the release of the insert fragment intact from the donor plasmid by the type IIS restriction enzyme for DNA assembly. The assembly vector on the other hand are prepared in methylation protection strain that expresses a different guide RNA guideY, resulting in selective methylation of outer, but not inner pair of type IIS restriction sites from methylation by M2.Eco31I. This enables the release of the negative selection marker from the vector backbone using the inner pair type IIS restriction sites to be replaced by the assembled insert fragments. Ligation of the vector backbone with the insert fragments generates fully assembled plasmid, in which the assembled insert is now flanked by the two outer type IIS restriction enzyme sites which cannot be cut by the type IIS enzyme because they are methylated. Thus the assembly is completed. The newly formed plasmid can then be transformed into a normal strain that does not express the dCas9-protection mechanism, which will remove all the specific methylation of the type IIS restriction sites for the given enzyme in the plasmid. The assembled plasmid can then be used for next stage assembly starting with transformation into the strain that expresses the insert-blocking methylation, dCas9 and guide RNA guideX, because the assembled insert is flanked by the dCas9-protectable type IIS restriction sites with the same seed sequence specificity as the donor plasmids before the assembly (FIG. 30). Alternatively, the assembly reaction mixture can be transformed directly into the strain that expresses the insert-blocking methylase (M2.Eco31I), dCas9 and guide RNA guideX, which yields assembled plasmids with the internal type IIS restriction sites blocked and the flanking sites protected by dCas9/guideX (FIG. 31). This assembled plasmid can be used directly as donor plasmid for next stage one-pot DNA assembly in the same system.

EXAMPLE 4—UNIVERSAL ASSEMBLY BASED ON DST1CAS9

[0596] Additionally, instead of dCas9 from the CRISPR-Cas9 system of Streptococcus pyogenes, other RNA-guided DNA binding proteins can be used as the specific DNA-binding protein for methylation protection of type IIS restriction sites within methylation-protectable restriction sites. Here we demonstrated that RNA-guided dST1Cas9 from the Streptococcus thermophiles CRISPR1-Cas9 system can be used for methylation protection of BsaI sites from methylation by M2.Eco31I in vivo using E. coli strains coexpressing dST1Cas9, guide RNA and M2.Eco31I methylase. Universal assembly systems based on dST1Cas9-based methylation protection and M.Sen0738I-based methylation switching were then designed and used for practical one-pot BsaI-based assembly of DNA fragments that contain internal BsaI sites.

[0597] Methods

[0598] Reagents

[0599] Enzymes for molecular biology are from NEB unless otherwise stated. A high-fidelity version of BsaI restriction enzyme named BsaI-HFv2 is used in place of BsaI for all the experiments.

[0600] Plasmids Construction

[0601] Plasmids for testing specific strains and for proof-of-principle DNA assembly were constructed using standard restriction enzyme/ligation-based cloning techniques. DNA fragments for cloning were generated by gene synthesis (Integrated DNA Technologies or Invitrogen), or by PCR with Q5 polymerase (NEB). Plasmids for functional testing, and the assembly vector for proof-of-principle DNA assembly were based on the ampicillin resistant MoClo vector pICH47732 (Addgene plasmid #48000; http://n2t.net/addgene:48000; RRID:Addgene_48000—Weber et al PLoS One. 2011 Feb. 18; 6(2):e16765. doi: 10.1371/journal.pone.0016765, which is herein incorporated by reference). Insert plasmids for proof-of-principle assembly were based on the kanamycin-resistant vector backbone described previously for MetClo proof-of-principle assembly (Lin D et al. Nucleic Acids Res. 2018 Nov. 2; 46(19):e113. doi: 10.1093/nar/gky596. PMID 29986052, which is herein incorporated by reference).

[0602] Plasmid pMOBKZ-2W276 or pMOBKZ-2W278 was constructed for coexpression of guide RNA guide #498 or guide #500 (under a J23119 promoter and an L3S2P21 terminator), dST1Cas9 (under a J23119 promoter, a B0034m* RBS and an L3S1P51 terminator), and M2.Eco31I (under a J23112 promoter, a B0034m* RBS and an L3S1P32 terminator). These plasmids were constructed in two stages. In stage one, an intermediate plasmid pMOBKZ-2W274 was constructed, which carries a LacZalpha selection cassette flanked by LguI restriction sites in place of the 20 bp guide RNA sequence in pMOBKZ-2W276 or pMOBKZ-2W278. Briefly, for the construction of pMOBKZ-2W274, the transcription units were each assembled from individual cloned DNA parts using BsaI-based MetClo with the MetClo vector set described previously (Lin D et al. Nucleic Acids Res. 2018 Nov. 2; 46(19):e113. doi: 10.1093/nar/gky596. PMID 29986052). The transcription units were then assembled using BsaI into a low copy number kanamycin-resistant vector with an F replication origin, insert-flanking arsB homologous sequences and a zeocin-selection marker. In each round of assembly, insert DNA fragments containing M2.Eco31I were generated by PCR to remove M2.Eco31I methylation in the DNA. The dST1Cas9 transcription unit for DNA assembly was generated by PCR using a flanking primer containing the J23119 promoter to avoid toxicity of the dST1Cas9 transcription unit in the p15A-based low copy plasmid. In the final round assembly of pMOBKZ-2W274, light blue colonies were screened and verified by sequencing.

[0603] In stage two, annealed oligo nucleotides designed based on the guide RNA sequences were cloned into LguI-digested pMOBKZ-2W274 to generate pMOBKZ-2W276 and pMOBKZ-2W278.

[0604] Strains

[0605] The E. coli strain that constitutively expresses M.Sen0738I and S.Sen0738I (DH10B-2W148R) was described in Example 1. The E. coli strains that deploy dST1Cas9-based methylation protection mechanisms (DH10B-2W276R and DH10B-2W278R) were generated by lambda-red recombineering into DH10B cells using a linear DNA fragment amplified by PCR from pMOBKZ-2W276 and pMOBKZ-2W278 respectively using primers TTCTGTTACCCATCCAATTGTTC (SEQ ID NO: 114) and CAGGCGCTTACCCGCTTCAT (SEQ ID NO: 115). Linear sequences of the PCR product used for recombineering to generate DH10B-2W276R and DH10B-2W278R are listed as 2W276R and 2W278R respectively.

[0606] DNA Assembly

[0607] The standard assembly reaction was 30 fmol of assembly vector, 60 fmol of each insert plasmid, 15 U T4 DNA ligase HC (Thermo Fisher) and 10 U BsaI-HFv2 (NEB) in 20 ul 1×T4 ligase buffer (NEB). The reaction condition was: 37° C. 15 min, followed by 45 cycles of 37° C. 2 min plus 16° C. 5 min, then 37° C. 20 min, and 80° C. 5 min. Assembly reactions were transformed into specific chemically competent cells, and plated on LB plates with AIX selection (ampicillin 100 μg/ml, IPTG 100 μM, X-gal 50 μg/ml) at 37° C. overnight. White colonies were expanded and screened by restriction digestion using DraIII or BsaI and by DNA sequencing.

[0608] For modular assembly of plasmid pMOP498_F5W using M.Sen0738I and a dST1Cas9-guide #498-based system, insert plasmids pMOK498_F5A, pMOK498_F5B, pMOK498_F5C and pMOK498_F5D were prepared in DH10B-2W276R from overnight culture in LB medium supplemented with 1% glucose and 30 μg/ml kanamycin. Assembly vector pMOP498_F5V was prepared in DH10B-2W148R from overnight culture in LB medium with 100 μg/ml ampicillin, and the assembly reaction was transformed into DH10B-2W276R. Assembled clones were cultured in LB medium supplemented with 1% glucose and 100 μg/ml ampicillin.

[0609] For modular assembly of plasmid pMOP500_F6W using M.Sen0738I and a dST1Cas9-guide #500-based system, insert plasmids pMOK500_F6A, pMOK500_F6B, pMOK500_F6C and pMOK500_F6D were prepared in DH10B-2W278R from overnight culture in LB medium supplemented with 1% glucose and 30 μg/ml kanamycin. Assembly vector pMOP500_F6V was prepared in DH10B-2W148R from overnight culture in LB medium with 100 μg/ml ampicillin, and the assembly reaction was transformed into DH10B-2W278R. Assembled clones were cultured in LB medium supplemented with 1% glucose and 100 μg/ml ampicillin.

[0610] Results

[0611] To develop practical Universal assembly systems based on RNA-guided DNA binding proteins other than dCas9, we designed systems using RNA-guided DNA binding protein dST1Cas9 as the specific DNA binding protein for methylation protection of BsaI restriction sites from methylation by M2.Eco31I. RNA guided DNA binding protein dST1Cas9 (also known as dCas9.sub.Sth1) is a variant of Cas9 from the CRISPR1 locus of Streptococcus thermophiles, carrying point mutations (D9A, H599A) that inactivate the nuclease activity (Rock J M et al. Nat Microbiol. 2017 Feb. 6; 2:16274. doi: 10.1038/nmicrobiol.2016.274. PMID 28165460, which is herein incorporated by reference). dST1Cas9 can recognise a 27 bp target sequence composed of a 20 bp sequence specified by the chimeric single guide RNA (sgRNA) and a 7 bp PAM motif (NNAGAAG, whereas N can be any of A, T, C or G) (Rock J M et al. Nat Microbiol. 2017 Feb. 6; 2:16274. doi: 10.1038/nmicrobiol.2016.274. PMID 28165460).

[0612] Methylation-protectable BsaI site can be designed by combining the 27 bp dST1Cas9 recognition sequence with a BsaI recognition sequence so that the two sequences overlap (FIG. 32). Two different designs were explored. In the first design (FIG. 32B), the last base of the 7 bp PAM motif in the dST1Cas9 recognition sequence overlaps with the first base pair of the BsaI restriction site, to form a methylation protectable BsaI site

TABLE-US-00021 (SEQ ID NO: 107)   (NNNNNNNNNNNNNNNNNNNN NNAGAA GTCTC,
with the 20 bp sequence that forms Watson-Crick base pairing with the guide RNA sequence underlined, 7 bp PAM motif in italic, and BsaI restriction site in bold). In this design the guide RNA pairs with the bottom strand of the methylation protectable BsaI site when the BsaI site lies at the right end of the methylation protectable BsaI site. In the second design (FIG. 32C), the first 6 bp of the 20 bp sequence specified by the guide RNA in the 27 bp dST1Cas9 recognition sequence contains the BsaI restriction site ‘GAGACC’ to form a 27 bp methylation protectable BsaI site

TABLE-US-00022 (SEQ ID NO: 108)   (GAGACCNNNNNNNNNNNNNNNNAGAAG,
with the 20 bp sequence that forms Watson-Crick base pairing with the guide RNA sequence underlined, 7 bp PAM motif in italic, and BsaI restriction site in bold, reverse complement as CTTCTNNNNNNNNNNNNNNNNGGTCTC (SEQ ID NO: 109), so that the guide RNA pairs with the top strand of the methylation protectable BsaI site when the BsaI site lies at the right end of the methylation protectable BsaI site.

[0613] To test the above designs of methylation-protectable BsaI sites for dST1Cas9 based methylation protection of BsaI sites from methylation by M2.Eco31I, we constructed E. coli strains that stably coexpress dST1Cas9, guide RNA and M2.Eco31I methylase, with both dST1Cas9 and guide RNA expression driven by the strong J23119 promoter, and M2.Eco31I driven by the weak J23112 promoter (FIG. 33A). We also constructed test plasmids that contain a BsaI site that overlaps with the dST1Cas9 recognition sequence corresponding to the guide RNA sequence (methylation-protectable BsaI site), and a standard BsaI site that does not overlap with the dST1Cas9 recognition sequence (FIG. 33B). For the first design in which the guide RNA pairs with the bottom strand of the methylation-protectable BsaI site, strain DH10B-2W276R was constructed, which expresses guide RNA guide #498 for dST1Cas9, which targets the methylation-protectable BsaI site (GTCTAGTATGCGGATGCCAGTGAGAAGGTCTC (SEQ ID NO: 110), with the BsaI recognition sequence underlined) in test plasmid pMOP_testN24. For the second design in which guide RNA pairs with the top strand of the methylation-protectable BsaI site, strain DH10B-2W278R was constructed, which expresses guide RNA guide #500 for dST1Cas9, which targets methylation-protectable BsaI site (CTTCTCAAGCCCCAGAATGGTGGTCTC (SEQ ID NO: 111), with the BsaI recognition sequence underlined) in test plasmid pMOP_testN32. For both designs, the test plasmid prepared in the corresponding strain can be cut at the BsaI site that overlaps with the guide RNA target sequence, but not the standard BsaI site (FIG. 33C). This demonstrates that dST1Cas9 can selectively protect BsaI sites that overlap with the specific RNA-guided dST1Cas9 binding sequence from methylation by M2.Eco31I.

[0614] The dST1Cas9-based methylation protection systems can also be combined with methylation switching mechanism to build one-pot Universal assembly systems. For example, both designs of dST1Cas9-based methylation-protectable BsaI sites can be combined with M.Sen0738I-based methylation switching mechanism to build dST1Cas9-protectable and M.Sen0738I-switchable BsaI sites (FIG. 34). The methylation-protectable BsaI sites targeted by dST1Cas9-guide #498 in strain DH10B-2W276R and dST1Cas9-guide #500 in DH10B-2W278R have indeed incorporated designs to make them methylation switchable by M.Sen0738I. These can then be used to build Universal assembly systems for one-pot DNA assembly, whereas the insert plasmids were prepared in strains that deploy dST1Cas9-based methylation protection, and the assembly vectors were prepared in strains that deploy methylation switching (FIG. 35-38).

[0615] The designed Universal assembly systems using dST1Cas9/M2.Eco31I-based methylation protection and M.Sen0738I-based methylation switching were then used for proof-of-principle assembly of a 3.6 kb DNA from the human MICA locus (Genbank sequence KF724576.1:23-3633) from 4 fragments, each of which contains an internal BsaI site that does not overlap with the RNA-guided dST1Cas9 binding sequence (FIG. 39A, Table 4). The insert plasmids carry the starting fragments flanked by dST1Cas9-protectable and M.Sen0738I-switchable BsaI sites that generates compatible adhesive ends for DNA assembly. Preparation of insert plasmids in strains that express dST1Cas9, M2.Eco31I, and the corresponding guide RNA for dST1Cas9-based methylation protection results in selective methylation of the internal BsaI sites, whereas the flanking BsaI sites were not methylated due to methylation protection. In both dST1Cas9-based methylation protection strains (DH10B-2W276R and DH10B-2W278R), the internal BsaI sites were fully methylated, and the percentage of insert plasmids with both flanking BsaI sites protected by dST1Cas9 from M2.Eco31I methylation exceeds 50% (FIG. 39B and FIG. 39C). One pot assembly using these insert plasmids prepared in corresponding strains for dST1Cas9-based methylation protection (DH10B-2W276R or DH10B-2W278R), and compatible assembly vector prepared in strains for M.Sen0738I-based methylation switching (DH10B-2W148R), followed by direct transformation into corresponding strains for dST1Cas9-based methylation protection (DH10B-2W276R or DH10B-2W278R), results in successful assembly of the 3.6 kb fragment (FIG. 39D). The assembly efficiency is 75% (6/8) for the proof-of-principle assembly using the dST1Cas9-guide #498/M2.Eco31I/M.Sen0738I universal assembly system, and 100% (8/8) for the dST1Cas9-guide #500/M2.Eco31I/M.Sen0738I system (FIG. 39D). As the assembly was transformed into corresponding methylation protection strains, the internal BsaI sites of the assembled plasmids were fully methylated, whereas the flanking BsaI sites were protected from methylation (FIG. 39E). The 3.6 kb assembled fragment can thus be released intact from the assembled plasmid, which enables the assembled plasmid to be used as insert plasmid for next stage of Universal assembly. The percentage of the successfully assembled plasmids with both flanking BsaI sites protected from M2.Eco31I methylation is higher for plasmids assembled in dST1Cas9-guide #498 based methylation protection strain (DH10B-2W276R) than dST1Cas9-guide #500 based strain (DH10B-2W278R) (FIG. 39E), suggesting strain DH10B-2W276R is more efficient for methylation protection.

[0616] These data together demonstrate that Universal assembly system based on dST1Cas9-based methylation protection can assemble DNA with internal BsaI sites in a one-pot reaction.

EXAMPLE 5—HIERARCHICAL ASSEMBLY OF ˜260 KB DNA

[0617] The set of scarless assembly vectors from the scarless assembly scheme described in Example 1 was also used to assemble a large piece of ˜260 kb DNA fragment.

[0618] Methods

[0619] Reagents

[0620] Enzymes for molecular biology are from NEB unless otherwise stated. A high-fidelity version of BsaI restriction enzyme named BsaI-HFv2 is used in place of BsaI for all the experiments.

[0621] Strains and Plasmids

[0622] E. coli strains DH10B-2W214R and DH10B-2W148R have been described in Example 1. The 10 insert plasmids carrying the starting fragments (pMOBK401_L3G1, pMOBK401_L3G2, pMOBK401_L3G3, pMOBK401_L3G4, pMOBK401_L3G5, pMOBK401_L3G6, pMOBK401_L3G7, pMOBK401_L3G8, pMOBK401_L3G9, pMOBK401_L3G10) are kanamycin-resistant plasmids with F replication origin. The assembly vectors (pMOBK401_VL, pMOBC401_VL, pMOBC401_VM, pMOBC401_VR) are the kanamycin (pMOBK401) or chloramphenicol (pMOBC401) resistant scarless assembly vectors with F replication origin from the scarless assembly scheme described in Example 1.

[0623] DNA Assembly

[0624] The 260 kb DNA was assembled in two stages using M.Sen0738I and dCas9-guide #401-based universal assembly system. In stage one, the insert fragments were assembled 2-4 fragments per group using 30 fmol of each insert plasmid prepared in DH10B-2W214R, 15 fmol of the recipient assembly vector prepared in DH10B-2W148R with 10 U BsaI-HFv2 (NEB) and 15 U T4 DNA ligase HC (Thermo Fisher) in a 20 ul assembly reaction in 1×T4 ligase buffer (NEB). The reaction condition was: 3TC 15 min, followed by 45 cycles of 3TC 2 min plus 16° C. 5 min, then 3TC 20 min, and 80° C. 5 min. 20 U BsaI-HFv2 was then added to the assembly reaction for incubation at 37° C. for 45 min followed by heat inactivation at 80° C. for 5 min. The assembly reaction was dialysed for 1 h with water, and then transformed into NEB10B competent cells by electroporation at 0.9 kV 100Ω 25 μF using 1 mm electroporation cuvettes a Gene Pulser electroporation device (Bio-Rad). 1 ml LB medium was then added to the cells, and cultured at 3TC for 1 h. Cells were then plated on LB plates supplemented with chloramphenicol 12.5 μg/ml, IPTG 100 μM, X-gal 50 μg/ml at 37° C. overnight. White colonies were screened by NotI restriction digest followed by pulsed-field electrophoresis. Plasmid DNA from positive clones were transformed into DH10B-2W214R, and then used as insert plasmids for the stage two assembly. In stage two, the assembly reaction set up and transformation procedure was the same as stage one. Transformed cells were plated onto LB plates supplemented with kanamycin 30 μg/ml, IPTG 100 μM, X-gal 50 μg/ml at 37° C. overnight. White colonies were screened by NotI digestion followed by pulsed-field electrophoresis.

[0625] Results

[0626] The set of scarless assembly vectors from the scarless assembly scheme (FIG. 22) was used to assemble a ˜260 kb DNA fragment from the human CLEC16A locus from 10 fragments in two stages (FIG. 40, Table 5 and Table 6). The 10 starting fragments cloned in insert plasmids with flanking dCas9/guide #401 methylation-protectable and M.Sen0738I-switchable BsaI sites carry suitable overhang sequences depending on the position the assembled fragment would take up in the next round of assembly, as designed based on the process illustrated in FIG. 26. In round one of the assembly, groups of 2-4 fragments were assembled into suitable assembly vectors depending on the position that the assembled fragment would take up in the next round of assembly. In round two, the three intermediate insert plasmids prepared in DH10B-2W214R were assembled in a single reaction into the final product. The success rate of the first round assembly exceeds 70%, and the success rate at the second round is around 17%. This demonstrates that the same set of scarless assembly vector from the scarless assembly scheme combined with Universal assembly can be used for scarless assembly of large DNA molecules up to ˜260 kb.

EXAMPLE 6—UNIVERSAL ASSEMBLY BASED ON IN VITRO METHYLATION SWITCHING AND IN VITRO METHYLATION PROTECTION

[0627] Here we describe the Universal Assembly approach based on in vitro methylation switching and in vitro methylation protection, using M.Osp807II methylation switching, and dCas9 with M2.Eco31I or M2.BsaI for methylation protection. Compared with the in vivo approach, the in vitro approach uses recombinant methylases in protein form to perform methylation switching and methylation protection of DNA directly in a tube. This eliminates the need for bacteria in this part of the assembly process and so eliminates the need to develop bacterial strains expressing particular methylases.

[0628] Methods

[0629] Reagents

[0630] Molecular biology reagents were from NEB unless otherwise stated. A high-fidelity version of BsaI restriction enzyme named BsaI-HFv2 is used in place of BsaI for all the experiments.

[0631] Plasmid Construction

[0632] Plasmids for recombinant protein expression were constructed by standard cloning techniques with PCR and restriction enzymes. Briefly, pET-M.Osp807II plasmid for expression of M.Osp807II with N terminal His tag was constructed by cloning a PCR product carrying E. coli-codon optimized M.Osp807II into pET-15b vector (Novagen) using NdeI/BamHI. pET-M2.Eco31I plasmid for expression of M2.Eco31I with deletion of the first 7 amino acids and with C terminal His tag was constructed by cloning a PCR product carrying E. coli-codon optimized M2.Eco31I into pET-30b (+) vector (Novagen) using NdeI/HindIII. pET-M2.BsaI plasmid for expression of M2.BsaI with C terminal His tag was constructed by cloning a PCR product carrying E. coli-codon optimized M2.BsaI into pET-30b (+) vector (Novagen) using NdeI/HindIII. Plasmid sequences for pET-M.Osp807II, pET-M2.Eco31I and pET-M2.BsaI plasmids are listed in the supplementary information.

[0633] Recombinant Protein Purification

[0634] Recombinant proteins were produced using the following protocol. E. coli strain BL21(DE3)pLysS carrying the plasmids for recombinant protein expression were cultured in 500 ml LB medium at 3TC until OD600˜0.5 to 0.7. Protein expression was induced with 0.5 mM IPTG at 20° C. for 16 h. Cell pellets were frozen at −20° C., thawed, and resuspended in 15 ml 1× binding buffer (50 mM sodium phosphate pH 8.0, 300 mM NaCl, 1 mM imidazole, 10% glycerol). The cells were sonicated on ice, and lysed cells were pelleted at 14,000 g at 4° C. for 30 min. Cleared supernatants containing soluble proteins were mixed with 2 ml Ni-NTA His Bind Resin (Merck) pre-equilibrated with 1× binding buffer, and incubated on ice for 1 hour. The resin/soluble protein were loaded onto a column, and the flowthrough were collected and loaded onto the column that contains the resin a second time. The resin was washed once with 25 ml 1× binding buffer, once with 30 ml 1× binding buffer with 10 mM imidazole, and once with 20 ml 1× binding buffer with 50 mM imidazole. The purified protein was eluted from the column using 8 ml elution buffer (1× binding buffer with 150 mM imidazole). Eluted fractions were desalted using PD-10 column using the desalting buffer (50 mM sodium phosphate pH7.4, 200 mM NaCl, 10% glycerol), and proteins were aliquoted and stored at −80° C.

[0635] Assays for Testing Methylase Activity

[0636] Methylation reaction was set up using 200 ng plasmid DNA substrate and 1 μL enzyme (˜2 μM concentration) in 1×BamHI methyltransferase buffer (NEB) supplemented with 160 μM SAM in a 20 μL reaction. The reaction was incubated at 37° C. for 60 min and heat-inactivated at 80° C. for 20 min. Following methylation, 3 μL 10× CutSmart Buffer (NEB), 4 μL 50 mM MgCl2 and 0.5 μL restriction enzyme and 2.5 μL water was added to the 20 μL methylation reaction, and incubated at 37° C. for 1 h. Samples were analyzed by 1% agarose gel electrophoresis.

[0637] In Vitro Methylation Switching of DNA Assembly Vector

[0638] 2 μg plasmid DNA were methylated using 2 μL 2.4 μM M.Osp807II in 1×BamHI methyltransferase buffer (NEB) supplemented with 160 μM SAM in a 20 μL reaction at 37° C. for 1 h followed by heat inactivation at 80° C. for 20 min. Methylated assembly vector plasmids were purified using Qiaquick PCR clean up kit (Qiagen).

[0639] In Vitro Methylation Protection of Insert DNA Plasmids

[0640] Cas9 sgRNA guide #360 were generated by in vitro transcription and purified using Precision gRNA Synthesis kit (ThermoFisher) following manufacture's protocol. For methylation protection reaction, 7.8 pmol dCas9 (NEB) was incubated with 7.8 pmol sgRNA and 3 μL 10×NEBuffer 3.1 (NEB) in a 15 μL reaction at 25° C. for 10 min, following which 780 fmol insert plasmids were added to the pre-formed dCas9/sgRNA reaction to a final volume 28 μL and incubated at 3TC for 15 min. 1 μL 2.4 μM M2.Eco31I and 1 μL 3.2 mM SAM were then added to the reaction, and incubated at 35° C. for 15 min, followed by heat inactivation at 80° C. for 20 min. Methylated insert DNA plasmids were purified using Qiaquick PCR clean up kit (Qiagen).

[0641] DNA Assembly

[0642] The DNA assembly reaction contains 60 fmol of each insert DNA plasmid (pMOK360_F3A, pMOK360_F3B, pMOK360_F3C and pMOK360_F3D) that has been subject to in vitro methylation protection using dCas9/sgRNA/M2.Eco31I or dCas9/sgRNA/M2.BsaI, 60 fmol DNA assembly vector pMOP360_F3V that has been subject to in vitro methylation switching using M.Osp807II, 1000 U NEB T4 DNA ligase, 5 U BsaI in 20 μL 1×T4 DNA ligase buffer (NEB). The reaction condition was 37° C. 15 min followed 45 cycles of 37° C. 2 min plus 16° C. 5 min, then 37° C. 20 min and 80° C. 5 min. 2 μL 10× CutSmart buffer and 10 U BsaI was added to the reaction and incubated at 37° C. for 3 h. The reaction was transformed into chemically competent DH10B cells and plated on LB agar plates with 100 μg/ml Ampicillin, 100 μM IPTG and 50 μg/ml X-Gal, and incubated at 37° C. overnight. White colonies were expanded and screened by restriction digestion using DraIII.

[0643] Results

[0644] Here we developed methods for Universal assembly by in vitro methylation using purified recombinant enzymes.

[0645] In Vitro Methylation Switching

[0646] The methylation switching step in Universal assembly can be carried out in vitro using recombinant switch methylase M.Osp807II, which selectively block M.Osp807II-methylation switchable BsaI sites from restriction by BsaI (FIG. 41).

[0647] In Vitro Methylation Protection

[0648] The methylation protection step can be carried out using recombinant dCas9 and recombinant methylase M2.Eco31I or M2.BsaI that methylates and blocks BsaI sites (FIG. 42 and FIG. 43) in a multi-step reaction (FIG. 44). DNA molecules were first incubated with dCas9/sgRNA, which selectively binds to the methylation-protectable BsaI sites, but not non-protectable standard BsaI sites (FIG. 44). Recombinant M2.Eco31I or M2.BsaI were then added to the reaction to methylate the BsaI sites in the DNA (FIG. 44). However, because the methylation-protectable BsaI sites were stably bound by dCas9/sgRNA in the previous step, this prevents the methylation-protectable BsaI sites from being methylated by M2.Eco31I or M2.BsaI in vitro, therefore results in selective methylation of non-protectable standard BsaI sites in the DNA. The reaction was then subject to heat inactivation and cleaned up using spin columns to remove stably bound dCas9/sgRNA and M2.Eco31I or M2.BsaI, generating purified DNA with selective methylation of non-protectable BsaI sites only (FIG. 44).

[0649] DNA Assembly Based on In Vitro Methylation Switching and Methylation Protection

[0650] The in vitro methylation switching and methylation protection methods can then be used for one pot assembly of DNA (FIG. 45). The assembly vector was subject to in vitro methylation switching by M.Osp807II to selectively methylate the pair of methylation-switchable BsaI sites located within the assembly vector backbone. The insert DNA plasmids were subject to in vitro methylation protection with dCas9/sgRNA and either M2.Eco31I or M2.BsaI to selectively methylate the internal BsaI sites located within the insert fragment, whereas the methylation-protectable BsaI sites that flanks the insert fragment remain unmethylated due to protection of dCas9/sgRNA. One pot assembly using methylated assembly vector and insert DNA plasmid generates plasmid carrying orderly assembled fragments in assembly vector backbone, in which all the BsaI sites were methylated. Transformation of the assembled plasmid into normal E. coli strains generates unmethylated assembled plasmids with the assembled fragment flanked by methylation-protectable BsaI sites ready to use as insert plasmid for next round of DNA assembly based on in vitro methylation protection.

[0651] Using M.Osp807II-based in vitro methylation switching, and dCas9/sgRNA/M2.Eco31I or M2.BsaI based in vitro methylation protection, proof-of-principle in vitro Universal assembly was then carried out for assembly of a ˜3.7 kb DNA from the MICA locus from 4 ˜1 kb insert fragments, each of which contain an internal BsaI sites (FIG. 46A). Over 99% of the resulting colonies after the transformation of the DNA assembly reaction were white. A selection of these were expanded and tested with digestion, with 80% (4/5) of the colonies verified by restriction digest for either M2.Eco31I or M2.BsaI-based system (FIG. 46B).

Tables

[0652] Table 1. Practical Universal Assembly systems
Table 2. Feasible designs of excision-type overhang sequences for scarless assembly based on M.Osp807II and M.Sen0738I-based methylation switching of BsaI sites
Table 3. Scarless assembly of a ˜90 kb DNA using Universal assembly
Table 4. Practical Universal Assembly systems using dST1Cas9-based methylation protection
Table 5. Scarless assembly of a ˜260 kb DNA using Universal assembly
Table 6. Insert plasmids for ˜260 kb DNA assembly

TABLE-US-00023 TABLE 1 Practical Universal Assembly systems Methylation Methylation protection switching for for insert vector preparation plasmid preparation Test Protection guide RNA Systems Methylase Strain plasmid Methylase mechanism sequence 1 M.Osp DH10B- pMOP_BsaNC M2.Eco311 dCas9 GTGCAGTAC 80711 MOsp80 CTCTCACGA 711 CT (SEQ ID NO: 105) 2 M.Sen DH10B- pMOP_testN10 M2.Eco311 dCas9 GAGTAATCA 07381 2W148R CGCCAGTGC AT (SEQ ID NO: 106) Proof of principle assembly Test Insert Assembled Systems Strain plasmid plasmids Vector plasmid 1 DH10B- pMOPtestN8 pMOK360_F3A, pMOP360_F3V pMOP360_F3W 2W213R pMOK360_F3B, pMOK360_F3C, pMOK360_F3D 2 DH10B- pMOPtestN20 pMOK401_F4A, pMOP401_F4V pMOP401_F4W 2W214R pMOK401_F4B, pMOK401_F4C, pMOK401_F4D

TABLE-US-00024 TABLE 2 Designs of excision-type overhang sequences for scarless assembly based on M.Osp807II and M.Sen0738I-based methylation switching of BsaI sites Excision- Pre-assembly Excised Bases type overhang adaptor excised design* sequence** sequence*** 4 bp GGTCTCA CTCA/TGAG CTCA/TGAG NGAGACC 4 bp GGTCTCC CTCC/GGAG CTCC/GGAG NGAGACC 4 bp GGTCTCG CTCG/CGAG CTCG/CGAG NGAGACC 4 bp GGTCTCT CTCT/AGAG CTCT/AGAG NGAGACC 5 bp GGTCTCN TCTC/GAGA TCTCN/NGAGA GAGACC 6 bp GGTCTNG GTCT/AGAC GTCTCN/NGAGAC AGACC *Methylated base due to methylation switching methylation opposite to the base highlighted in bold **Forward and reverse-complement overhang sequence forward and reverse complement orientations ***Adaptor sequence to be excised from the end of the insert DNA in

TABLE-US-00025 TABLE 3 Scarless assembly of a ~90 kb DNA using Universal assembly Size of Round of Assembly Assembled assembled Success assembly Insert plasmids vector plasmid fragment rate First pMOLC401_F1, pMOLC401_F2, pMOLC401_F3, pMOBK401_ pMOBK401_G1 25.7 kb  83% pMOLC401_F4 VL (5/6) First pMOLC401_F5, pMOLC401_F6, pMOLC401_F7, pMOBK401_ pMOBK401_G2 25.7 kb 100% pMOLC401_F8 VM (6/6) First pMOLC401_F9, pMOLC401_F10, pMOLC401_F11, pMOBK401_ pMOBK401_G3 26.5 kb  83% pMOLC401_F12 VM (5/6) First pMOLC401_F13, pMOLC401_F14 pMOBK401_ pMOBK401_G4 12.6 kb 100% VR (6/6) Second pMOBK401_G1, pMOBK401_G2, pMOBK401_G3, pMOBC401_ pMOBC401_H1 90.2 kb 100% pMOBK401_G4 VM (12/12)

TABLE-US-00026 TABLE 4 Practical Universal Assembly systems using dST1Cas9-based methylation protection Methylation protection Methylation switching for insert plasmid for vector preparation preparation Test Protection guide RNA Systems Methylase Strain plasmid Methylase mechanism sequence 3 M.Sen0738I DH10B- pMOPtest M2.Eco31I dST1Cas9 GTCTAGTATG 2W148R N10 CGGATGCCAG 4 M.Sen0738I DH10B- pMOPtest M2.Eco31I dST1Cas9 GAGACCACCA 2W148R N10 TTCTGGGGCT Methylation protection for insert plasmid preparation Proof of principle assembly Test Insert Assembled Systems Strain plasmid plasmids Vector plasmid 3 DH10B- pMOP_test pMOK498_F5A, pMOP498_F5V pMOP498_F5W 2W276R N24 pMOK498_F5B, pMOK498_F5C, pMOK498_F5D 4 DH10B- pMOP_test pMOK500_F6A, PMOP500_F6V pMOP500_F6W 2W278R N32 pMOK500_F6B, pMOK500_F6C, pMOK500_F6D

TABLE-US-00027 TABLE 5 Scarless assembly of a ~260 kb DNA using Universal assembly Round of Size of assembled Success assembly Insert plasmids Assembly vector Assembled plasmid fragment rate First pMOBK401_L3G1, pMOBK401_L3G2 pMOBC401_VL pMOBC401_L3H1  51.4 kb 100% (6/6) First pMOBK401_L3G3, pMOBK401_L3G4, pMOBC401_VM pMOBC401_L3H2 104.8 kb 100% (4/4) pMOBK401_L3G5, pMOBK401_L3G6 First pMOBK401_L3G7, pMOBK401_L3G8, pMOBC401_VR pMOBC401_L3H3 104.6 kb  78% (7/9) pMOBK401_L3G9, pMOBK401_L3G10 Second pMOBC401_L3H1, pMOBC401_L3H2, pMOBK401_VL pMOBK401_L3I1 260.7 kb  17% (2/12) pMOBC401_L3H3

TABLE-US-00028 TABLE 6 Insert plasmids for ~260 kb DNA assembly Plasmid Insert fragment coordinates pMOBK401_L3G1 NC_000016.10:10923586-10952567 pMOBK401_L3G2 NC_000016.10:10952564-10974968 pMOBK401_L3G3 NC_000016.10:10974965-11004845 pMOBK401_L3G4 NC_000016.10:11004842-11027273 pMOBK401_L3G5 NC_000016.10:11027270-11057197 pMOBK401_L3G6 NC_000016.10:11057194-11079729 pMOBK401_L3G7 NC_000016.10:11079726-11102053 pMOBK401_L3G8 NC_000016.10:11102050-11132052 pMOBK401_L3G9 NC_000016.10:11132049-11161958 pMOBK401_L3G10 NC_000016.10:11161955-11184328 pMOBC401_L3H1 NC_000016.10:10923586-10974968 pMOBC401_L3H2 NC_000016.10:10974965-11079729 pMOBC401_L3H3 NC_000016.10:11079726-11184328 pMOBK401_L3I1 NC_000016.10:10923586-11184328

[0653] Sequences for Generation of Modified E. coli Strains:

[0654] The modified bacterial strain described herein may be modified/transformed with, or comprise, nucleic acid comprising one or more of the following sequences. It is understood that the sequences may or may not be limited with a specific guide sequences (as identified by underlining) used. In some embodiment, the specific guide RNA sequences (as identified by underlining) may be of any sequence. All polynucleotides and their sequences described herein, including functional variants thereof, may be considered to be an aspect or embodiment of the invention.

[0655] >2W148R (Linear DNA sequence for recombineering to generate the stable E. coli strain DH10B-2W148R) (SEQ ID NO: 116)

TABLE-US-00029 TTCTGTTACCCATCCAATTGTTCAAAATTCTTGCTGATGAAACCCGTCTG GGCATCGTTTTACTGCTCAGCGAACTGGGAGAGTTATGCGTCTGCGATCT CTGCACTGCTCTCGACCAGTCGCAGCCCAAGATCTCCCGCCACCTGGCAT TGCTGCGTGAAAGCGGGCTATTGCTGGACCGCAAGCAAGGTAAGTGGGTT CATTACCGCTTATCACCGCATATTCCAGCATGGGCGGCGAAAATTATTGA TGAGGCCTGGCGATGTGAACAGGAAAAGGTTCAGGCGATTGTCCGCAACC TGGCTCGACAAAACTGTTCCGGGGACAGTAAGAACATTTGCAGTTAAAAA TTTAGCTAAACACATATGAATTTTCAGATGTGTTTTATCCGGGAGGCATT ATGTTACTGGCAGGCGCTATCTTTGTCCTGACCATCGTATTGGTTATCTG GCAGCCGAAAGGTTTAGGCATCGGCTGGAGTGCAACGCTCGGCGCAGTAC TGGCGTTAGTTACGGGCGTGGTCCATCCGGGTGATATTCCGGTGGTGTGG AATATCGTCTGGAACGCGACGGCTGCGTTTATCGCCGTCATTATCATCAG CCTGCTGCTGGATGAGTCCGGCTTTTTTGAATGGGCGGCGCTGCACGTCT CACGCTGGGGTAATGGTCGTGGTCGCTTGCTGTTTACCTGGATTGTCCTG CTCGGTGCTGCCGTTGCCGCCCTGTTTGCCAATGATGGCGCGGCGCTTAT TTTGACACCGATTGTCATCGCCATGCTGCTGGCTTTAGGGTTCAGTAAAG GCACTACGCTGGCGTTCGTGATGGCGGCCGGATTCATTGCCGATACCGCC AGCCTGCCGCTTATTGTCTCCAACCTGGTGAATATCGTTTCCGCTGATTT CTTTGGCCTCGGCTTTCGCGAATACGCCTCGGTGATGGTGCCGGTGGATA TCGCCGCGATTGTTGCCACGCTGGTGATGTTACATCTCTATTTTCGCAAA GATATTCCGCAGAACTACGATATGGCGCTGCTGAAATCTCCCGCAGAAGC GATCAAAGATCCTGCTACGTTCAAAACTGGGCAAGGAGTTGACGGCTAGC TCAGTCCTAGGTACAGTGCTAGCTACTAGAGAAAGAGGAGAAATACAAAT GTCAATCTCATCTGCTATTAAAAGCTTGCAAGACATCATGCGCAAAGATG CCGGTGTTGATGGTGATGCCCAGCGTTTGGGCCAGCTTTCTTGGTTACTG TTTTTAAAAATCTTTGATGCACAGGAACAGGCACTGGAGATTGAACAAGA AAAGTATCGTCTGCCTATGCCAGAACGCTATTTATGGCGCAATTGGGCCG CCGATAATGAAGGAATTACAGGGGATAAATTGTTGGCGTTCGTCAACGAT GATCTTTTTCCCACCTTGAAGGACTTGCCCGCCCAGATCGACATCAACCC CCGCGGGTATGTAGTAAAGCAGGCTTTCTCCGACGCTTATAACTACATGA AAAACGGGACGTTGCTGCGCCAAGTTATCAACAAGTTAAATGAGATTGAC TTTACACGTGCTTCAGAGCGCCATCTTTTTGGAGATATCTACGAGCAAAT TTTGCGTGACTTGCAGGCTGCCGGTAATGCTGGTGAATTTTATACGCCTC GCGCTGTTACTCGCTTCATGGTTGAACGTGTAGACCCTAAACTGGGTGAG TCGATCATGGATCCGGCTTGTGGTACGGGGGGATTTTTGGCATGCGCCTT CGACCACGTTAAAAACCATTATGCCCATACTGTGACTGACCATCAAATCT TGCAGAAACAAATTCATGGGGTCGAAAAGAAGCAATTACCACACTTGTTG TGTACGACCAACATGCTGCTTCACGGCATCGAAGTCCCCGTTCAAATCCG CCACGACAATACTCTTAATAAACCACTTTCATCGTGGGACGAGCAAATGG ATGTTATCATCACTAACCCCCCATTCGGGGGTACTGAGGAAGATGGCATT GAAAAGAACTTCCCCTCTGACATGCAGACACGTGAGACGGCGGACTTATT TCTTCAGCTTATTATTGAGGTCTTAGCTAAGAACGGTCGTGCCGCCGTGG TGTTGCCCGATGGTACGCTTTTTGGTGAAGGTGTTAAAACTAAGATTAAA AAGTTGCTGACCGAAGAGTGCAATTTGCACACAATTGTCCGTTTACCTAA CGGCGTTTTTAACCCCTATACGGGGATCAAAACTAACCTGTTATTTTTCA CGAAAGGGCAGCCGACCAAGGAGATCTGGTTTTATGAACACCCTTACCCC GCAGGTGTTAAAAACTACAGCAAGACGAAACCAATGAAATTTGAGGAGTT TCAGGCAGAAATTGATTGGTGGGGTAACGAAGCGGATGGGTTTGCAAGTC GCGTCGAGAATGAGCAGGCCTGGAAAGTCTCTATTGATGAAGTTATCGCC CGTAACTTCAATCTTGATATTAAGAACCCCCACCAGGCAGAAACCGTATC CCATGACCCAGACGAGCTGTTAGCACAATACGCCAAACAGCAAGAGGCCA TCCAAACTCTTCGCCATCAGCTTCGTGACATCTTAGGAACTGCGTTGAGT GGTAAAGAGGCGAACTAAGCTTCTCGGTACCAAATTCCAGAAAAGAGGCC TCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCCGCTAGGTGGAGTTGAC GGCTAGCTCAGTCCTAGGTACAGTGCTAGCTACTAGAGAAAGAGGAGAAA TACAAATGGCGGTGGAAAAACTTATTGTAGATCATATCGACACCTGGACT ACCGCCCTTCAGACGCGCTCCACGGCCGGTCGCGGGAGCAGCGGCAAAAT CGACTTGTATGGAATTAAGAAGCTTCGCGAACTTATTTTAGAACTGGCCG TACGTGGCAAGCTGGTCCCGCAAGACCCAAATGACAAGCCCGCTTCGGTG CTTTTAGAACGCATCGCGACAGAAAAAGCGGAGCTGGTGAAACAAGGTAA GATTAAAAAGCAGAAGCCCTTGCCAGAAATCTCTGAGGAGGAAAAGCCAT TTGAACTGCCAGCGGGCTGGGAATGGGCTCGTTTAAATGAATTAGCTCCT ATGGGAATCATTGACGGGGATTGGATCGAGTCAAAAGACCAGGACCCGTC AGGCGCTTACCGTTTGATTCAGTTAGCTGATGTTGGCGTGGGAGATTTCA AGGACAAGTCCGATCGCTATATCAACACTTCGACGTTTCATCGTTTGAAC TGCCATCAACTGATGGAGGGTGATATCCTTATCGCGCGCTTACCGAACCC GATCGGACGCGCGTGCATCTTTCCTAAGCTTTCCCAAAGCGCTATCACTG TAGTAGATATTGCTACAATGCGCCCTTCAGGAAATTATAGTGCGGAATAT ATTATTTCCGCCATTAACAGTTTAACATTTCGTCAGCAAGTAGAATCGTT TGGAAAGGGCGCGACGCGCTTCCGCATCGCAACCGGGCACCTGAAAACAT TGCTTTTGCCAATTCCACCCGTCCAGGAACAATATAGTATCTTCAAAAAA ATTAAGGAGCTGATGTCCCTTTGCGATCAGTTAGAACAATATAGCCTGAC TTCCCTTGATGCACATCAACAATTAGTGGAAACGTTGTTAACCACCTTGA CGGACAGCCAGAATGCAGATGAGTTAGCTGAAAATTGGGCCCGTATCAGC GAACACTTCGATACTCTTTTTACAACTGAAGTCAGTATCGACGCCCTGAA GCAGACGATCCTGCAACTGGCTGTCATGGGTAAGCTGGTGCCACAGGACC CAAACGATGAACCAGCATCTGAACTGCTGAAGCGTATTGCACAGGAAAAA GCACAGTTGGTAAAGGATGGAAAGATGAAGAAGCAAAAGCCATTACCGCC GATTAGTGATGAGGAAAAACCATTCGAGTTACCATCGGGTTGGGAATGGT GTTTATTTGAGGACGTCGTAGATATTCAATCGGGCATTACCAAGGGCCGC AACTTAGCAAATCGCAAGCTTATTTCTATCCCGTATTTGCGCGTAGCCAA CGTGCAGCGCGGTTATTTGGACCTTTCAGAAGTTAAAGAAATCGACATTC CCGAAGAAGAGAAGGATAAGTACCACGTGATCAAGGGCGACTTGTTAATC ACGGAAGGCGGCGATTGGGACACAGTAGGGCGTACTACTGTTTGGTGCCA CGACTGGTATATCGCCAATCAAAACCACGTGTTTAAGGGACGTATTATCG GGCAGGACATCGATCCCTATTGGCTTGAGACGTACATGAACTCTCCTTAC GCCCGTGATTACTTCGCGAGTGCCTCTAAACAAACTACCAACCTGGCGAG TATCAACAAGACGCAACTTCGTGGATGTCCAGTGGCTATTCCACCTAGTA GCGAAGCAGAAAAAATCATGCTTAAATTAAACGATTTTAATGAACTGTGT GAAAAATTAAAGCTGCAGATTCAGAGCGCTCAACAGACGCAGCTTCATCT TGCGGACGCCCTGACTGATGCCGCAATTAACTAAGCTTAAAAAAAAAAAA GGCCTCCCAAATCGGGGGGCCTTTTTTATTGATAACAAAACGCTACTAAC TGTCTATGCCTGGGAAAGGGTGGGCAGGAGATGGGGCAGTGCAGGAAAAG TGGCACTATGAACCCTGCAGCCCTAGTTTGACAATTAATCATTGGCATAG TATATCTGCATAGTATAATACAACTCACTATAGCAATTGTACTAACCTTC TTCTCTTTCCTCTCCTGACAGGAGGAGCCATCATGGCCAAGTTGACCAGT GCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTG GACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCG GTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAG GTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGA GCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCT CCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTC GCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTTGTGGCAGAGGAGCA GGACTGATTACCTGTAACAGAGCATTAGCGCAAGGTGATTTTTGTCTTCT TGCGCTAATTTTTTCTGGGTTGTTTTACTGCTTCTGCTGGTGGGATTTTT CGTCCTGGAACCGCTCGGCATTCCGGTGAGCGCCATTGCAGCTGTGGGCG CGCTGATATTATTTGTCGTCGCTAAACGCGGTCATGCGATTAATACGGGT AAAGTCCTGCGCGGTGCCCCCTGGCAGATTGTCATCTTCTCGCTCGGCAT GTATCTGGTGGTTTATGGCCTGCGCAATGCCGGATTAACGGAATATCTTT CTGGCGTACTCAACGTGCTGGCGGATAACGGCCTGTGGGCCGCGACGCTC GGCACCGGATTCCTCACCGCCTTCCTCTCTTCTATTATGAACAATATGCC GACGGTACTGGTTGGCGCGTTGTCCATTGATGGCAGCACGGCATCTGGCG TTATCAAAGAAGCGATGGTTTATGCCAATGTGATTGGCTGCGATTTGGGA CCGAAAATTACCCCAATTGGTAGCCTGGCTACGCTACTCTGGCTGCACGT ACTTTCGCAGAAGAATATGACTATCAGCTGGGGATATTACTTCCGTACAG GGATTATCATGACCCTGCCTGTGCTGTTTGTGACGCTGGCTGCGCTGGCG CTACGTCTCTCTTTCACTTTGTAATGAGATACTGATATGAGCAACATTAC CATTTATCACAACCCGGCCTGCGGCACGTCGCGTAATACGCTGGAGATGA TCCGCAACAGCGGCACAGAACCGACTATTATCCATTATCTGGAAACTCCG CCAACGCGCGATGAACTGGTCAAACTCATTGCCGATATGGGGATTTCCGT ACGCGCGCTGCTGCGTAAAAACGTCGAACCGTATGAGGAGCTGGGCCTTG CGGAAGATAAATTTACTGACGATCGGTTAATCGACTTTATGCTTCAGCAC CCGATTCTGATTAATCGCCCGATTGTGGTGACGCCGCTGGGAACTCGCCT GTGCCGCCCTTCAGAAGTGGTGCTGGAAATTCTGCCAGATGCGCAAAAAG GCGCATTCTCCAAGGAAGATGGCGAGAAAGTGGTTGATGAAGCGGGTAAG CGCCTG

[0656] >2W213R (Linear DNA sequence for recombineering to generate the stable E. coli strain DH10B-2W213R, with the 20 bp sequence corresponding to the guide RNA sequence guide #360 that specifies dCas9 specificity underlined). (SEQ ID NO: 117)

TABLE-US-00030 TTCTGTTACCCATCCAATTGTTCAAAATTCTTGCTGATGAAACCCGTCTG GGCATCGTTTTACTGCTCAGCGAACTGGGAGAGTTATGCGTCTGCGATCT CTGCACTGCTCTCGACCAGTCGCAGCCCAAGATCTCCCGCCACCTGGCAT TGCTGCGTGAAAGCGGGCTATTGCTGGACCGCAAGCAAGGTAAGTGGGTT CATTACCGCTTATCACCGCATATTCCAGCATGGGCGGCGAAAATTATTGA TGAGGCCTGGCGATGTGAACAGGAAAAGGTTCAGGCGATTGTCCGCAACC TGGCTCGACAAAACTGTTCCGGGGACAGTAAGAACATTTGCAGTTAAAAA TTTAGCTAAACACATATGAATTTTCAGATGTGTTTTATCCGGGAGGCATT ATGTTACTGGCAGGCGCTATCTTTGTCCTGACCATCGTATTGGTTATCTG GCAGCCGAAAGGTTTAGGCATCGGCTGGAGTGCAACGCTCGGCGCAGTAC TGGCGTTAGTTACGGGCGTGGTCCATCCGGGTGATATTCCGGTGGTGTGG AATATCGTCTGGAACGCGACGGCTGCGTTTATCGCCGTCATTATCATCAG CCTGCTGCTGGATGAGTCCGGCTTTTTTGAATGGGCGGCGCTGCACGTCT CACGCTGGGGTAATGGTCGTGGTCGCTTGCTGTTTACCTGGATTGTCCTG CTCGGTGCTGCCGTTGCCGCCCTGTTTGCCAATGATGGCGCGGCGCTTAT TTTGACACCGATTGTCATCGCCATGCTGCTGGCTTTAGGGTTCAGTAAAG GCACTACGCTGGCGTTCGTGATGGCGGCCGGATTCATTGCCGATACCGCC AGCCTGCCGCTTATTGTCTCCAACCTGGTGAATATCGTTTCCGCTGATTT CTTTGGCCTCGGCTTTCGCGAATACGCCTCGGTGATGGTGCCGGTGGATA TCGCCGCGATTGTTGCCACGCTGGTGATGTTACATCTCTATTTTCGCAAA GATATTCCGCAGAACTACGATATGGCGCTGCTGAAATCTCCCGCAGAAGC GATCAAAGATCCTGCTACGTTCAAAACTGGGCAAGGAGTTGACAGCTAGC TCAGTCCTAGGTATAATGCTAGCGTGCAGTACCTCTCACGACTGTTTTAG AGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAA AGTGGCACCGAGTCGGTGCTTTTTTTGAAGCTTGGGCCCGAACAAAAAGC TTCTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTT TTCGTTTTGGTCCCGCTAGGTTTGACAGCTAGCTCAGTCCTAGGTATAAT GCTAGCAGAGAAAGAGGAGAAATACAAATGGATAAGAAATACTCAATAGG CTTAGCCATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAAT ATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCAC AGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGAC AGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTC GGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCG AAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGA AGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATG AAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAA TTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTT AGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAA ATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACC TACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGC TAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATC TCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTC ATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTT GGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATT TAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTG GCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGT AAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCT ACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAA CAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGG ATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAAT TTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTG AAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGG CTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAA GACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAA AAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGG CAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCC CATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTT ATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACT ACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGA CAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCA GGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAA AGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTT TTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTA GGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGA TAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCT TATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCAC CTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGG TTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAAT CTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGC AATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACAT TCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTG CAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTA AAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAA TATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGA AAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTA GGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAA TGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGG ACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATGCCATT GTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAAC GCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAG TAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTA ATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTT GAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTC GCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACT AAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTT AAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAG TACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCC GTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTT TGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGT CTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAAT ATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCG CAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGG ATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAA GTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGA GTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAG ACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTAT TCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAA ATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTG AAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAA AAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAA CGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATG AGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCAT TATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTT TGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTG AATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTT AGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAA TATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTA AATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAA GTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAAC ACGCATTGATTTGAGTCAGCTAGGAGGTGACTAAGCTTAAAAAAAAAAAA GGCCTCCCAAATCGGGGGGCCTTTTTTATTGATAACAAAACGCTTGCCGG AGCTGATAGCTAGCTCAGTCCTAGGGATTATGCTAGCTACTAGAGAAAGA GGAGAAATACAAATGACCAAGTCTGAAACATTCATGATTCCAAACCACAA GGCCGCCAAATTAAGTGAGCTGGATATGATGATCGTTAACTCTGTCCCGC CTGGGGGAAACTGGAAGAATATTCCCTTGGATGTACCATCGAAACGTATC GAACAGATTCGTGACAGCTATGCTCAAGGAAAAGGGTCGCGCAGCACATA CTACGGGCGTTTATTGCCCGATATGCCAGCTTATACGATCAATACTTATT TCAATCGTCCTGGCAACGGATGCCACATTCACTACGAGCAAGATCGCGTA CTTTCACAACGCGAAGCTGCACGTCTGCAGTCGTTCCCTGATGACTTTAT CTTTTTTGGAGGTCAAACGGCGATTAATACGCAAATCGGTAATGCCGTGC CTCCCTTTCTTGCGTTTCTTATTGCAAAAGAAATTGAAAAAGCGATCGGT AATACCGGCTACTACATTGACTTATTCAGTGGTGCAGGCGGATTGGGGTT GGGCTTTAAGTGGGCCGGGTGGACTCCATTGTTAGCTAATGACATTGAGG AAAAGTACTTACAGACATACTCGAACAACGTACACAAAGAAGTTTTGTGC GGAAGCATTTCGGACAACGAAACTTTTTCTAAGATCGCAGACAAGATTTC TGGCTTTAAGAAATTATATTTTGATAAACAGCTGTGGATTCTGGGCGGGC CTCCGTGCCAGGGATTTAGCACGGCTGGCAACGCGCGTACAATGGACGAC CCACGCAACAGTCTGTTTATGCACTACAAGTCGCTGCTTAACGAGATTAA GCCGAATGGATTCATTTTCGAGAACGTCGCCGGCCTGTTGAACATGGAAA AAGGAAAGGTCTTTGAACGTGTTAAGGAGGAATTCTCGTCCACAATGAAA ACCATGAATGGTTGGATTTTAAATTCGGAACATTACGCAATTCCACAACG CCGTAAGCGTGTAATTCTTGTGGGCAGCAATGATCCGCTGTTCTCGATCG AACCACCTCAGAAGCTGACGGAAGATAAAGAGTCTTGGGTGTCAGTAAAA GATGCGTTATCTGACCTTCCCCCATTACAACACGGCGAGGATGGATCTGG TAAATACTATATCCACCACCCGGAAAATGATTACCAGTTGTTTATGCGTG GAAACATTACACCCTCAGAGTATTATGAACGCAACATTAAGCCGTCGCTT TAAGCTTGACGAACAATAAGGCCTCCCAAATCGGGGGGCCTTTTTATTTT TCAACAAAACGCTACTAACTGTCTATGCCTGGGAAAGGGTGGGCAGGAGA TGGGGCAGTGCAGGAAAAGTGGCACTATGAACCCTGCAGCCCTAGTTTGA CAATTAATCATTGGCATAGTATATCTGCATAGTATAATACAACTCACTAT AGCAATTGTACTAACCTTCTTCTCTTTCCTCTCCTGACAGGAGGAGCCAT CATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCG CCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTC GTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCAT CAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGT GGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCC ACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCA GCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGC ACTTTGTGGCAGAGGAGCAGGACTGATTACCTGTAACAGAGCATTAGCGC AAGGTGATTTTTGTCTTCTTGCGCTAATTTTTTCTGGGTTGTTTTACTGC TTCTGCTGGTGGGATTTTTCGTCCTGGAACCGCTCGGCATTCCGGTGAGC GCCATTGCAGCTGTGGGCGCGCTGATATTATTTGTCGTCGCTAAACGCGG TCATGCGATTAATACGGGTAAAGTCCTGCGCGGTGCCCCCTGGCAGATTG TCATCTTCTCGCTCGGCATGTATCTGGTGGTTTATGGCCTGCGCAATGCC GGATTAACGGAATATCTTTCTGGCGTACTCAACGTGCTGGCGGATAACGG CCTGTGGGCCGCGACGCTCGGCACCGGATTCCTCACCGCCTTCCTCTCTT CTATTATGAACAATATGCCGACGGTACTGGTTGGCGCGTTGTCCATTGAT GGCAGCACGGCATCTGGCGTTATCAAAGAAGCGATGGTTTATGCCAATGT GATTGGCTGCGATTTGGGACCGAAAATTACCCCAATTGGTAGCCTGGCTA CGCTACTCTGGCTGCACGTACTTTCGCAGAAGAATATGACTATCAGCTGG GGATATTACTTCCGTACAGGGATTATCATGACCCTGCCTGTGCTGTTTGT GACGCTGGCTGCGCTGGCGCTACGTCTCTCTTTCACTTTGTAATGAGATA CTGATATGAGCAACATTACCATTTATCACAACCCGGCCTGCGGCACGTCG CGTAATACGCTGGAGATGATCCGCAACAGCGGCACAGAACCGACTATTAT CCATTATCTGGAAACTCCGCCAACGCGCGATGAACTGGTCAAACTCATTG CCGATATGGGGATTTCCGTACGCGCGCTGCTGCGTAAAAACGTCGAACCG TATGAGGAGCTGGGCCTTGCGGAAGATAAATTTACTGACGATCGGTTAAT CGACTTTATGCTTCAGCACCCGATTCTGATTAATCGCCCGATTGTGGTGA CGCCGCTGGGAACTCGCCTGTGCCGCCCTTCAGAAGTGGTGCTGGAAATT CTGCCAGATGCGCAAAAAGGCGCATTCTCCAAGGAAGATGGCGAGAAAGT GGTTGATGAAGCGGGTAAGCGCCTG

[0657] >2W214R (Linear DNA sequence for recombineering to generate the stable E. coli strain DH10B-2W214R, with the 20 bp sequence corresponding to the guide RNA sequence guide #401 that specifies dCas9 specificity underlined). (SEQ ID NO: 118)

TABLE-US-00031 TTCTGTTACCCATCCAATTGTTCAAAATTCTTGCTGATGAAACCCGTCTG GGCATCGTTTTACTGCTCAGCGAACTGGGAGAGTTATGCGTCTGCGATCT CTGCACTGCTCTCGACCAGTCGCAGCCCAAGATCTCCCGCCACCTGGCAT TGCTGCGTGAAAGCGGGCTATTGCTGGACCGCAAGCAAGGTAAGTGGGTT CATTACCGCTTATCACCGCATATTCCAGCATGGGCGGCGAAAATTATTGA TGAGGCCTGGCGATGTGAACAGGAAAAGGTTCAGGCGATTGTCCGCAACC TGGCTCGACAAAACTGTTCCGGGGACAGTAAGAACATTTGCAGTTAAAAA TTTAGCTAAACACATATGAATTTTCAGATGTGTTTTATCCGGGAGGCATT ATGTTACTGGCAGGCGCTATCTTTGTCCTGACCATCGTATTGGTTATCTG GCAGCCGAAAGGTTTAGGCATCGGCTGGAGTGCAACGCTCGGCGCAGTAC TGGCGTTAGTTACGGGCGTGGTCCATCCGGGTGATATTCCGGTGGTGTGG AATATCGTCTGGAACGCGACGGCTGCGTTTATCGCCGTCATTATCATCAG CCTGCTGCTGGATGAGTCCGGCTTTTTTGAATGGGCGGCGCTGCACGTCT CACGCTGGGGTAATGGTCGTGGTCGCTTGCTGTTTACCTGGATTGTCCTG CTCGGTGCTGCCGTTGCCGCCCTGTTTGCCAATGATGGCGCGGCGCTTAT TTTGACACCGATTGTCATCGCCATGCTGCTGGCTTTAGGGTTCAGTAAAG GCACTACGCTGGCGTTCGTGATGGCGGCCGGATTCATTGCCGATACCGCC AGCCTGCCGCTTATTGTCTCCAACCTGGTGAATATCGTTTCCGCTGATTT CTTTGGCCTCGGCTTTCGCGAATACGCCTCGGTGATGGTGCCGGTGGATA TCGCCGCGATTGTTGCCACGCTGGTGATGTTACATCTCTATTTTCGCAAA GATATTCCGCAGAACTACGATATGGCGCTGCTGAAATCTCCCGCAGAAGC GATCAAAGATCCTGCTACGTTCAAAACTGGGCAAGGAGTTGACAGCTAGC TCAGTCCTAGGTATAATGCTAGCGAGTAATCACGCCAGTGCATGTTTTAG AGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAA AGTGGCACCGAGTCGGTGCTTTTTTTGAAGCTTGGGCCCGAACAAAAAGC TTCTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTT TTCGTTTTGGTCCCGCTAGGTTTGACAGCTAGCTCAGTCCTAGGTATAAT GCTAGCAGAGAAAGAGGAGAAATACAAATGGATAAGAAATACTCAATAGG CTTAGCCATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAAT ATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCAC AGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGAC AGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTC GGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCG AAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGA AGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATG AAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAA TTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTT AGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAA ATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACC TACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGC TAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATC TCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTC ATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTT GGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATT TAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTG GCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGT AAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCT ACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAA CAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGG ATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAAT TTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTG AAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGG CTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAA GACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAA AAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGG CAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCC CATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTT ATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACT ACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGA CAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCA GGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAA AGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTT TTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTA GGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGA TAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCT TATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCAC CTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGG TTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAAT CTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGC AATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACAT TCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTG CAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTA AAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAA TATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGA AAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTA GGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAA TGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGG ACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATGCCATT GTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAAC GCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAG TAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTA ATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTT GAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTC GCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACT AAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTT AAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAG TACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCC GTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTT TGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGT CTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAAT ATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCG CAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGG ATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAA GTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGA GTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAG ACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTAT TCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAA ATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTG AAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAA AAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAA CGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATG AGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCAT TATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTT TGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTG AATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTT AGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAA TATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTA AATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAA GTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAAC ACGCATTGATTTGAGTCAGCTAGGAGGTGACTAAGCTTAAAAAAAAAAAA GGCCTCCCAAATCGGGGGGCCTTTTTTATTGATAACAAAACGCTTGCCGG AGCTGATAGCTAGCTCAGTCCTAGGGATTATGCTAGCTACTAGAGAAAGA GGAGAAATACAAATGACCAAGTCTGAAACATTCATGATTCCAAACCACAA GGCCGCCAAATTAAGTGAGCTGGATATGATGATCGTTAACTCTGTCCCGC CTGGGGGAAACTGGAAGAATATTCCCTTGGATGTACCATCGAAACGTATC GAACAGATTCGTGACAGCTATGCTCAAGGAAAAGGGTCGCGCAGCACATA CTACGGGCGTTTATTGCCCGATATGCCAGCTTATACGATCAATACTTATT TCAATCGTCCTGGCAACGGATGCCACATTCACTACGAGCAAGATCGCGTA CTTTCACAACGCGAAGCTGCACGTCTGCAGTCGTTCCCTGATGACTTTAT CTTTTTTGGAGGTCAAACGGCGATTAATACGCAAATCGGTAATGCCGTGC CTCCCTTTCTTGCGTTTCTTATTGCAAAAGAAATTGAAAAAGCGATCGGT AATACCGGCTACTACATTGACTTATTCAGTGGTGCAGGCGGATTGGGGTT GGGCTTTAAGTGGGCCGGGTGGACTCCATTGTTAGCTAATGACATTGAGG AAAAGTACTTACAGACATACTCGAACAACGTACACAAAGAAGTTTTGTGC GGAAGCATTTCGGACAACGAAACTTTTTCTAAGATCGCAGACAAGATTTC TGGCTTTAAGAAATTATATTTTGATAAACAGCTGTGGATTCTGGGCGGGC CTCCGTGCCAGGGATTTAGCACGGCTGGCAACGCGCGTACAATGGACGAC CCACGCAACAGTCTGTTTATGCACTACAAGTCGCTGCTTAACGAGATTAA GCCGAATGGATTCATTTTCGAGAACGTCGCCGGCCTGTTGAACATGGAAA AAGGAAAGGTCTTTGAACGTGTTAAGGAGGAATTCTCGTCCACAATGAAA ACCATGAATGGTTGGATTTTAAATTCGGAACATTACGCAATTCCACAACG CCGTAAGCGTGTAATTCTTGTGGGCAGCAATGATCCGCTGTTCTCGATCG AACCACCTCAGAAGCTGACGGAAGATAAAGAGTCTTGGGTGTCAGTAAAA GATGCGTTATCTGACCTTCCCCCATTACAACACGGCGAGGATGGATCTGG TAAATACTATATCCACCACCCGGAAAATGATTACCAGTTGTTTATGCGTG GAAACATTACACCCTCAGAGTATTATGAACGCAACATTAAGCCGTCGCTT TAAGCTTGACGAACAATAAGGCCTCCCAAATCGGGGGGCCTTTTTATTTT TCAACAAAACGCTACTAACTGTCTATGCCTGGGAAAGGGTGGGCAGGAGA TGGGGCAGTGCAGGAAAAGTGGCACTATGAACCCTGCAGCCCTAGTTTGA CAATTAATCATTGGCATAGTATATCTGCATAGTATAATACAACTCACTAT AGCAATTGTACTAACCTTCTTCTCTTTCCTCTCCTGACAGGAGGAGCCAT CATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCG CCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTC GTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCAT CAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGT GGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCC ACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCA GCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGC ACTTTGTGGCAGAGGAGCAGGACTGATTACCTGTAACAGAGCATTAGCGC AAGGTGATTTTTGTCTTCTTGCGCTAATTTTTTCTGGGTTGTTTTACTGC TTCTGCTGGTGGGATTTTTCGTCCTGGAACCGCTCGGCATTCCGGTGAGC GCCATTGCAGCTGTGGGCGCGCTGATATTATTTGTCGTCGCTAAACGCGG TCATGCGATTAATACGGGTAAAGTCCTGCGCGGTGCCCCCTGGCAGATTG TCATCTTCTCGCTCGGCATGTATCTGGTGGTTTATGGCCTGCGCAATGCC GGATTAACGGAATATCTTTCTGGCGTACTCAACGTGCTGGCGGATAACGG CCTGTGGGCCGCGACGCTCGGCACCGGATTCCTCACCGCCTTCCTCTCTT CTATTATGAACAATATGCCGACGGTACTGGTTGGCGCGTTGTCCATTGAT GGCAGCACGGCATCTGGCGTTATCAAAGAAGCGATGGTTTATGCCAATGT GATTGGCTGCGATTTGGGACCGAAAATTACCCCAATTGGTAGCCTGGCTA CGCTACTCTGGCTGCACGTACTTTCGCAGAAGAATATGACTATCAGCTGG GGATATTACTTCCGTACAGGGATTATCATGACCCTGCCTGTGCTGTTTGT GACGCTGGCTGCGCTGGCGCTACGTCTCTCTTTCACTTTGTAATGAGATA CTGATATGAGCAACATTACCATTTATCACAACCCGGCCTGCGGCACGTCG CGTAATACGCTGGAGATGATCCGCAACAGCGGCACAGAACCGACTATTAT CCATTATCTGGAAACTCCGCCAACGCGCGATGAACTGGTCAAACTCATTG CCGATATGGGGATTTCCGTACGCGCGCTGCTGCGTAAAAACGTCGAACCG TATGAGGAGCTGGGCCTTGCGGAAGATAAATTTACTGACGATCGGTTAAT CGACTTTATGCTTCAGCACCCGATTCTGATTAATCGCCCGATTGTGGTGA CGCCGCTGGGAACTCGCCTGTGCCGCCCTTCAGAAGTGGTGCTGGAAATT CTGCCAGATGCGCAAAAAGGCGCATTCTCCAAGGAAGATGGCGAGAAAGT GGTTGATGAAGCGGGTAAGCGCCTG

[0658] >2W89R (Linear DNA sequence for recombineering to generate the stable E. coli strain DH10B-2W89R) (SEQ ID NO: 119)

TABLE-US-00032 TTCTGTTACCCATCCAATTGTTCAAAATTC TTGCTGATGAAACCCGTCTGGGCATCGTTTTACTGCTCAGCGAACTGGGA GAGTTATGCGTCTGCGATCTCTGCACTGCTCTCGACCAGTCGCAGCCCAA GATCTCCCGCCACCTGGCATTGCTGCGTGAAAGCGGGCTATTGCTGGACC GCAAGCAAGGTAAGTGGGTTCATTACCGCTTATCACCGCATATTCCAGCA TGGGCGGCGAAAATTATTGATGAGGCCTGGCGATGTGAACAGGAAAAGGT TCAGGCGATTGTCCGCAACCTGGCTCGACAAAACTGTTCCGGGGACAGTA AGAACATTTGCAGTTAAAAATTTAGCTAAACACATATGAATTTTCAGATG TGTTTTATCCGGGAGGCATTATGTTACTGGCAGGCGCTATCTTTGTCCTG ACCATCGTATTGGTTATCTGGCAGCCGAAAGGTTTAGGCATCGGCTGGAG TGCAACGCTCGGCGCAGTACTGGCGTTAGTTACGGGCGTGGTCCATCCGG GTGATATTCCGGTGGTGTGGAATATCGTCTGGAACGCGACGGCTGCGTTT ATCGCCGTCATTATCATCAGCCTGCTGCTGGATGAGTCCGGCTTTTTTGA ATGGGCGGCGCTGCACGTCTCACGCTGGGGTAATGGTCGTGGTCGCTTGC TGTTTACCTGGATTGTCCTGCTCGGTGCTGCCGTTGCCGCCCTGTTTGCC AATGATGGCGCGGCGCTTATTTTGACACCGATTGTCATCGCCATGCTGCT GGCTTTAGGGTTCAGTAAAGGCACTACGCTGGCGTTCGTGATGGCGGCCG GATTCATTGCCGATACCGCCAGCCTGCCGCTTATTGTCTCCAACCTGGTG AATATCGTTTCCGCTGATTTCTTTGGCCTCGGCTTTCGCGAATACGCCTC GGTGATGGTGCCGGTGGATATCGCCGCGATTGTTGCCACGCTGGTGATGT TACATCTCTATTTTCGCAAAGATATTCCGCAGAACTACGATATGGCGCTG CTGAAATCTCCCGCAGAAGCGATCAAAGATCCTGCTACGTTCAAAACTGG GCAAGGAG CTGATAGCTAGCTCAGTCCTAGGGATTATGCTAGCTACTAG AGAAAGAGGAGAAATACAAATGGAGGAGATCTTCTATATGAAACACATCC ATTTAATCAACAGCCTGTCGCTGGACGAAACTACTAAGTTCACGAAAAAA GCTACCGGTAAATACTACACCGACCCCAAAATCGCGCTGTTAATGATCGA GAAGTTACTTCCGCTGATTAACTCCTGTGATAAAAAGAGCTATAACGTTG CTGATCCCTTTTCAGGTGACGGTCGTCTGATCACTCTTTTGATCAAGCAG TGGATGATTAACGGCTTCCCCGATGTCGAATGGAACGTCTACCTGTTTGA CATTGAGAACACCGGTCTGACTTACGCCAAAAACGCTCTGTCGGAATTGA AGTTAGCCGGCGCTAATATCAATATTACAATTAAGAACTCGGATGTATTT TACGAATTTAAGAAGTATGTAGACTACTTCGACTGTGTGATCACAAATCC TCCCTGGGAGAACATTAAACCTGATTCTCGTGAGCTTGATTTTTTCGAAC CAAGCATGAAATCCATGTATATTGACAGCCTTCGTGAATTTGATGATTAC CTTTCACGTGTGCTTCCCTATAGTCAGCCGAAGCGCAAGTTCGCTGGCTG GGGTACGAACTTAAGTCGTGTTGGTGCAGAGTTATCTCTTGAGATTTGCA ACAAAAATGGGTTAGTAGCGATCGTCATGCCCGCCAGCTTTTTTGCGGAC GAGCAATCCTATATTTTACGTGAGAAGTTTTTCAATTCCGGACGTATCGA CTGTATTAACTATTATCCAGCCGAAGCAAAACTTTTCGGTGGAGCAGATG TGAGCTCCTGCTCCTTCATTTTTAACAAGGGAGAATCCTTGAATGATAAT ATTCAATTAAGCGTCTACGATAAAAATTTGAACATCAAGTCATTGGGTTT TTTCGATTTGTCAAGCATCGATTCCCAATATCTGTCGATCCCCGTGTCTC AAGGCGTGCATGCGGTCCATCTTTTGCGCAAACTTCAAGAGGGTTACCCA ACGTGGGGTAGTTTAGAAAAGAACGGCGAGATTTGGGCGGGCCGTGAAAT CGACGAAACTGGCTCGTCGGATTGGACTCAGAAGTCGGGTGGGGGGCTGT TGTTTATTAAGGGCAAAATGATTGGCCGCTACAATTTCCACAATGAAAAG TCTCTGCGCATTACGAAAAAGATTGACAAGGTGCTTTCCAATAGCAACTT CGTCCGCATCGCCTGGCGCGATATTTCGCGCCCAAGCCAAAAACGCCGCA TGATCGCAACTATCATTCCGCCTAACTCGTTGGCTGGTAACTCATTGGGT GTAGTATACTACAAATCCGGGTCCCAGGATTCCCTGTTTTCCTTGCTTGG AATTATTAACTCTCTGTGCTTCGAATTTCAATTGCGCTCCTTTTTGGCTA CTGGGCATGTTAGTCTGTCTGCTCTTCGTAAAACCGCGATCCCTAGCGAA AAGATCTTACTGCAACACAGTGAGTTGAAACAGCTGGTAATTAGCTGCAT TGAGGGGTGCTGCGATGCGGAATTAAAGATTGAAGCATATGTGGCGAAGA ACATTTACAAACTTGACCTGAATGAGTTCAACAAATTGCTTAGTAGCTTC GACAAAATCGAGTTGGCAGAAAAAGAGTCTTTGTTACGCATCTTCCAGCA CTACGATTAAGCTTCTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAA GGGGGGCCTTTTTTCGTTTTGGTCCCGCTAGGTGGAGTTGACGGCTAGCT CAGTCCTAGGTACAGTGCTAGCTACTAGAGAAAGAGGAGAAATACAAATG GCTATCACTAACTTCGTAAAGCGCATCCAAGATGTGATGCGTAATGACGC TGGAGTTAATGGGGATGCACAACGCATCGAGCAAATCGTATGGATCTTAT TTTTAAAGATTTATGATGCAAAGGAAGAGGAGTGGGAGTTGGTAAACGAA AAGTATTCCTCGATTATCCCGGAGGAATGTAAATGGCGTAATTGGGCCGT AGATGAAAAGGACGGTGAAGCTCTGACAGGCGACGCGATTTTAAACTTTG TCAATAACACACTGTTCCCTATCCTGAAAAATTTAGAGATTGATGACGAA ACCCCGATGAATCAGGTCATCGTGAAGTACGCTTTCGAGGACGCCAACAA TTATATGAAAGATGGAGTTCTTCTTCGCCAGGTAGTTAATATCATTGATG AGATTGATTTCACTGAATATGAAGAGCGCCATGCATTTGGTACGATTTAT GAGAGCTTTTTAAAAGATCTTCAAAGTGCGGGTAACGCTGGTGAGTTCTA CACGCCCCGCGCCGTGACCGACTTTATGGTCGAAGTGATCAAACCAAAGC TTGGAGAGAAAGTAGCCGACTTTGCTTGCGGCACAGGGGGGTTCTTAGTG TCTGCGTTGAATGCTCTTGAGAAACAGGTTGGGAATTCACTTGAGAATCG CGAGATCTATAATAATTCTATTTACGGTGTGGAAAAGAAAGGGCTGCCCC ATATTCTGTGTGTGACAAATATGTTGTTGCATGACATCGACAACCCGGAC ATCATTCACGGGAACACACTGGAAACAGATTACAAGGAGTACCGCCGTAT GGAACAATTCGACATTGTACTGATGAACCCGCCATACGGTGGGAATGAAA AGGAGAGTGTTAAGGCTAACTTCCCTTCGGATCTTCGCTCCTCCGAGACA GCTGACTTATTTATCGATATTATTATGTTTCGTTTGAAGAAAAATGGGCG TTGTGCGATCATTCTTCCTGACGGCTTTCTGTTTGGTACTGATAACGCTA AGGTCAACATTAAGAAGAAGTTACTTGGGGAATTTAACTTGCACACAATC GTCCGCCTTCCCCACTCTGTGTTCGCGCCTTACACCTCGATTACGACTAA TATTCTTTTTTTCGACAATACGCACCCTACCGAAGAAACATGGTTTTATC GCCTGGACATGCCCGATGGGTATAAGAATTTTTCTAAAACAAAACCGATG AAGTTAGAGCACTTTGGGTCGGCAATTGAATGGTGGGACAACCGTGAGGA GATCGAAGTTGATGGATTTCCGAAAGCGAAGAAATACACCGTCGAGGACA TTGAAAACTTAGGGTACAACTTGGACTTGTGTGGCTTTCCCCACGAGGAA GAGGAGATTTTGGACCCCATGGACTTAATTCGCGAGTACCAGGAGAAGCG CGCCTCCTTAAACGCAGAGATTGACCATGTGCTGGAGAAGATTACCTCTA TGTTAGGAGGGAACTAAGCTTAAAAAAAAAAAAGGCCTCCCAAATCGGGG GGCCTTTTTTATTGATAACAAAACGCTTGCCGGAGTTGACGGCTAGCTCA GTCCTAGGTACAGTGCTAGCTACTAGAGAAAGAGGAGAAATACAAATGAC AGCACAAGATTTAAAGAACAGTATCTTGCAGCTTGCGATTCAAGGAAAAT TAGTCGAACAGCGTGCCGAAGAAGGCACCGCGAAGGAGCTTTTAGAGCAG ATCAAGACCGAAAAGGAACGTTTAATCAATGAAAAAAAGATCAAAAAAGA AAAGCCGCTTAGTGAAATTACGGAAGATGAGATTCCGTTTGAGATCCCGG AGTCGTGGGAATGGGTCCGCTTGAATGAGATCTACAATTTTATTGATTAT CGTGGGAAAACACCCAATAAAATCGATGAAGGAGTCGTTTTGATCACGGC GTCGAACGTGCGTAAGGGCTACTTAGACTTTAGTAAGTCGGATTTTATTT CCGAAAATGAGTATAAGGAGCGTATGACGCGCGGGATTACGCGCAAGGGT GACCTTCTGTTCACCACCGAGGCACCACTTGGTTACGTGGCGGTGAACAC GATTGAAATTGCTTCTTGCGGACAACGCGTGATCACATTTCAACAGTACG GCGTTAACGTATTTTGTAACGAGCTGATGTGTTATTTCATCCAGTCACCA TTCTTCCAGAACGAGCTGTTAGTCAAAGCTACAGGGACTACGGCGAAAGG CATCAAGGCTGAAAAATTAAAGTATTTGCTGATCCCTGTGCCGCCATTAG AAGAGCAACAACGCATTATTGCTAAAATCGAGGACATTTTACCTTACATT GAACAATACGACAAAGCCTATTCAAAACTGGAGGTATTTAACAAGAAATT CCCTGAGGATATGCAAAAGAGTATTCTGCAATACGCGATTCAGGGGAAGC TGGTCGAACAGCGTCAGGAAGAAGGAACGGCGGAAGAGCTGTACGAACAG ATCCAGGCAGAAAAGGAGCGCCTTATCAAAGAGGGGAAAATCAAGAAAGA AAAACCATTAAGCGAGTTTACGGAGGAGGAAATTCCTTATGAGATTCCTG ACTCTTGGAAATGGGTTAAGCTTGGCGATGTATTCCAAATTAACCCTCGT AATTCGATCGAGGATAAGATCGAAGTCTCGTTTATTCCAATGACGTTGTT GCAGGAAGGGTATGTATCTAAGTTCACATTTGAAATCAAAAAATGGGGCG ATGTCAAGAAAGGTTTCACACATTTTAAGGATAACGACATCATCTTCGCT AAAATCACCCCTTGTTTCCAGAATTTAAAATCAGCCATTATGGAGAACCT GAAAAATGGATATGGCGCAGGCACGACAGAATTGCACGTCTTACGCTGTT ACAAAATGCTTAGTTTAGAATATTTTCTGTGGTTCGTAAAGAGCCCTTAC TTCATGTCGTTCTGCGAAGCCAACATGAGCGGTACAGCCGGACAACAACG TGTGGGTACGGACATTGTCAAGAATGTTCTTTTACCATTACCCCCGTTAG AGGAGCAAAAACGTATCGTATATGTAATTGAAAAGTATTTCCCATTTTGT CAGCAATTGCGTAAATAAGCTTGACGAACAATAAGGCCTCCCAAATCGGG GGGCCTTTTTATTTTTCAACAAAACGCT ACTAACTGTCTATGCCTGGGA AAGGGTGGGCAGGAGATGGGGCAGTGCAGGAAAAGTGGCACTATGAACCC TGCAGCCCTAGTTTGACAATTAATCATTGGCATAGTATATCTGCATAGTA TAATACAACTCACTATAGCAATTGTACTAACCTTCTTCTCTTTCCTCTCC TGACAGGAGGAGCCATCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTC ACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGG GTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACG ACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAAC ACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTG GTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGA CCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCG GCCGGCAACTGCGTGCACTTTGTGGCAGAGGAGCAGGACTGATTACCTGT AACAGAGCATTAGCGCAAGGTGATTTTTGTCTTCTTGCGCTAATTTTTTC TGGGTTGTTTTACTGCTTCTGCTGGTGGGATTTTTCGTCCTGGAACCGCT CGGCATTCCGGTGAGCGCCATTGCAGCTGTGGGCGCGCTGATATTATTTG TCGTCGCTAAACGCGGTCATGCGATTAATACGGGTAAAGTCCTGCGCGGT GCCCCCTGGCAGATTGTCATCTTCTCGCTCGGCATGTATCTGGTGGTTTA TGGCCTGCGCAATGCCGGATTAACGGAATATCTTTCTGGCGTACTCAACG TGCTGGCGGATAACGGCCTGTGGGCCGCGACGCTCGGCACCGGATTCCTC ACCGCCTTCCTCTCTTCTATTATGAACAATATGCCGACGGTACTGGTTGG CGCGTTGTCCATTGATGGCAGCACGGCATCTGGCGTTATCAAAGAAGCGA TGGTTTATGCCAATGTGATTGGCTGCGATTTGGGACCGAAAATTACCCCA ATTGGTAGCCTGGCTACGCTACTCTGGCTGCACGTACTTTCGCAGAAGAA TATGACTATCAGCTGGGGATATTACTTCCGTACAGGGATTATCATGACCC TGCCTGTGCTGTTTGTGACGCTGGCTGCGCTGGCGCTACGTCTCTCTTTC ACTTTGTAATGAGATACTGATATGAGCAACATTACCATTTATCACAACCC GGCCTGCGGCACGTCGCGTAATACGCTGGAGATGATCCGCAACAGCGGCA CAGAACCGACTATTATCCATTATCTGGAAACTCCGCCAACGCGCGATGAA CTGGTCAAACTCATTGCCGATATGGGGATTTCCGTACGCGCGCTGCTGCG TAAAAACGTCGAACCGTATGAGGAGCTGGGCCTTGCGGAAGATAAATTTA CTGACGATCGGTTAATCGACTTTATGCTTCAGCACCCGATTCTGATTAAT CGCCCGATTGTGGTGACGCCGCTGGGAACTCGCCTGTGCCGCCCTTCAGA AGTGGTGCTGGAAATTCTGCCAGATGCGCAAAAAGGCGCATTCTCCAAGG AAGATGGCGAGAAAGTGGTTGATGAAGCGGGTAAGCGCCTG

[0659] >2W91R (Linear DNA sequence for recombineering to generate the stable E. coli strain DH10B-2W91R) (SEQ ID NO: 120)

TABLE-US-00033 TTCTGTTACCCATCCAATTGTTCAAAATTC TTGCTGATGAAACCCGTCTGGGCATCGTTTTACTGCTCAGCGAACTGGGA GAGTTATGCGTCTGCGATCTCTGCACTGCTCTCGACCAGTCGCAGCCCAA GATCTCCCGCCACCTGGCATTGCTGCGTGAAAGCGGGCTATTGCTGGACC GCAAGCAAGGTAAGTGGGTTCATTACCGCTTATCACCGCATATTCCAGCA TGGGCGGCGAAAATTATTGATGAGGCCTGGCGATGTGAACAGGAAAAGGT TCAGGCGATTGTCCGCAACCTGGCTCGACAAAACTGTTCCGGGGACAGTA AGAACATTTGCAGTTAAAAATTTAGCTAAACACATATGAATTTTCAGATG TGTTTTATCCGGGAGGCATTATGTTACTGGCAGGCGCTATCTTTGTCCTG ACCATCGTATTGGTTATCTGGCAGCCGAAAGGTTTAGGCATCGGCTGGAG TGCAACGCTCGGCGCAGTACTGGCGTTAGTTACGGGCGTGGTCCATCCGG GTGATATTCCGGTGGTGTGGAATATCGTCTGGAACGCGACGGCTGCGTTT ATCGCCGTCATTATCATCAGCCTGCTGCTGGATGAGTCCGGCTTTTTTGA ATGGGCGGCGCTGCACGTCTCACGCTGGGGTAATGGTCGTGGTCGCTTGC TGTTTACCTGGATTGTCCTGCTCGGTGCTGCCGTTGCCGCCCTGTTTGCC AATGATGGCGCGGCGCTTATTTTGACACCGATTGTCATCGCCATGCTGCT GGCTTTAGGGTTCAGTAAAGGCACTACGCTGGCGTTCGTGATGGCGGCCG GATTCATTGCCGATACCGCCAGCCTGCCGCTTATTGTCTCCAACCTGGTG AATATCGTTTCCGCTGATTTCTTTGGCCTCGGCTTTCGCGAATACGCCTC GGTGATGGTGCCGGTGGATATCGCCGCGATTGTTGCCACGCTGGTGATGT TACATCTCTATTTTCGCAAAGATATTCCGCAGAACTACGATATGGCGCTG CTGAAATCTCCCGCAGAAGCGATCAAAGATCCTGCTACGTTCAAAACTGG GCAAGGAG TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCTACTAG AGAAAGAGGAGAAATACAAATGACCAAGTCTGAAACATTCATGATTCCAA ACCACAAGGCCGCCAAATTAAGTGAGCTGGATATGATGATCGTTAACTCT GTCCCGCCTGGGGGAAACTGGAAGAATATTCCCTTGGATGTACCATCGAA ACGTATCGAACAGATTCGTGACAGCTATGCTCAAGGAAAAGGGTCGCGCA GCACATACTACGGGCGTTTATTGCCCGATATGCCAGCTTATACGATCAAT ACTTATTTCAATCGTCCTGGCAACGGATGCCACATTCACTACGAGCAAGA TCGCGTACTTTCACAACGCGAAGCTGCACGTCTGCAGTCGTTCCCTGATG ACTTTATCTTTTTTGGAGGTCAAACGGCGATTAATACGCAAATCGGTAAT GCCGTGCCTCCCTTTCTTGCGTTTCTTATTGCAAAAGAAATTGAAAAAGC GATCGGTAATACCGGCTACTACATTGACTTATTCAGTGGTGCAGGCGGAT TGGGGTTGGGCTTTAAGTGGGCCGGGTGGACTCCATTGTTAGCTAATGAC ATTGAGGAAAAGTACTTACAGACATACTCGAACAACGTACACAAAGAAGT TTTGTGCGGAAGCATTTCGGACAACGAAACTTTTTCTAAGATCGCAGACA AGATTTCTGGCTTTAAGAAATTATATTTTGATAAACAGCTGTGGATTCTG GGCGGGCCTCCGTGCCAGGGATTTAGCACGGCTGGCAACGCGCGTACAAT GGACGACCCACGCAACAGTCTGTTTATGCACTACAAGTCGCTGCTTAACG AGATTAAGCCGAATGGATTCATTTTCGAGAACGTCGCCGGCCTGTTGAAC ATGGAAAAAGGAAAGGTCTTTGAACGTGTTAAGGAGGAATTCTCGTCCAC AATGAAAACCATGAATGGTTGGATTTTAAATTCGGAACATTACGCAATTC CACAACGCCGTAAGCGTGTAATTCTTGTGGGCAGCAATGATCCGCTGTTC TCGATCGAACCACCTCAGAAGCTGACGGAAGATAAAGAGTCTTGGGTGTC AGTAAAAGATGCGTTATCTGACCTTCCCCCATTACAACACGGCGAGGATG GATCTGGTAAATACTATATCCACCACCCGGAAAATGATTACCAGTTGTTT ATGCGTGGAAACATTACACCCTCAGAGTATTATGAACGCAACATTAAGCC GTCGCTTTAAGCTTCTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAA GGGGGGCCTTTTTTCGTTTTGGTCCCGCTAGGTGGAGTTGACGGCTAGCT CAGTCCTAGGTACAGTGCTAGCTACTAGAGAAAGAGGAGAAATACAAATG GCTATCACTAACTTCGTAAAGCGCATCCAAGATGTGATGCGTAATGACGC TGGAGTTAATGGGGATGCACAACGCATCGAGCAAATCGTATGGATCTTAT TTTTAAAGATTTATGATGCAAAGGAAGAGGAGTGGGAGTTGGTAAACGAA AAGTATTCCTCGATTATCCCGGAGGAATGTAAATGGCGTAATTGGGCCGT AGATGAAAAGGACGGTGAAGCTCTGACAGGCGACGCGATTTTAAACTTTG TCAATAACACACTGTTCCCTATCCTGAAAAATTTAGAGATTGATGACGAA ACCCCGATGAATCAGGTCATCGTGAAGTACGCTTTCGAGGACGCCAACAA TTATATGAAAGATGGAGTTCTTCTTCGCCAGGTAGTTAATATCATTGATG AGATTGATTTCACTGAATATGAAGAGCGCCATGCATTTGGTACGATTTAT GAGAGCTTTTTAAAAGATCTTCAAAGTGCGGGTAACGCTGGTGAGTTCTA CACGCCCCGCGCCGTGACCGACTTTATGGTCGAAGTGATCAAACCAAAGC TTGGAGAGAAAGTAGCCGACTTTGCTTGCGGCACAGGGGGGTTCTTAGTG TCTGCGTTGAATGCTCTTGAGAAACAGGTTGGGAATTCACTTGAGAATCG CGAGATCTATAATAATTCTATTTACGGTGTGGAAAAGAAAGGGCTGCCCC ATATTCTGTGTGTGACAAATATGTTGTTGCATGACATCGACAACCCGGAC ATCATTCACGGGAACACACTGGAAACAGATTACAAGGAGTACCGCCGTAT GGAACAATTCGACATTGTACTGATGAACCCGCCATACGGTGGGAATGAAA AGGAGAGTGTTAAGGCTAACTTCCCTTCGGATCTTCGCTCCTCCGAGACA GCTGACTTATTTATCGATATTATTATGTTTCGTTTGAAGAAAAATGGGCG TTGTGCGATCATTCTTCCTGACGGCTTTCTGTTTGGTACTGATAACGCTA AGGTCAACATTAAGAAGAAGTTACTTGGGGAATTTAACTTGCACACAATC GTCCGCCTTCCCCACTCTGTGTTCGCGCCTTACACCTCGATTACGACTAA TATTCTTTTTTTCGACAATACGCACCCTACCGAAGAAACATGGTTTTATC GCCTGGACATGCCCGATGGGTATAAGAATTTTTCTAAAACAAAACCGATG AAGTTAGAGCACTTTGGGTCGGCAATTGAATGGTGGGACAACCGTGAGGA GATCGAAGTTGATGGATTTCCGAAAGCGAAGAAATACACCGTCGAGGACA TTGAAAACTTAGGGTACAACTTGGACTTGTGTGGCTTTCCCCACGAGGAA GAGGAGATTTTGGACCCCATGGACTTAATTCGCGAGTACCAGGAGAAGCG CGCCTCCTTAAACGCAGAGATTGACCATGTGCTGGAGAAGATTACCTCTA TGTTAGGAGGGAACTAAGCTTAAAAAAAAAAAAGGCCTCCCAAATCGGGG GGCCTTTTTTATTGATAACAAAACGCTTGCCGGAGTTGACGGCTAGCTCA GTCCTAGGTACAGTGCTAGCTACTAGAGAAAGAGGAGAAATACAAATGAC AGCACAAGATTTAAAGAACAGTATCTTGCAGCTTGCGATTCAAGGAAAAT TAGTCGAACAGCGTGCCGAAGAAGGCACCGCGAAGGAGCTTTTAGAGCAG ATCAAGACCGAAAAGGAACGTTTAATCAATGAAAAAAAGATCAAAAAAGA AAAGCCGCTTAGTGAAATTACGGAAGATGAGATTCCGTTTGAGATCCCGG AGTCGTGGGAATGGGTCCGCTTGAATGAGATCTACAATTTTATTGATTAT CGTGGGAAAACACCCAATAAAATCGATGAAGGAGTCGTTTTGATCACGGC GTCGAACGTGCGTAAGGGCTACTTAGACTTTAGTAAGTCGGATTTTATTT CCGAAAATGAGTATAAGGAGCGTATGACGCGCGGGATTACGCGCAAGGGT GACCTTCTGTTCACCACCGAGGCACCACTTGGTTACGTGGCGGTGAACAC GATTGAAATTGCTTCTTGCGGACAACGCGTGATCACATTTCAACAGTACG GCGTTAACGTATTTTGTAACGAGCTGATGTGTTATTTCATCCAGTCACCA TTCTTCCAGAACGAGCTGTTAGTCAAAGCTACAGGGACTACGGCGAAAGG CATCAAGGCTGAAAAATTAAAGTATTTGCTGATCCCTGTGCCGCCATTAG AAGAGCAACAACGCATTATTGCTAAAATCGAGGACATTTTACCTTACATT GAACAATACGACAAAGCCTATTCAAAACTGGAGGTATTTAACAAGAAATT CCCTGAGGATATGCAAAAGAGTATTCTGCAATACGCGATTCAGGGGAAGC TGGTCGAACAGCGTCAGGAAGAAGGAACGGCGGAAGAGCTGTACGAACAG ATCCAGGCAGAAAAGGAGCGCCTTATCAAAGAGGGGAAAATCAAGAAAGA AAAACCATTAAGCGAGTTTACGGAGGAGGAAATTCCTTATGAGATTCCTG ACTCTTGGAAATGGGTTAAGCTTGGCGATGTATTCCAAATTAACCCTCGT AATTCGATCGAGGATAAGATCGAAGTCTCGTTTATTCCAATGACGTTGTT GCAGGAAGGGTATGTATCTAAGTTCACATTTGAAATCAAAAAATGGGGCG ATGTCAAGAAAGGTTTCACACATTTTAAGGATAACGACATCATCTTCGCT AAAATCACCCCTTGTTTCCAGAATTTAAAATCAGCCATTATGGAGAACCT GAAAAATGGATATGGCGCAGGCACGACAGAATTGCACGTCTTACGCTGTT ACAAAATGCTTAGTTTAGAATATTTTCTGTGGTTCGTAAAGAGCCCTTAC TTCATGTCGTTCTGCGAAGCCAACATGAGCGGTACAGCCGGACAACAACG TGTGGGTACGGACATTGTCAAGAATGTTCTTTTACCATTACCCCCGTTAG AGGAGCAAAAACGTATCGTATATGTAATTGAAAAGTATTTCCCATTTTGT CAGCAATTGCGTAAATAAGCTTGACGAACAATAAGGCCTCCCAAATCGGG GGGCCTTTTTATTTTTCAACAAAACGCT ACTAACTGTCTATGCCTGGGA AAGGGTGGGCAGGAGATGGGGCAGTGCAGGAAAAGTGGCACTATGAACCC TGCAGCCCTAGTTTGACAATTAATCATTGGCATAGTATATCTGCATAGTA TAATACAACTCACTATAGCAATTGTACTAACCTTCTTCTCTTTCCTCTCC TGACAGGAGGAGCCATCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTC ACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGG GTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACG ACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAAC ACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTG GTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGA CCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCG GCCGGCAACTGCGTGCACTTTGTGGCAGAGGAGCAGGACTGATTACCTGT AACAGAGCATTAGCGCAAGGTGATTTTTGTCTTCTTGCGCTAATTTTTTC TGGGTTGTTTTACTGCTTCTGCTGGTGGGATTTTTCGTCCTGGAACCGCT CGGCATTCCGGTGAGCGCCATTGCAGCTGTGGGCGCGCTGATATTATTTG TCGTCGCTAAACGCGGTCATGCGATTAATACGGGTAAAGTCCTGCGCGGT GCCCCCTGGCAGATTGTCATCTTCTCGCTCGGCATGTATCTGGTGGTTTA TGGCCTGCGCAATGCCGGATTAACGGAATATCTTTCTGGCGTACTCAACG TGCTGGCGGATAACGGCCTGTGGGCCGCGACGCTCGGCACCGGATTCCTC ACCGCCTTCCTCTCTTCTATTATGAACAATATGCCGACGGTACTGGTTGG CGCGTTGTCCATTGATGGCAGCACGGCATCTGGCGTTATCAAAGAAGCGA TGGTTTATGCCAATGTGATTGGCTGCGATTTGGGACCGAAAATTACCCCA ATTGGTAGCCTGGCTACGCTACTCTGGCTGCACGTACTTTCGCAGAAGAA TATGACTATCAGCTGGGGATATTACTTCCGTACAGGGATTATCATGACCC TGCCTGTGCTGTTTGTGACGCTGGCTGCGCTGGCGCTACGTCTCTCTTTC ACTTTGTAATGAGATACTGATATGAGCAACATTACCATTTATCACAACCC GGCCTGCGGCACGTCGCGTAATACGCTGGAGATGATCCGCAACAGCGGCA CAGAACCGACTATTATCCATTATCTGGAAACTCCGCCAACGCGCGATGAA CTGGTCAAACTCATTGCCGATATGGGGATTTCCGTACGCGCGCTGCTGCG TAAAAACGTCGAACCGTATGAGGAGCTGGGCCTTGCGGAAGATAAATTTA CTGACGATCGGTTAATCGACTTTATGCTTCAGCACCCGATTCTGATTAAT CGCCCGATTGTGGTGACGCCGCTGGGAACTCGCCTGTGCCGCCCTTCAGA AGTGGTGCTGGAAATTCTGCCAGATGCGCAAAAAGGCGCATTCTCCAAGG AAGATGGCGAGAAAGTGGTTGATGAAGCGGGTAAGCGCCTG

[0660] >2W92R (Linear DNA sequence for recombineering to generate the stable E. coli strain DH10B-2W92R) (SEQ ID NO: 121)

TABLE-US-00034 TTCTGTTACCCATCCAATTGTTCAAAATTC TTGCTGATGAAACCCGTCTGGGCATCGTTTTACTGCTCAGCGAACTGGGA GAGTTATGCGTCTGCGATCTCTGCACTGCTCTCGACCAGTCGCAGCCCAA GATCTCCCGCCACCTGGCATTGCTGCGTGAAAGCGGGCTATTGCTGGACC GCAAGCAAGGTAAGTGGGTTCATTACCGCTTATCACCGCATATTCCAGCA TGGGCGGCGAAAATTATTGATGAGGCCTGGCGATGTGAACAGGAAAAGGT TCAGGCGATTGTCCGCAACCTGGCTCGACAAAACTGTTCCGGGGACAGTA AGAACATTTGCAGTTAAAAATTTAGCTAAACACATATGAATTTTCAGATG TGTTTTATCCGGGAGGCATTATGTTACTGGCAGGCGCTATCTTTGTCCTG ACCATCGTATTGGTTATCTGGCAGCCGAAAGGTTTAGGCATCGGCTGGAG TGCAACGCTCGGCGCAGTACTGGCGTTAGTTACGGGCGTGGTCCATCCGG GTGATATTCCGGTGGTGTGGAATATCGTCTGGAACGCGACGGCTGCGTTT ATCGCCGTCATTATCATCAGCCTGCTGCTGGATGAGTCCGGCTTTTTTGA ATGGGCGGCGCTGCACGTCTCACGCTGGGGTAATGGTCGTGGTCGCTTGC TGTTTACCTGGATTGTCCTGCTCGGTGCTGCCGTTGCCGCCCTGTTTGCC AATGATGGCGCGGCGCTTATTTTGACACCGATTGTCATCGCCATGCTGCT GGCTTTAGGGTTCAGTAAAGGCACTACGCTGGCGTTCGTGATGGCGGCCG GATTCATTGCCGATACCGCCAGCCTGCCGCTTATTGTCTCCAACCTGGTG AATATCGTTTCCGCTGATTTCTTTGGCCTCGGCTTTCGCGAATACGCCTC GGTGATGGTGCCGGTGGATATCGCCGCGATTGTTGCCACGCTGGTGATGT TACATCTCTATTTTCGCAAAGATATTCCGCAGAACTACGATATGGCGCTG CTGAAATCTCCCGCAGAAGCGATCAAAGATCCTGCTACGTTCAAAACTGG GCAAGGAG TTTATGGCTAGCTCAGTCCTAGGTACAATGCTAGCTACTAG AGAAAGAGGAGAAATACAAATGACCAAGTCTGAAACATTCATGATTCCAA ACCACAAGGCCGCCAAATTAAGTGAGCTGGATATGATGATCGTTAACTCT GTCCCGCCTGGGGGAAACTGGAAGAATATTCCCTTGGATGTACCATCGAA ACGTATCGAACAGATTCGTGACAGCTATGCTCAAGGAAAAGGGTCGCGCA GCACATACTACGGGCGTTTATTGCCCGATATGCCAGCTTATACGATCAAT ACTTATTTCAATCGTCCTGGCAACGGATGCCACATTCACTACGAGCAAGA TCGCGTACTTTCACAACGCGAAGCTGCACGTCTGCAGTCGTTCCCTGATG ACTTTATCTTTTTTGGAGGTCAAACGGCGATTAATACGCAAATCGGTAAT GCCGTGCCTCCCTTTCTTGCGTTTCTTATTGCAAAAGAAATTGAAAAAGC GATCGGTAATACCGGCTACTACATTGACTTATTCAGTGGTGCAGGCGGAT TGGGGTTGGGCTTTAAGTGGGCCGGGTGGACTCCATTGTTAGCTAATGAC ATTGAGGAAAAGTACTTACAGACATACTCGAACAACGTACACAAAGAAGT TTTGTGCGGAAGCATTTCGGACAACGAAACTTTTTCTAAGATCGCAGACA AGATTTCTGGCTTTAAGAAATTATATTTTGATAAACAGCTGTGGATTCTG GGCGGGCCTCCGTGCCAGGGATTTAGCACGGCTGGCAACGCGCGTACAAT GGACGACCCACGCAACAGTCTGTTTATGCACTACAAGTCGCTGCTTAACG AGATTAAGCCGAATGGATTCATTTTCGAGAACGTCGCCGGCCTGTTGAAC ATGGAAAAAGGAAAGGTCTTTGAACGTGTTAAGGAGGAATTCTCGTCCAC AATGAAAACCATGAATGGTTGGATTTTAAATTCGGAACATTACGCAATTC CACAACGCCGTAAGCGTGTAATTCTTGTGGGCAGCAATGATCCGCTGTTC TCGATCGAACCACCTCAGAAGCTGACGGAAGATAAAGAGTCTTGGGTGTC AGTAAAAGATGCGTTATCTGACCTTCCCCCATTACAACACGGCGAGGATG GATCTGGTAAATACTATATCCACCACCCGGAAAATGATTACCAGTTGTTT ATGCGTGGAAACATTACACCCTCAGAGTATTATGAACGCAACATTAAGCC GTCGCTTTAAGCTTCTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAA GGGGGGCCTTTTTTCGTTTTGGTCCCGCTAGGTGGAGTTGACGGCTAGCT CAGTCCTAGGTACAGTGCTAGCTACTAGAGAAAGAGGAGAAATACAAATG GCTATCACTAACTTCGTAAAGCGCATCCAAGATGTGATGCGTAATGACGC TGGAGTTAATGGGGATGCACAACGCATCGAGCAAATCGTATGGATCTTAT TTTTAAAGATTTATGATGCAAAGGAAGAGGAGTGGGAGTTGGTAAACGAA AAGTATTCCTCGATTATCCCGGAGGAATGTAAATGGCGTAATTGGGCCGT AGATGAAAAGGACGGTGAAGCTCTGACAGGCGACGCGATTTTAAACTTTG TCAATAACACACTGTTCCCTATCCTGAAAAATTTAGAGATTGATGACGAA ACCCCGATGAATCAGGTCATCGTGAAGTACGCTTTCGAGGACGCCAACAA TTATATGAAAGATGGAGTTCTTCTTCGCCAGGTAGTTAATATCATTGATG AGATTGATTTCACTGAATATGAAGAGCGCCATGCATTTGGTACGATTTAT GAGAGCTTTTTAAAAGATCTTCAAAGTGCGGGTAACGCTGGTGAGTTCTA CACGCCCCGCGCCGTGACCGACTTTATGGTCGAAGTGATCAAACCAAAGC TTGGAGAGAAAGTAGCCGACTTTGCTTGCGGCACAGGGGGGTTCTTAGTG TCTGCGTTGAATGCTCTTGAGAAACAGGTTGGGAATTCACTTGAGAATCG CGAGATCTATAATAATTCTATTTACGGTGTGGAAAAGAAAGGGCTGCCCC ATATTCTGTGTGTGACAAATATGTTGTTGCATGACATCGACAACCCGGAC ATCATTCACGGGAACACACTGGAAACAGATTACAAGGAGTACCGCCGTAT GGAACAATTCGACATTGTACTGATGAACCCGCCATACGGTGGGAATGAAA AGGAGAGTGTTAAGGCTAACTTCCCTTCGGATCTTCGCTCCTCCGAGACA GCTGACTTATTTATCGATATTATTATGTTTCGTTTGAAGAAAAATGGGCG TTGTGCGATCATTCTTCCTGACGGCTTTCTGTTTGGTACTGATAACGCTA AGGTCAACATTAAGAAGAAGTTACTTGGGGAATTTAACTTGCACACAATC GTCCGCCTTCCCCACTCTGTGTTCGCGCCTTACACCTCGATTACGACTAA TATTCTTTTTTTCGACAATACGCACCCTACCGAAGAAACATGGTTTTATC GCCTGGACATGCCCGATGGGTATAAGAATTTTTCTAAAACAAAACCGATG AAGTTAGAGCACTTTGGGTCGGCAATTGAATGGTGGGACAACCGTGAGGA GATCGAAGTTGATGGATTTCCGAAAGCGAAGAAATACACCGTCGAGGACA TTGAAAACTTAGGGTACAACTTGGACTTGTGTGGCTTTCCCCACGAGGAA GAGGAGATTTTGGACCCCATGGACTTAATTCGCGAGTACCAGGAGAAGCG CGCCTCCTTAAACGCAGAGATTGACCATGTGCTGGAGAAGATTACCTCTA TGTTAGGAGGGAACTAAGCTTAAAAAAAAAAAAGGCCTCCCAAATCGGGG GGCCTTTTTTATTGATAACAAAACGCTTGCCGGAGTTGACGGCTAGCTCA GTCCTAGGTACAGTGCTAGCTACTAGAGAAAGAGGAGAAATACAAATGAC AGCACAAGATTTAAAGAACAGTATCTTGCAGCTTGCGATTCAAGGAAAAT TAGTCGAACAGCGTGCCGAAGAAGGCACCGCGAAGGAGCTTTTAGAGCAG ATCAAGACCGAAAAGGAACGTTTAATCAATGAAAAAAAGATCAAAAAAGA AAAGCCGCTTAGTGAAATTACGGAAGATGAGATTCCGTTTGAGATCCCGG AGTCGTGGGAATGGGTCCGCTTGAATGAGATCTACAATTTTATTGATTAT CGTGGGAAAACACCCAATAAAATCGATGAAGGAGTCGTTTTGATCACGGC GTCGAACGTGCGTAAGGGCTACTTAGACTTTAGTAAGTCGGATTTTATTT CCGAAAATGAGTATAAGGAGCGTATGACGCGCGGGATTACGCGCAAGGGT GACCTTCTGTTCACCACCGAGGCACCACTTGGTTACGTGGCGGTGAACAC GATTGAAATTGCTTCTTGCGGACAACGCGTGATCACATTTCAACAGTACG GCGTTAACGTATTTTGTAACGAGCTGATGTGTTATTTCATCCAGTCACCA TTCTTCCAGAACGAGCTGTTAGTCAAAGCTACAGGGACTACGGCGAAAGG CATCAAGGCTGAAAAATTAAAGTATTTGCTGATCCCTGTGCCGCCATTAG AAGAGCAACAACGCATTATTGCTAAAATCGAGGACATTTTACCTTACATT GAACAATACGACAAAGCCTATTCAAAACTGGAGGTATTTAACAAGAAATT CCCTGAGGATATGCAAAAGAGTATTCTGCAATACGCGATTCAGGGGAAGC TGGTCGAACAGCGTCAGGAAGAAGGAACGGCGGAAGAGCTGTACGAACAG ATCCAGGCAGAAAAGGAGCGCCTTATCAAAGAGGGGAAAATCAAGAAAGA AAAACCATTAAGCGAGTTTACGGAGGAGGAAATTCCTTATGAGATTCCTG ACTCTTGGAAATGGGTTAAGCTTGGCGATGTATTCCAAATTAACCCTCGT AATTCGATCGAGGATAAGATCGAAGTCTCGTTTATTCCAATGACGTTGTT GCAGGAAGGGTATGTATCTAAGTTCACATTTGAAATCAAAAAATGGGGCG ATGTCAAGAAAGGTTTCACACATTTTAAGGATAACGACATCATCTTCGCT AAAATCACCCCTTGTTTCCAGAATTTAAAATCAGCCATTATGGAGAACCT GAAAAATGGATATGGCGCAGGCACGACAGAATTGCACGTCTTACGCTGTT ACAAAATGCTTAGTTTAGAATATTTTCTGTGGTTCGTAAAGAGCCCTTAC TTCATGTCGTTCTGCGAAGCCAACATGAGCGGTACAGCCGGACAACAACG TGTGGGTACGGACATTGTCAAGAATGTTCTTTTACCATTACCCCCGTTAG AGGAGCAAAAACGTATCGTATATGTAATTGAAAAGTATTTCCCATTTTGT CAGCAATTGCGTAAATAAGCTTGACGAACAATAAGGCCTCCCAAATCGGG GGGCCTTTTTATTTTTCAACAAAACGCT ACTAACTGTCTATGCCTGGGA AAGGGTGGGCAGGAGATGGGGCAGTGCAGGAAAAGTGGCACTATGAACCC TGCAGCCCTAGTTTGACAATTAATCATTGGCATAGTATATCTGCATAGTA TAATACAACTCACTATAGCAATTGTACTAACCTTCTTCTCTTTCCTCTCC TGACAGGAGGAGCCATCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTC ACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGG GTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACG ACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAAC ACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTG GTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGA CCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCG GCCGGCAACTGCGTGCACTTTGTGGCAGAGGAGCAGGACTGATTACCTGT AACAGAGCATTAGCGCAAGGTGATTTTTGTCTTCTTGCGCTAATTTTTTC TGGGTTGTTTTACTGCTTCTGCTGGTGGGATTTTTCGTCCTGGAACCGCT CGGCATTCCGGTGAGCGCCATTGCAGCTGTGGGCGCGCTGATATTATTTG TCGTCGCTAAACGCGGTCATGCGATTAATACGGGTAAAGTCCTGCGCGGT GCCCCCTGGCAGATTGTCATCTTCTCGCTCGGCATGTATCTGGTGGTTTA TGGCCTGCGCAATGCCGGATTAACGGAATATCTTTCTGGCGTACTCAACG TGCTGGCGGATAACGGCCTGTGGGCCGCGACGCTCGGCACCGGATTCCTC ACCGCCTTCCTCTCTTCTATTATGAACAATATGCCGACGGTACTGGTTGG CGCGTTGTCCATTGATGGCAGCACGGCATCTGGCGTTATCAAAGAAGCGA TGGTTTATGCCAATGTGATTGGCTGCGATTTGGGACCGAAAATTACCCCA ATTGGTAGCCTGGCTACGCTACTCTGGCTGCACGTACTTTCGCAGAAGAA TATGACTATCAGCTGGGGATATTACTTCCGTACAGGGATTATCATGACCC TGCCTGTGCTGTTTGTGACGCTGGCTGCGCTGGCGCTACGTCTCTCTTTC ACTTTGTAATGAGATACTGATATGAGCAACATTACCATTTATCACAACCC GGCCTGCGGCACGTCGCGTAATACGCTGGAGATGATCCGCAACAGCGGCA CAGAACCGACTATTATCCATTATCTGGAAACTCCGCCAACGCGCGATGAA CTGGTCAAACTCATTGCCGATATGGGGATTTCCGTACGCGCGCTGCTGCG TAAAAACGTCGAACCGTATGAGGAGCTGGGCCTTGCGGAAGATAAATTTA CTGACGATCGGTTAATCGACTTTATGCTTCAGCACCCGATTCTGATTAAT CGCCCGATTGTGGTGACGCCGCTGGGAACTCGCCTGTGCCGCCCTTCAGA AGTGGTGCTGGAAATTCTGCCAGATGCGCAAAAAGGCGCATTCTCCAAGG AAGATGGCGAGAAAGTGGTTGATGAAGCGGGTAAGCGCCTG

[0661] >2W94R (Linear DNA sequence for recombineering to generate the stable E. coli strain DH10B-2W94R) (SEQ ID NO: 122)

TABLE-US-00035 TTCTGTTACCCATCCAATTGTTCAAAATTC TTGCTGATGAAACCCGTCTGGGCATCGTTTTACTGCTCAGCGAACTGGGA GAGTTATGCGTCTGCGATCTCTGCACTGCTCTCGACCAGTCGCAGCCCAA GATCTCCCGCCACCTGGCATTGCTGCGTGAAAGCGGGCTATTGCTGGACC GCAAGCAAGGTAAGTGGGTTCATTACCGCTTATCACCGCATATTCCAGCA TGGGCGGCGAAAATTATTGATGAGGCCTGGCGATGTGAACAGGAAAAGGT TCAGGCGATTGTCCGCAACCTGGCTCGACAAAACTGTTCCGGGGACAGTA AGAACATTTGCAGTTAAAAATTTAGCTAAACACATATGAATTTTCAGATG TGTTTTATCCGGGAGGCATTATGTTACTGGCAGGCGCTATCTTTGTCCTG ACCATCGTATTGGTTATCTGGCAGCCGAAAGGTTTAGGCATCGGCTGGAG TGCAACGCTCGGCGCAGTACTGGCGTTAGTTACGGGCGTGGTCCATCCGG GTGATATTCCGGTGGTGTGGAATATCGTCTGGAACGCGACGGCTGCGTTT ATCGCCGTCATTATCATCAGCCTGCTGCTGGATGAGTCCGGCTTTTTTGA ATGGGCGGCGCTGCACGTCTCACGCTGGGGTAATGGTCGTGGTCGCTTGC TGTTTACCTGGATTGTCCTGCTCGGTGCTGCCGTTGCCGCCCTGTTTGCC AATGATGGCGCGGCGCTTATTTTGACACCGATTGTCATCGCCATGCTGCT GGCTTTAGGGTTCAGTAAAGGCACTACGCTGGCGTTCGTGATGGCGGCCG GATTCATTGCCGATACCGCCAGCCTGCCGCTTATTGTCTCCAACCTGGTG AATATCGTTTCCGCTGATTTCTTTGGCCTCGGCTTTCGCGAATACGCCTC GGTGATGGTGCCGGTGGATATCGCCGCGATTGTTGCCACGCTGGTGATGT TACATCTCTATTTTCGCAAAGATATTCCGCAGAACTACGATATGGCGCTG CTGAAATCTCCCGCAGAAGCGATCAAAGATCCTGCTACGTTCAAAACTGG GCAAGGAG CTGATAGCTAGCTCAGTCCTAGGGATTATGCTAGCTACTAG AGAAAGAGGAGAAATACAAATGACCAAGTCTGAAACATTCATGATTCCAA ACCACAAGGCCGCCAAATTAAGTGAGCTGGATATGATGATCGTTAACTCT GTCCCGCCTGGGGGAAACTGGAAGAATATTCCCTTGGATGTACCATCGAA ACGTATCGAACAGATTCGTGACAGCTATGCTCAAGGAAAAGGGTCGCGCA GCACATACTACGGGCGTTTATTGCCCGATATGCCAGCTTATACGATCAAT ACTTATTTCAATCGTCCTGGCAACGGATGCCACATTCACTACGAGCAAGA TCGCGTACTTTCACAACGCGAAGCTGCACGTCTGCAGTCGTTCCCTGATG ACTTTATCTTTTTTGGAGGTCAAACGGCGATTAATACGCAAATCGGTAAT GCCGTGCCTCCCTTTCTTGCGTTTCTTATTGCAAAAGAAATTGAAAAAGC GATCGGTAATACCGGCTACTACATTGACTTATTCAGTGGTGCAGGCGGAT TGGGGTTGGGCTTTAAGTGGGCCGGGTGGACTCCATTGTTAGCTAATGAC ATTGAGGAAAAGTACTTACAGACATACTCGAACAACGTACACAAAGAAGT TTTGTGCGGAAGCATTTCGGACAACGAAACTTTTTCTAAGATCGCAGACA AGATTTCTGGCTTTAAGAAATTATATTTTGATAAACAGCTGTGGATTCTG GGCGGGCCTCCGTGCCAGGGATTTAGCACGGCTGGCAACGCGCGTACAAT GGACGACCCACGCAACAGTCTGTTTATGCACTACAAGTCGCTGCTTAACG AGATTAAGCCGAATGGATTCATTTTCGAGAACGTCGCCGGCCTGTTGAAC ATGGAAAAAGGAAAGGTCTTTGAACGTGTTAAGGAGGAATTCTCGTCCAC AATGAAAACCATGAATGGTTGGATTTTAAATTCGGAACATTACGCAATTC CACAACGCCGTAAGCGTGTAATTCTTGTGGGCAGCAATGATCCGCTGTTC TCGATCGAACCACCTCAGAAGCTGACGGAAGATAAAGAGTCTTGGGTGTC AGTAAAAGATGCGTTATCTGACCTTCCCCCATTACAACACGGCGAGGATG GATCTGGTAAATACTATATCCACCACCCGGAAAATGATTACCAGTTGTTT ATGCGTGGAAACATTACACCCTCAGAGTATTATGAACGCAACATTAAGCC GTCGCTTTAAGCTTCTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAA GGGGGGCCTTTTTTCGTTTTGGTCCCGCTAGGTGGAGTTGACGGCTAGCT CAGTCCTAGGTACAGTGCTAGCTACTAGAGAAAGAGGAGAAATACAAATG GCTATCACTAACTTCGTAAAGCGCATCCAAGATGTGATGCGTAATGACGC TGGAGTTAATGGGGATGCACAACGCATCGAGCAAATCGTATGGATCTTAT TTTTAAAGATTTATGATGCAAAGGAAGAGGAGTGGGAGTTGGTAAACGAA AAGTATTCCTCGATTATCCCGGAGGAATGTAAATGGCGTAATTGGGCCGT AGATGAAAAGGACGGTGAAGCTCTGACAGGCGACGCGATTTTAAACTTTG TCAATAACACACTGTTCCCTATCCTGAAAAATTTAGAGATTGATGACGAA ACCCCGATGAATCAGGTCATCGTGAAGTACGCTTTCGAGGACGCCAACAA TTATATGAAAGATGGAGTTCTTCTTCGCCAGGTAGTTAATATCATTGATG AGATTGATTTCACTGAATATGAAGAGCGCCATGCATTTGGTACGATTTAT GAGAGCTTTTTAAAAGATCTTCAAAGTGCGGGTAACGCTGGTGAGTTCTA CACGCCCCGCGCCGTGACCGACTTTATGGTCGAAGTGATCAAACCAAAGC TTGGAGAGAAAGTAGCCGACTTTGCTTGCGGCACAGGGGGGTTCTTAGTG TCTGCGTTGAATGCTCTTGAGAAACAGGTTGGGAATTCACTTGAGAATCG CGAGATCTATAATAATTCTATTTACGGTGTGGAAAAGAAAGGGCTGCCCC ATATTCTGTGTGTGACAAATATGTTGTTGCATGACATCGACAACCCGGAC ATCATTCACGGGAACACACTGGAAACAGATTACAAGGAGTACCGCCGTAT GGAACAATTCGACATTGTACTGATGAACCCGCCATACGGTGGGAATGAAA AGGAGAGTGTTAAGGCTAACTTCCCTTCGGATCTTCGCTCCTCCGAGACA GCTGACTTATTTATCGATATTATTATGTTTCGTTTGAAGAAAAATGGGCG TTGTGCGATCATTCTTCCTGACGGCTTTCTGTTTGGTACTGATAACGCTA AGGTCAACATTAAGAAGAAGTTACTTGGGGAATTTAACTTGCACACAATC GTCCGCCTTCCCCACTCTGTGTTCGCGCCTTACACCTCGATTACGACTAA TATTCTTTTTTTCGACAATACGCACCCTACCGAAGAAACATGGTTTTATC GCCTGGACATGCCCGATGGGTATAAGAATTTTTCTAAAACAAAACCGATG AAGTTAGAGCACTTTGGGTCGGCAATTGAATGGTGGGACAACCGTGAGGA GATCGAAGTTGATGGATTTCCGAAAGCGAAGAAATACACCGTCGAGGACA TTGAAAACTTAGGGTACAACTTGGACTTGTGTGGCTTTCCCCACGAGGAA GAGGAGATTTTGGACCCCATGGACTTAATTCGCGAGTACCAGGAGAAGCG CGCCTCCTTAAACGCAGAGATTGACCATGTGCTGGAGAAGATTACCTCTA TGTTAGGAGGGAACTAAGCTTAAAAAAAAAAAAGGCCTCCCAAATCGGGG GGCCTTTTTTATTGATAACAAAACGCTTGCCGGAGTTGACGGCTAGCTCA GTCCTAGGTACAGTGCTAGCTACTAGAGAAAGAGGAGAAATACAAATGAC AGCACAAGATTTAAAGAACAGTATCTTGCAGCTTGCGATTCAAGGAAAAT TAGTCGAACAGCGTGCCGAAGAAGGCACCGCGAAGGAGCTTTTAGAGCAG ATCAAGACCGAAAAGGAACGTTTAATCAATGAAAAAAAGATCAAAAAAGA AAAGCCGCTTAGTGAAATTACGGAAGATGAGATTCCGTTTGAGATCCCGG AGTCGTGGGAATGGGTCCGCTTGAATGAGATCTACAATTTTATTGATTAT CGTGGGAAAACACCCAATAAAATCGATGAAGGAGTCGTTTTGATCACGGC GTCGAACGTGCGTAAGGGCTACTTAGACTTTAGTAAGTCGGATTTTATTT CCGAAAATGAGTATAAGGAGCGTATGACGCGCGGGATTACGCGCAAGGGT GACCTTCTGTTCACCACCGAGGCACCACTTGGTTACGTGGCGGTGAACAC GATTGAAATTGCTTCTTGCGGACAACGCGTGATCACATTTCAACAGTACG GCGTTAACGTATTTTGTAACGAGCTGATGTGTTATTTCATCCAGTCACCA TTCTTCCAGAACGAGCTGTTAGTCAAAGCTACAGGGACTACGGCGAAAGG CATCAAGGCTGAAAAATTAAAGTATTTGCTGATCCCTGTGCCGCCATTAG AAGAGCAACAACGCATTATTGCTAAAATCGAGGACATTTTACCTTACATT GAACAATACGACAAAGCCTATTCAAAACTGGAGGTATTTAACAAGAAATT CCCTGAGGATATGCAAAAGAGTATTCTGCAATACGCGATTCAGGGGAAGC TGGTCGAACAGCGTCAGGAAGAAGGAACGGCGGAAGAGCTGTACGAACAG ATCCAGGCAGAAAAGGAGCGCCTTATCAAAGAGGGGAAAATCAAGAAAGA AAAACCATTAAGCGAGTTTACGGAGGAGGAAATTCCTTATGAGATTCCTG ACTCTTGGAAATGGGTTAAGCTTGGCGATGTATTCCAAATTAACCCTCGT AATTCGATCGAGGATAAGATCGAAGTCTCGTTTATTCCAATGACGTTGTT GCAGGAAGGGTATGTATCTAAGTTCACATTTGAAATCAAAAAATGGGGCG ATGTCAAGAAAGGTTTCACACATTTTAAGGATAACGACATCATCTTCGCT AAAATCACCCCTTGTTTCCAGAATTTAAAATCAGCCATTATGGAGAACCT GAAAAATGGATATGGCGCAGGCACGACAGAATTGCACGTCTTACGCTGTT ACAAAATGCTTAGTTTAGAATATTTTCTGTGGTTCGTAAAGAGCCCTTAC TTCATGTCGTTCTGCGAAGCCAACATGAGCGGTACAGCCGGACAACAACG TGTGGGTACGGACATTGTCAAGAATGTTCTTTTACCATTACCCCCGTTAG AGGAGCAAAAACGTATCGTATATGTAATTGAAAAGTATTTCCCATTTTGT CAGCAATTGCGTAAATAAGCTTGACGAACAATAAGGCCTCCCAAATCGGG GGGCCTTTTTATTTTTCAACAAAACGCT ACTAACTGTCTATGCCTGGGA AAGGGTGGGCAGGAGATGGGGCAGTGCAGGAAAAGTGGCACTATGAACCC TGCAGCCCTAGTTTGACAATTAATCATTGGCATAGTATATCTGCATAGTA TAATACAACTCACTATAGCAATTGTACTAACCTTCTTCTCTTTCCTCTCC TGACAGGAGGAGCCATCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTC ACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGG GTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACG ACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAAC ACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTG GTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGA CCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCG GCCGGCAACTGCGTGCACTTTGTGGCAGAGGAGCAGGACTGATTACCTGT AACAGAGCATTAGCGCAAGGTGATTTTTGTCTTCTTGCGCTAATTTTTTC TGGGTTGTTTTACTGCTTCTGCTGGTGGGATTTTTCGTCCTGGAACCGCT CGGCATTCCGGTGAGCGCCATTGCAGCTGTGGGCGCGCTGATATTATTTG TCGTCGCTAAACGCGGTCATGCGATTAATACGGGTAAAGTCCTGCGCGGT GCCCCCTGGCAGATTGTCATCTTCTCGCTCGGCATGTATCTGGTGGTTTA TGGCCTGCGCAATGCCGGATTAACGGAATATCTTTCTGGCGTACTCAACG TGCTGGCGGATAACGGCCTGTGGGCCGCGACGCTCGGCACCGGATTCCTC ACCGCCTTCCTCTCTTCTATTATGAACAATATGCCGACGGTACTGGTTGG CGCGTTGTCCATTGATGGCAGCACGGCATCTGGCGTTATCAAAGAAGCGA TGGTTTATGCCAATGTGATTGGCTGCGATTTGGGACCGAAAATTACCCCA ATTGGTAGCCTGGCTACGCTACTCTGGCTGCACGTACTTTCGCAGAAGAA TATGACTATCAGCTGGGGATATTACTTCCGTACAGGGATTATCATGACCC TGCCTGTGCTGTTTGTGACGCTGGCTGCGCTGGCGCTACGTCTCTCTTTC ACTTTGTAATGAGATACTGATATGAGCAACATTACCATTTATCACAACCC GGCCTGCGGCACGTCGCGTAATACGCTGGAGATGATCCGCAACAGCGGCA CAGAACCGACTATTATCCATTATCTGGAAACTCCGCCAACGCGCGATGAA CTGGTCAAACTCATTGCCGATATGGGGATTTCCGTACGCGCGCTGCTGCG TAAAAACGTCGAACCGTATGAGGAGCTGGGCCTTGCGGAAGATAAATTTA CTGACGATCGGTTAATCGACTTTATGCTTCAGCACCCGATTCTGATTAAT CGCCCGATTGTGGTGACGCCGCTGGGAACTCGCCTGTGCCGCCCTTCAGA AGTGGTGCTGGAAATTCTGCCAGATGCGCAAAAAGGCGCATTCTCCAAGG AAGATGGCGAGAAAGTGGTTGATGAAGCGGGTAAGCGCCTG

[0662] >2W276R (Linear DNA sequence for recombineering to generate the stable E. coli strain DH10B-2W276R, with the 20 bp sequence corresponding to the guide RNA sequence guide #498 that specifies dST1Cas9 specificity underlined). (SEQ ID NO: 123)

TABLE-US-00036 TTCTGTTACCCATCCAATTGTTCAAAATTC TTGCTGATGAAACCCGTCTGGGCATCGTTTTACTGCTCAGCGAACTGGGA GAGTTATGCGTCTGCGATCTCTGCACTGCTCTCGACCAGTCGCAGCCCAA GATCTCCCGCCACCTGGCATTGCTGCGTGAAAGCGGGCTATTGCTGGACC GCAAGCAAGGTAAGTGGGTTCATTACCGCTTATCACCGCATATTCCAGCA TGGGCGGCGAAAATTATTGATGAGGCCTGGCGATGTGAACAGGAAAAGGT TCAGGCGATTGTCCGCAACCTGGCTCGACAAAACTGTTCCGGGGACAGTA AGAACATTTGCAGTTAAAAATTTAGCTAAACACATATGAATTTTCAGATG TGTTTTATCCGGGAGGCATTATGTTACTGGCAGGCGCTATCTTTGTCCTG ACCATCGTATTGGTTATCTGGCAGCCGAAAGGTTTAGGCATCGGCTGGAG TGCAACGCTCGGCGCAGTACTGGCGTTAGTTACGGGCGTGGTCCATCCGG GTGATATTCCGGTGGTGTGGAATATCGTCTGGAACGCGACGGCTGCGTTT ATCGCCGTCATTATCATCAGCCTGCTGCTGGATGAGTCCGGCTTTTTTGA ATGGGCGGCGCTGCACGTCTCACGCTGGGGTAATGGTCGTGGTCGCTTGC TGTTTACCTGGATTGTCCTGCTCGGTGCTGCCGTTGCCGCCCTGTTTGCC AATGATGGCGCGGCGCTTATTTTGACACCGATTGTCATCGCCATGCTGCT GGCTTTAGGGTTCAGTAAAGGCACTACGCTGGCGTTCGTGATGGCGGCCG GATTCATTGCCGATACCGCCAGCCTGCCGCTTATTGTCTCCAACCTGGTG AATATCGTTTCCGCTGATTTCTTTGGCCTCGGCTTTCGCGAATACGCCTC GGTGATGGTGCCGGTGGATATCGCCGCGATTGTTGCCACGCTGGTGATGT TACATCTCTATTTTCGCAAAGATATTCCGCAGAACTACGATATGGCGCTG CTGAAATCTCCCGCAGAAGCGATCAAAGATCCTGCTACGTTCAAAACTGG GCAAGGAGTTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCGTCTAG TATGCGGATGCCAGGTTTTTGTACTCGAAAGAAGCTACAAAGATAAGGC TTCATGCCGAAATCAACACCCTGTCATTTTATGGCAGGGTGTTTTTGAAG CTTGGGCCCGAACAAAAAGCTTCTCGGTACCAAATTCCAGAAAAGAGGCC TCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCCGCTAGGTTTGACAGCT AGCTCAGTCCTAGGTATAATGCTAGCAGAGAAAGAGGAGAAATACAAATG AGTGACCTGGTATTAGGTCTGGCGATTGGCATTGGGTCTGTTGGCGTCGG AATTTTGAACAAAGTTACCGGAGAGATTATCCACAAAAACTCGCGTATTT TCCCAGCTGCCCAAGCTGAAAATAACTTGGTTCGCCGTACAAATCGCCAG GGTCGTCGTTTGGCTCGCCGCAAGAAACACCGCCGCGTGCGCCTTAATCG CCTGTTCGAAGAATCCGGTTTAATCACCGACTTCACGAAGATCTCGATTA ATCTTAATCCATACCAATTACGCGTAAAGGGGCTTACGGATGAGTTAAGC AACGAGGAACTGTTCATTGCGTTGAAAAATATGGTCAAACATCGCGGTAT TAGCTATTTAGATGACGCCAGCGACGACGGTAATTCATCGGTGGGCGATT ATGCCCAAATCGTAAAAGAAAACTCGAAGCAACTTGAGACAAAAACGCCT GGCCAAATTCAACTTGAGCGCTATCAGACCTATGGACAGTTGCGCGGTGA TTTCACTGTCGAAAAAGACGGAAAAAAACACCGCCTGATTAATGTATTTC CCACCTCGGCTTACCGCAGTGAAGCTTTGCGCATTCTTCAGACCCAACAA GAGTTTAATCCCCAGATCACAGACGAGTTCATCAATCGTTATCTGGAAAT CCTGACTGGCAAACGTAAATATTATCATGGACCCGGCAACGAGAAGTCCC GCACTGACTATGGCCGTTATCGCACCAGTGGTGAGACTCTTGATAATATT TTTGGCATTTTAATTGGGAAGTGTACTTTCTATCCGGATGAGTTTCGTGC TGCCAAGGCCTCTTATACGGCGCAAGAATTTAATTTATTGAACGATCTTA ATAATCTTACGGTCCCTACTGAAACTAAGAAATTATCAAAGGAGCAGAAG AATCAAATCATCAATTACGTAAAAAATGAAAAGGCTATGGGGCCGGCTAA ATTGTTCAAATACATTGCGAAATTACTGAGTTGCGACGTTGCCGATATTA AGGGGTACCGCATTGATAAAAGCGGGAAGGCGGAGATTCACACTTTCGAA GCCTATCGCAAAATGAAAACGTTGGAAACTTTAGACATCGAACAAATGGA TCGTGAAACGTTGGACAAGTTGGCTTACGTCCTTACTCTGAATACAGAAC GCGAAGGCATTCAGGAAGCCTTGGAGCATGAATTCGCAGATGGAAGTTTC TCTCAAAAGCAAGTAGACGAGTTAGTTCAATTCCGCAAAGCGAACTCGTC AATTTTTGGAAAGGGCTGGCACAATTTCAGTGTGAAGTTAATGATGGAAC TTATTCCAGAATTGTATGAGACGAGTGAGGAGCAAATGACGATCTTAACG CGCCTTGGTAAGCAAAAAACGACATCTTCCAGCAATAAAACAAAATACAT CGATGAGAAACTTTTGACCGAGGAAATTTATAATCCGGTCGTTGCAAAGA GTGTTCGCCAAGCAATCAAGATTGTCAATGCCGCCATCAAAGAATATGGA GATTTTGATAATATCGTTATCGAGATGGCACGCGAGACAAACGAGGACGA CGAGAAGAAAGCGATTCAGAAGATTCAGAAGGCAAACAAAGACGAAAAGG ATGCAGCCATGCTGAAAGCTGCAAATCAATATAATGGTAAGGCTGAGCTT CCTCATAGTGTGTTTCACGGGCATAAACAGTTGGCGACGAAGATCCGTCT GTGGCATCAGCAGGGAGAGCGTTGCTTATACACCGGCAAGACTATCAGTA TTCACGACTTGATTAACAATAGTAACCAATTCGAAGTTGATGCTATTCTT CCTTTATCAATCACATTCGACGACTCTCTGGCTAATAAAGTCTTGGTATA CGCGACAGCCAACCAAGAAAAAGGACAGCGTACCCCATATCAAGCTTTGG ATTCAATGGATGACGCCTGGTCCTTCCGTGAGCTGAAGGCTTTCGTGCGC GAGTCCAAAACCTTGTCTAACAAAAAAAAGGAGTACTTATTGACGGAGGA GGACATTAGTAAATTCGACGTACGCAAAAAGTTTATTGAGCGCAACCTTG TTGATACTCGTTACGCATCTCGTGTTGTCCTTAACGCCTTACAGGAGCAT TTTCGCGCACACAAGATCGACACCAAGGTGTCTGTCGTACGTGGACAATT CACCAGCCAACTTCGCCGCCACTGGGGGATTGAAAAGACCCGTGACACAT ATCACCACCATGCCGTTGACGCTCTTATCATTGCTGCGAGTTCCCAGTTG AATTTATGGAAGAAACAAAAGAACACACTGGTCAGTTATTCTGAAGATCA ACTTCTGGACATTGAAACTGGAGAGCTGATTTCTGACGATGAATATAAGG AGTCAGTTTTCAAAGCACCATATCAGCATTTTGTAGATACGCTGAAATCT AAAGAGTTCGAGGACTCAATCTTGTTCTCGTATCAGGTTGACTCTAAATT CAATCGCAAGATCAGTGATGCCACTATCTACGCCACTCGCCAAGCGAAGG TGGGGAAAGATAAAGCCGACGAGACATATGTCTTAGGGAAAATCAAAGAT ATCTACACACAGGATGGGTACGATGCTTTCATGAAAATTTATAAGAAAGA CAAAAGTAAATTCTTAATGTATCGCCACGATCCTCAGACTTTCGAGAAGG TGATCGAGCCTATTCTTGAAAATTATCCTAATAAACAGATCAACGACAAG GGTAAGGAAGTGCCATGCAATCCTTTTCTGAAGTATAAAGAAGAACACGG TTACATCCGTAAGTATTCAAAAAAGGGGAATGGACCGGAGATCAAGAGTC TGAAATACTACGACAGTAAACTTGGGAATCACATTGATATCACCCCGAAG GATTCCAACAACAAGGTGGTCCTTCAGTCTGTTTCGCCTTGGCGCGCTGA CGTTTATTTCAATAAGACGACGGGCAAATATGAGATTTTGGGTCTTAAAT ATGCAGATTTGCAATTTGATAAAGGAACTGGGACATACAAAATTTCACAA GAAAAATACAATGACATTAAGAAAAAGGAGGGGGTAGACAGTGATTCAGA GTTTAAGTTTACCTTATATAAAAATGATCTTTTGTTAGTCAAGGACACCG AGACGAAGGAACAACAGCTGTTTCGCTTCTTATCGCGCACAATGCCTAAA CAAAAACATTATGTTGAACTGAAGCCCTACGACAAACAAAAATTTGAGGG AGGTGAAGCATTAATCAAGGTCCTTGGGAATGTAGCTAACTCGGGCCAGT GTAAGAAGGGTCTTGGCAAATCTAACATCTCTATCTATAAGGTTCGCACG GATGTCTTGGGAAACCAACATATCATTAAGAACGAAGGAGACAAACCAAA GTTGGACTTCTAAGCTTAAAAAAAAAAAAGGCCTCCCAAATCGGGGGGCC TTTTTTATTGATAACAAAACGCTTGCCGGAGCTGATAGCTAGCTCAGTCC TAGGGATTATGCTAGCTACTAGAGAAAGAGGAGAAATACAAATGACCAAG TCTGAAACATTCATGATTCCAAACCACAAGGCCGCCAAATTAAGTGAGCT GGATATGATGATCGTTAACTCTGTCCCGCCTGGGGGAAACTGGAAGAATA TTCCCTTGGATGTACCATCGAAACGTATCGAACAGATTCGTGACAGCTAT GCTCAAGGAAAAGGGTCGCGCAGCACATACTACGGGCGTTTATTGCCCGA TATGCCAGCTTATACGATCAATACTTATTTCAATCGTCCTGGCAACGGAT GCCACATTCACTACGAGCAAGATCGCGTACTTTCACAACGCGAAGCTGCA CGTCTGCAGTCGTTCCCTGATGACTTTATCTTTTTTGGAGGTCAAACGGC GATTAATACGCAAATCGGTAATGCCGTGCCTCCCTTTCTTGCGTTTCTTA TTGCAAAAGAAATTGAAAAAGCGATCGGTAATACCGGCTACTACATTGAC TTATTCAGTGGTGCAGGCGGATTGGGGTTGGGCTTTAAGTGGGCCGGGTG GACTCCATTGTTAGCTAATGACATTGAGGAAAAGTACTTACAGACATACT CGAACAACGTACACAAAGAAGTTTTGTGCGGAAGCATTTCGGACAACGAA ACTTTTTCTAAGATCGCAGACAAGATTTCTGGCTTTAAGAAATTATATTT TGATAAACAGCTGTGGATTCTGGGCGGGCCTCCGTGCCAGGGATTTAGCA CGGCTGGCAACGCGCGTACAATGGACGACCCACGCAACAGTCTGTTTATG CACTACAAGTCGCTGCTTAACGAGATTAAGCCGAATGGATTCATTTTCGA GAACGTCGCCGGCCTGTTGAACATGGAAAAAGGAAAGGTCTTTGAACGTG TTAAGGAGGAATTCTCGTCCACAATGAAAACCATGAATGGTTGGATTTTA AATTCGGAACATTACGCAATTCCACAACGCCGTAAGCGTGTAATTCTTGT GGGCAGCAATGATCCGCTGTTCTCGATCGAACCACCTCAGAAGCTGACGG AAGATAAAGAGTCTTGGGTGTCAGTAAAAGATGCGTTATCTGACCTTCCC CCATTACAACACGGCGAGGATGGATCTGGTAAATACTATATCCACCACCC GGAAAATGATTACCAGTTGTTTATGCGTGGAAACATTACACCCTCAGAGT ATTATGAACGCAACATTAAGCCGTCGCTTTAAGCTTGACGAACAATAAGG CCTCCCAAATCGGGGGGCCTTTTTATTTTTCAACAAAACGCTACTAACTG TCTATGCCTGGGAAAGGGTGGGCAGGAGATGGGGCAGTGCAGGAAAAGTG GCACTATGAACCCTGCAGCCCTAGTTTGACAATTAATCATTGGCATAGTA TATCTGCATAGTATAATACAACTCACTATAGCAATTGTACTAACCTTCTT CTCTTTCCTCTCCTGACAGGAGGAGCCATCATGGCCAAGTTGACCAGTGC CGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGA CCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGT GTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGT GGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGC TGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCC GGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGC CCTGCGCGACCCGGCCGGCAACTGCGTGCACTTTGTGGCAGAGGAGCAGG ACTGATTACCTGTAACAGAGCATTAGCGCAAGGTGATTTTTGTCTTCTTG CGCTAATTTTTTCTGGGTTGTTTTACTGCTTCTGCTGGTGGGATTTTTCG TCCTGGAACCGCTCGGCATTCCGGTGAGCGCCATTGCAGCTGTGGGCGCG CTGATATTATTTGTCGTCGCTAAACGCGGTCATGCGATTAATACGGGTAA AGTCCTGCGCGGTGCCCCCTGGCAGATTGTCATCTTCTCGCTCGGCATGT ATCTGGTGGTTTATGGCCTGCGCAATGCCGGATTAACGGAATATCTTTCT GGCGTACTCAACGTGCTGGCGGATAACGGCCTGTGGGCCGCGACGCTCGG CACCGGATTCCTCACCGCCTTCCTCTCTTCTATTATGAACAATATGCCGA CGGTACTGGTTGGCGCGTTGTCCATTGATGGCAGCACGGCATCTGGCGTT ATCAAAGAAGCGATGGTTTATGCCAATGTGATTGGCTGCGATTTGGGACC GAAAATTACCCCAATTGGTAGCCTGGCTACGCTACTCTGGCTGCACGTAC TTTCGCAGAAGAATATGACTATCAGCTGGGGATATTACTTCCGTACAGGG ATTATCATGACCCTGCCTGTGCTGTTTGTGACGCTGGCTGCGCTGGCGCT ACGTCTCTCTTTCACTTTGTAATGAGATACTGATATGAGCAACATTACCA TTTATCACAACCCGGCCTGCGGCACGTCGCGTAATACGCTGGAGATGATC CGCAACAGCGGCACAGAACCGACTATTATCCATTATCTGGAAACTCCGCC AACGCGCGATGAACTGGTCAAACTCATTGCCGATATGGGGATTTCCGTAC GCGCGCTGCTGCGTAAAAACGTCGAACCGTATGAGGAGCTGGGCCTTGCG GAAGATAAATTTACTGACGATCGGTTAATCGACTTTATGCTTCAGCACCC GATTCTGATTAATCGCCCGATTGTGGTGACGCCGCTGGGAACTCGCCTGT GCCGCCCTTCAGAAGTGGTGCTGGAAATTCTGCCAGATGCGCAAAAAGGC GCATTCTCCAAGGAAGATGGCGAGAAAGTGGTTGATGAAGCGGGTAAGCG CCTG

[0663] >2W278R (Linear DNA sequence for recombineering to generate the stable E. coli strain DH10B-2W278R, with the 20 bp sequence corresponding to the guide RNA sequence guide #500 that specifies dST1Cas9 specificity underlined). (SEQ ID NO: 124)

TABLE-US-00037 TTCTGTTACCCATCCAATTGTTCAAAATTC TTGCTGATGAAACCCGTCTGGGCATCGTTTTACTGCTCAGCGAACTGGGA GAGTTATGCGTCTGCGATCTCTGCACTGCTCTCGACCAGTCGCAGCCCAA GATCTCCCGCCACCTGGCATTGCTGCGTGAAAGCGGGCTATTGCTGGACC GCAAGCAAGGTAAGTGGGTTCATTACCGCTTATCACCGCATATTCCAGCA TGGGCGGCGAAAATTATTGATGAGGCCTGGCGATGTGAACAGGAAAAGGT TCAGGCGATTGTCCGCAACCTGGCTCGACAAAACTGTTCCGGGGACAGTA AGAACATTTGCAGTTAAAAATTTAGCTAAACACATATGAATTTTCAGATG TGTTTTATCCGGGAGGCATTATGTTACTGGCAGGCGCTATCTTTGTCCTG ACCATCGTATTGGTTATCTGGCAGCCGAAAGGTTTAGGCATCGGCTGGAG TGCAACGCTCGGCGCAGTACTGGCGTTAGTTACGGGCGTGGTCCATCCGG GTGATATTCCGGTGGTGTGGAATATCGTCTGGAACGCGACGGCTGCGTTT ATCGCCGTCATTATCATCAGCCTGCTGCTGGATGAGTCCGGCTTTTTTGA ATGGGCGGCGCTGCACGTCTCACGCTGGGGTAATGGTCGTGGTCGCTTGC TGTTTACCTGGATTGTCCTGCTCGGTGCTGCCGTTGCCGCCCTGTTTGCC AATGATGGCGCGGCGCTTATTTTGACACCGATTGTCATCGCCATGCTGCT GGCTTTAGGGTTCAGTAAAGGCACTACGCTGGCGTTCGTGATGGCGGCCG GATTCATTGCCGATACCGCCAGCCTGCCGCTTATTGTCTCCAACCTGGTG AATATCGTTTCCGCTGATTTCTTTGGCCTCGGCTTTCGCGAATACGCCTC GGTGATGGTGCCGGTGGATATCGCCGCGATTGTTGCCACGCTGGTGATGT TACATCTCTATTTTCGCAAAGATATTCCGCAGAACTACGATATGGCGCTG CTGAAATCTCCCGCAGAAGCGATCAAAGATCCTGCTACGTTCAAAACTGG GCAAGGAGTTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCGAGACC ACCATTCTGGGGCTGTTTTTGTACTCGAAAGAAGCTACAAAGATAAGGC TTCATGCCGAAATCAACACCCTGTCATTTTATGGCAGGGTGTTTTTGAAG CTTGGGCCCGAACAAAAAGCTTCTCGGTACCAAATTCCAGAAAAGAGGCC TCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCCGCTAGGTTTGACAGCT AGCTCAGTCCTAGGTATAATGCTAGCAGAGAAAGAGGAGAAATACAAATG AGTGACCTGGTATTAGGTCTGGCGATTGGCATTGGGTCTGTTGGCGTCGG AATTTTGAACAAAGTTACCGGAGAGATTATCCACAAAAACTCGCGTATTT TCCCAGCTGCCCAAGCTGAAAATAACTTGGTTCGCCGTACAAATCGCCAG GGTCGTCGTTTGGCTCGCCGCAAGAAACACCGCCGCGTGCGCCTTAATCG CCTGTTCGAAGAATCCGGTTTAATCACCGACTTCACGAAGATCTCGATTA ATCTTAATCCATACCAATTACGCGTAAAGGGGCTTACGGATGAGTTAAGC AACGAGGAACTGTTCATTGCGTTGAAAAATATGGTCAAACATCGCGGTAT TAGCTATTTAGATGACGCCAGCGACGACGGTAATTCATCGGTGGGCGATT ATGCCCAAATCGTAAAAGAAAACTCGAAGCAACTTGAGACAAAAACGCCT GGCCAAATTCAACTTGAGCGCTATCAGACCTATGGACAGTTGCGCGGTGA TTTCACTGTCGAAAAAGACGGAAAAAAACACCGCCTGATTAATGTATTTC CCACCTCGGCTTACCGCAGTGAAGCTTTGCGCATTCTTCAGACCCAACAA GAGTTTAATCCCCAGATCACAGACGAGTTCATCAATCGTTATCTGGAAAT CCTGACTGGCAAACGTAAATATTATCATGGACCCGGCAACGAGAAGTCCC GCACTGACTATGGCCGTTATCGCACCAGTGGTGAGACTCTTGATAATATT TTTGGCATTTTAATTGGGAAGTGTACTTTCTATCCGGATGAGTTTCGTGC TGCCAAGGCCTCTTATACGGCGCAAGAATTTAATTTATTGAACGATCTTA ATAATCTTACGGTCCCTACTGAAACTAAGAAATTATCAAAGGAGCAGAAG AATCAAATCATCAATTACGTAAAAAATGAAAAGGCTATGGGGCCGGCTAA ATTGTTCAAATACATTGCGAAATTACTGAGTTGCGACGTTGCCGATATTA AGGGGTACCGCATTGATAAAAGCGGGAAGGCGGAGATTCACACTTTCGAA GCCTATCGCAAAATGAAAACGTTGGAAACTTTAGACATCGAACAAATGGA TCGTGAAACGTTGGACAAGTTGGCTTACGTCCTTACTCTGAATACAGAAC GCGAAGGCATTCAGGAAGCCTTGGAGCATGAATTCGCAGATGGAAGTTTC TCTCAAAAGCAAGTAGACGAGTTAGTTCAATTCCGCAAAGCGAACTCGTC AATTTTTGGAAAGGGCTGGCACAATTTCAGTGTGAAGTTAATGATGGAAC TTATTCCAGAATTGTATGAGACGAGTGAGGAGCAAATGACGATCTTAACG CGCCTTGGTAAGCAAAAAACGACATCTTCCAGCAATAAAACAAAATACAT CGATGAGAAACTTTTGACCGAGGAAATTTATAATCCGGTCGTTGCAAAGA GTGTTCGCCAAGCAATCAAGATTGTCAATGCCGCCATCAAAGAATATGGA GATTTTGATAATATCGTTATCGAGATGGCACGCGAGACAAACGAGGACGA CGAGAAGAAAGCGATTCAGAAGATTCAGAAGGCAAACAAAGACGAAAAGG ATGCAGCCATGCTGAAAGCTGCAAATCAATATAATGGTAAGGCTGAGCTT CCTCATAGTGTGTTTCACGGGCATAAACAGTTGGCGACGAAGATCCGTCT GTGGCATCAGCAGGGAGAGCGTTGCTTATACACCGGCAAGACTATCAGTA TTCACGACTTGATTAACAATAGTAACCAATTCGAAGTTGATGCTATTCTT CCTTTATCAATCACATTCGACGACTCTCTGGCTAATAAAGTCTTGGTATA CGCGACAGCCAACCAAGAAAAAGGACAGCGTACCCCATATCAAGCTTTGG ATTCAATGGATGACGCCTGGTCCTTCCGTGAGCTGAAGGCTTTCGTGCGC GAGTCCAAAACCTTGTCTAACAAAAAAAAGGAGTACTTATTGACGGAGGA GGACATTAGTAAATTCGACGTACGCAAAAAGTTTATTGAGCGCAACCTTG TTGATACTCGTTACGCATCTCGTGTTGTCCTTAACGCCTTACAGGAGCAT TTTCGCGCACACAAGATCGACACCAAGGTGTCTGTCGTACGTGGACAATT CACCAGCCAACTTCGCCGCCACTGGGGGATTGAAAAGACCCGTGACACAT ATCACCACCATGCCGTTGACGCTCTTATCATTGCTGCGAGTTCCCAGTTG AATTTATGGAAGAAACAAAAGAACACACTGGTCAGTTATTCTGAAGATCA ACTTCTGGACATTGAAACTGGAGAGCTGATTTCTGACGATGAATATAAGG AGTCAGTTTTCAAAGCACCATATCAGCATTTTGTAGATACGCTGAAATCT AAAGAGTTCGAGGACTCAATCTTGTTCTCGTATCAGGTTGACTCTAAATT CAATCGCAAGATCAGTGATGCCACTATCTACGCCACTCGCCAAGCGAAGG TGGGGAAAGATAAAGCCGACGAGACATATGTCTTAGGGAAAATCAAAGAT ATCTACACACAGGATGGGTACGATGCTTTCATGAAAATTTATAAGAAAGA CAAAAGTAAATTCTTAATGTATCGCCACGATCCTCAGACTTTCGAGAAGG TGATCGAGCCTATTCTTGAAAATTATCCTAATAAACAGATCAACGACAAG GGTAAGGAAGTGCCATGCAATCCTTTTCTGAAGTATAAAGAAGAACACGG TTACATCCGTAAGTATTCAAAAAAGGGGAATGGACCGGAGATCAAGAGTC TGAAATACTACGACAGTAAACTTGGGAATCACATTGATATCACCCCGAAG GATTCCAACAACAAGGTGGTCCTTCAGTCTGTTTCGCCTTGGCGCGCTGA CGTTTATTTCAATAAGACGACGGGCAAATATGAGATTTTGGGTCTTAAAT ATGCAGATTTGCAATTTGATAAAGGAACTGGGACATACAAAATTTCACAA GAAAAATACAATGACATTAAGAAAAAGGAGGGGGTAGACAGTGATTCAGA GTTTAAGTTTACCTTATATAAAAATGATCTTTTGTTAGTCAAGGACACCG AGACGAAGGAACAACAGCTGTTTCGCTTCTTATCGCGCACAATGCCTAAA CAAAAACATTATGTTGAACTGAAGCCCTACGACAAACAAAAATTTGAGGG AGGTGAAGCATTAATCAAGGTCCTTGGGAATGTAGCTAACTCGGGCCAGT GTAAGAAGGGTCTTGGCAAATCTAACATCTCTATCTATAAGGTTCGCACG GATGTCTTGGGAAACCAACATATCATTAAGAACGAAGGAGACAAACCAAA GTTGGACTTCTAAGCTTAAAAAAAAAAAAGGCCTCCCAAATCGGGGGGCC TTTTTTATTGATAACAAAACGCTTGCCGGAGCTGATAGCTAGCTCAGTCC TAGGGATTATGCTAGCTACTAGAGAAAGAGGAGAAATACAAATGACCAAG TCTGAAACATTCATGATTCCAAACCACAAGGCCGCCAAATTAAGTGAGCT GGATATGATGATCGTTAACTCTGTCCCGCCTGGGGGAAACTGGAAGAATA TTCCCTTGGATGTACCATCGAAACGTATCGAACAGATTCGTGACAGCTAT GCTCAAGGAAAAGGGTCGCGCAGCACATACTACGGGCGTTTATTGCCCGA TATGCCAGCTTATACGATCAATACTTATTTCAATCGTCCTGGCAACGGAT GCCACATTCACTACGAGCAAGATCGCGTACTTTCACAACGCGAAGCTGCA CGTCTGCAGTCGTTCCCTGATGACTTTATCTTTTTTGGAGGTCAAACGGC GATTAATACGCAAATCGGTAATGCCGTGCCTCCCTTTCTTGCGTTTCTTA TTGCAAAAGAAATTGAAAAAGCGATCGGTAATACCGGCTACTACATTGAC TTATTCAGTGGTGCAGGCGGATTGGGGTTGGGCTTTAAGTGGGCCGGGTG GACTCCATTGTTAGCTAATGACATTGAGGAAAAGTACTTACAGACATACT CGAACAACGTACACAAAGAAGTTTTGTGCGGAAGCATTTCGGACAACGAA ACTTTTTCTAAGATCGCAGACAAGATTTCTGGCTTTAAGAAATTATATTT TGATAAACAGCTGTGGATTCTGGGCGGGCCTCCGTGCCAGGGATTTAGCA CGGCTGGCAACGCGCGTACAATGGACGACCCACGCAACAGTCTGTTTATG CACTACAAGTCGCTGCTTAACGAGATTAAGCCGAATGGATTCATTTTCGA GAACGTCGCCGGCCTGTTGAACATGGAAAAAGGAAAGGTCTTTGAACGTG TTAAGGAGGAATTCTCGTCCACAATGAAAACCATGAATGGTTGGATTTTA AATTCGGAACATTACGCAATTCCACAACGCCGTAAGCGTGTAATTCTTGT GGGCAGCAATGATCCGCTGTTCTCGATCGAACCACCTCAGAAGCTGACGG AAGATAAAGAGTCTTGGGTGTCAGTAAAAGATGCGTTATCTGACCTTCCC CCATTACAACACGGCGAGGATGGATCTGGTAAATACTATATCCACCACCC GGAAAATGATTACCAGTTGTTTATGCGTGGAAACATTACACCCTCAGAGT ATTATGAACGCAACATTAAGCCGTCGCTTTAAGCTTGACGAACAATAAGG CCTCCCAAATCGGGGGGCCTTTTTATTTTTCAACAAAACGCTACTAACTG TCTATGCCTGGGAAAGGGTGGGCAGGAGATGGGGCAGTGCAGGAAAAGTG GCACTATGAACCCTGCAGCCCTAGTTTGACAATTAATCATTGGCATAGTA TATCTGCATAGTATAATACAACTCACTATAGCAATTGTACTAACCTTCTT CTCTTTCCTCTCCTGACAGGAGGAGCCATCATGGCCAAGTTGACCAGTGC CGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGA CCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGT GTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGT GGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGC TGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCC GGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGC CCTGCGCGACCCGGCCGGCAACTGCGTGCACTTTGTGGCAGAGGAGCAGG ACTGATTACCTGTAACAGAGCATTAGCGCAAGGTGATTTTTGTCTTCTTG CGCTAATTTTTTCTGGGTTGTTTTACTGCTTCTGCTGGTGGGATTTTTCG TCCTGGAACCGCTCGGCATTCCGGTGAGCGCCATTGCAGCTGTGGGCGCG CTGATATTATTTGTCGTCGCTAAACGCGGTCATGCGATTAATACGGGTAA AGTCCTGCGCGGTGCCCCCTGGCAGATTGTCATCTTCTCGCTCGGCATGT ATCTGGTGGTTTATGGCCTGCGCAATGCCGGATTAACGGAATATCTTTCT GGCGTACTCAACGTGCTGGCGGATAACGGCCTGTGGGCCGCGACGCTCGG CACCGGATTCCTCACCGCCTTCCTCTCTTCTATTATGAACAATATGCCGA CGGTACTGGTTGGCGCGTTGTCCATTGATGGCAGCACGGCATCTGGCGTT ATCAAAGAAGCGATGGTTTATGCCAATGTGATTGGCTGCGATTTGGGACC GAAAATTACCCCAATTGGTAGCCTGGCTACGCTACTCTGGCTGCACGTAC TTTCGCAGAAGAATATGACTATCAGCTGGGGATATTACTTCCGTACAGGG ATTATCATGACCCTGCCTGTGCTGTTTGTGACGCTGGCTGCGCTGGCGCT ACGTCTCTCTTTCACTTTGTAATGAGATACTGATATGAGCAACATTACCA TTTATCACAACCCGGCCTGCGGCACGTCGCGTAATACGCTGGAGATGATC CGCAACAGCGGCACAGAACCGACTATTATCCATTATCTGGAAACTCCGCC AACGCGCGATGAACTGGTCAAACTCATTGCCGATATGGGGATTTCCGTAC GCGCGCTGCTGCGTAAAAACGTCGAACCGTATGAGGAGCTGGGCCTTGCG GAAGATAAATTTACTGACGATCGGTTAATCGACTTTATGCTTCAGCACCC GATTCTGATTAATCGCCCGATTGTGGTGACGCCGCTGGGAACTCGCCTGT GCCGCCCTTCAGAAGTGGTGCTGGAAATTCTGCCAGATGCGCAAAAAGGC GCATTCTCCAAGGAAGATGGCGAGAAAGTGGTTGATGAAGCGGGTAAGCG CCTG

[0664] Further Plasmid Sequences

[0665] >pET-M.Osp807II (for expression of recombinant M.Osp807II with N terminal His tag) (SEQ ID NO: 127)

TABLE-US-00038 TTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATG TCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAAT GTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTA TCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAA GGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTT GCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGT AAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGG ATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTT CCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCG TGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGA ATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGC ATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACAC TGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCG CTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAA CCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCC TGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTA CTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTT GCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGA TAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGG GGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGT CAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTC ACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTT AGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATC CTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCA CTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTT TTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCA GCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGT AACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGC CGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTC GCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTG TCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGT CGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACC TACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCT TCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTAT AGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATG CTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTT TACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCG TTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGA TACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGG AAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGT ATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCC GCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATG GCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTG TCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCT GCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTG CGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTG TTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGC TTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACT GATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGAT GAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCC GGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGG GACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGA TGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGA ACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACA CGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCA GCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAAC CAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACG ATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGATGCGCCGCGT GCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGG TTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGA GTGGTGAATCCGTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTG GCCCGGCTCCATGCACCGCGACGCAACGCGGGGAGGCAGACAAGGTATAG GGCGGCGCCTACAATCCATGCCAACCCGTTCCATGTGCTCGCCGAGGCGG CATAAATCGCCGTGACGATCAGCGGTCCAGTGATCGAAGTTAGGCTGGTA AGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCTG CCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGA GAAGAATCATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGC AAGACGTAGCCCAGCGCGTCGGCCGCCATGCCGGCGATAATGGCCTGCTT CTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGG CGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTC CAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTG TCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAG TCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAG GGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAAT TGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGC TGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGG CGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGC CCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTT TGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATA ACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCAC CAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATC TGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCAT TTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTT CCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCC AGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGAT TTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGT CTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCA AGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATC CTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGA GAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACC ATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGC CGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGC CAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGA ATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGC AGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGA CACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACC ACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGT TTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGAC TCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGC CGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGG CCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCG AAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCC AGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGT AGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGG AATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTT AAGAAGGAGATATACCATGGGCAGCAGCCATCATCATCATCATCACAGCA GCGGCCTGGTGCCGCGCGGCAGCCATATGGCAAAGAAGGACACAAACCTT AAGCTTTCCCACCTTTATAAGGGTAACGTATCGGAAGTCTACGGGCGCTG GCCTAGTCCGGACTTAATTGTATCGGATGGGGCATACGGTGTTCGTGGAT TTCGCGGGGACACCGTTGACGCTGCCGGGTTGACGGACTGGTATAAGCCA CACGTGCTGGCGTGGGCCAAAGCGGCCAAGCCTTCCACTTCTTTGTGGTT TTGGAACACAGAAGTAGGCTGGGCCACGGTCCACCCTTTATTATTGTCCA CCGGATGGGAATACGTGCAGTTAGCTGTCTGGGATAAAGGACTGGCGCAC ATTGCCGGCAATGTTAACGGGAAAACAATCCGTCAATTGCCCGTGGTAAC AGAGGTGGCGGCCCTTTATCGCCGTACGGTGTACCTGGAAACTGGAGACG GACTTACATTAAACGCAAAGTCATGGTTGCGCGCAGAGTGGCGTCGCTCG GGCCTGTCCCTTTCAAAGTCGAATGAAGCGTGTGGTGTGAAGAACGCTGC GACCCGTAAATATTTGACAGCAGACTGGCTGTGGTACTGGCCGCCTGGTG ACGCGGTCCAAAAGATGGCGGAGTACTGCATGCAATATGGTAAAAAAACT TCTTGGCCCTATTTCAGTCTGGATGGCAAAACTATGATTTCAGCTCATGA TTGGGATAGTTTACGCACTACATGGAATCACCGCAATGGAGTCACAAACG TGTGGAGCCGTCCGCCGTTGGCCGATAGTGAGCGTTTGAAAGGGACAATG GAACGCTCCGCACCTCGCACGTATAAACCCACCAAACAGTCAGCAGCACA TCTGAACCAAAAGCCCCTGGACCTGATGCTTACTCAGGTAGCAGCAGCCA GTAATGTCGGCGATACAGTCTGGGAACCATTTGGAGGTCTGTGCTCGGCT AGTGTGGCATCGTCATTACTTGGTCGCCGCAGTTACGCTGCAGAAATTGA CGACACATTTTACAAGTTAGCAGCGGCCCGCCTGAATGAGGCAAATGCCT ATTTTGAATCGAACGGTGTTTACGAGTTTAAAGAAGGGGAATAAGGATCC GGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTG AGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGT TTTTTGCTGAAAGGAGGAACTATATCCGGATATCCCGCAAGAGGCCCGGC AGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCC GAGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGC GTTAGCAATTTAACTGTGATAAACTACCGCATTAAAGCTTATCGATGATA AGCTGTCAAACATGAGAA

[0666] >pET-M2.Eco31I (for expression of recombinant M2.Eco31I with 7 amino acid N terminal truncation and with C terminal His tag) (SEQ ID NO: 128)

TABLE-US-00039 TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGG TGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCT CCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCG TCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTAC GGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGG CCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTT CTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCT CGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGG TTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAAT ATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAA CCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATG AATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTAT TCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAAT GAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTA TCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCC CCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACT GAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTC AACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAAC CGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTG TTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACAC TGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATA CCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCA TCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGT CAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTAC CTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAAT CGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATA CCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAG ACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATG TAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTT CGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGA GATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACC GCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTC CGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTA GTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTAC ATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATA AGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCG CAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCG AACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCG CCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGG GTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTA TCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTT TGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCG GCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTT TCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGT GAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTG AGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCT GTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTC TGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTG GGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGAC GGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCC GGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAG GCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGT CTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAAT GTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTT GGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGA TACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAA CATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGAT GCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTA ATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAG ATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTA CGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACG TTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTC TGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAG GAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCT GCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCG AGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGC GCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCA CCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACG ATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCT CAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACAT TAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGC CAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTAT TGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGA TTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCT GGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGA TATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATGTCC GCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGC CATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCA GCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCC CGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCC AGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCG CGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTA CCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGAC ATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGG CATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGC GCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTC TACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAA TCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCA ACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTT GGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTT TCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAA GAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATT CACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAA AGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATG CGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCA CCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCC CCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAG CCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGG CGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCG GCGTAGAGGATCGAGATCGATCTCGATCCCGCGAAATTAATACGACTCAC TATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGT TTAACTTTAAGAAGGAGATATACATATGATTCCAAACCACAAGGCCGCCA AATTAAGTGAGCTGGATATGATGATCGTTAACTCTGTCCCGCCTGGGGGA AACTGGAAGAATATTCCCTTGGATGTACCATCGAAACGTATCGAACAGAT TCGTGACAGCTATGCTCAAGGAAAAGGGTCGCGCAGCACATACTACGGGC GTTTATTGCCCGATATGCCAGCTTATACGATCAATACTTATTTCAATCGT CCTGGCAACGGATGCCACATTCACTACGAGCAAGATCGCGTACTTTCACA ACGCGAAGCTGCACGTCTGCAGTCGTTCCCTGATGACTTTATCTTTTTTG GAGGTCAAACGGCGATTAATACGCAAATCGGTAATGCCGTGCCTCCCTTT CTTGCGTTTCTTATTGCAAAAGAAATTGAAAAAGCGATCGGTAATACCGG CTACTACATTGACTTATTCAGTGGTGCAGGCGGATTGGGGTTGGGCTTTA AGTGGGCCGGGTGGACTCCATTGTTAGCTAATGACATTGAGGAAAAGTAC TTACAGACATACTCGAACAACGTACACAAAGAAGTTTTGTGCGGAAGCAT TTCGGACAACGAAACTTTTTCTAAGATCGCAGACAAGATTTCTGGCTTTA AGAAATTATATTTTGATAAACAGCTGTGGATTCTGGGCGGGCCTCCGTGC CAGGGATTTAGCACGGCTGGCAACGCGCGTACAATGGACGACCCACGCAA CAGTCTGTTTATGCACTACAAGTCGCTGCTTAACGAGATTAAGCCGAATG GATTCATTTTCGAGAACGTCGCCGGCCTGTTGAACATGGAAAAAGGAAAG GTCTTTGAACGTGTTAAGGAGGAATTCTCGTCCACAATGAAAACCATGAA TGGTTGGATTTTAAATTCGGAACATTACGCAATTCCACAACGCCGTAAGC GTGTAATTCTTGTGGGCAGCAATGATCCGCTGTTCTCGATCGAACCACCT CAGAAGCTGACGGAAGATAAAGAGTCTTGGGTGTCAGTAAAAGATGCGTT ATCTGACCTTCCCCCATTACAACACGGCGAGGATGGATCTGGTAAATACT ATATCCACCACCCGGAAAATGATTACCAGTTGTTTATGCGTGGAAACATT ACACCCTCAGAGTATTATGAACGCAACATTAAGCCGTCGCTTAAGCTTGC GGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACA AAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTA GCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAA AGGAGGAACTATATCCGGAT

[0667] >pET-M2.BsaI (for expression of M2.BsaI with C terminal His tag) (SEQ ID NO: 129)

TABLE-US-00040 TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGG TGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCT CCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCG TCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTAC GGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGG CCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTT CTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCT CGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGG TTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAAT ATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAA CCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATG AATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTAT TCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAAT GAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTA TCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCC CCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACT GAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTC AACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAAC CGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTG TTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACAC TGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATA CCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCA TCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGT CAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTAC CTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAAT CGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATA CCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAG ACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATG TAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTT CGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGA GATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACC GCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTC CGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTA GTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTAC ATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATA AGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCG CAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCG AACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCG CCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGG GTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTA TCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTT TGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCG GCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTT TCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGT GAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTG AGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCT GTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTC TGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTG GGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGAC GGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCC GGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAG GCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGT CTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAAT GTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTT GGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGA TACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAA CATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGAT GCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTA ATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAG ATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTA CGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACG TTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTC TGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAG GAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCT GCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCG AGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGC GCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCA CCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACG ATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCT CAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACAT TAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGC CAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTAT TGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGA TTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCT GGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGA TATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATGTCC GCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGC CATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCA GCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCC CGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCC AGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCG CGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTA CCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGAC ATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGG CATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGC GCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTC TACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAA TCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCA ACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTT GGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTT TCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAA GAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATT CACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAA AGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATG CGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCA CCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCC CCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAG CCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGG CGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCG GCGTAGAGGATCGAGATCGATCTCGATCCCGCGAAATTAATACGACTCAC TATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGT TTAACTTTAAGAAGGAGATATACATATGATTCCCAACCACGTATCTTCCA AGTTAAGTGAACTTGACATGCTTATCATTAAACACGTGCCGCCCGGAGGA AATTGGAAAGACATCCCCGAATGGGTGCCGTCTAAGCGTTTGGAGCAAAT TCGTAAGTCTTATGCTGAGGGAAAAGGATCACGCTCAACTTACTATGGCC GCCTGTTACCAGATATGCCAAGCTACACTATCAACACGTACTTCAACCGT CCAGGTAATGGGTGCCACATCCATTATGAACAGGATCGCACTCTGTCACA GCGTGAGGCCGCGCGCTTGCAGTCTTTCCCTGACGATTTCATCTTCTACG GTAGCAAGACGGCCATCAACAACCAGATCGGCAACGCTGTTCCGCCCCTT CTTGCTTATCAGATCGCCAAGGCATTTCCCTTCAAAGGCCAATTTGTCGA TCTTTTTAGTGGGGCGGGGGGCCTTTCCTTAGGTTTCCTTTGGGCCGGGT GGAAACCGATCATCGCAAATGACATCGATAAATGGGCGCTGACCACCTAC ATGAATAATATTCACAATGAAGTAGTGCTTGGGGATATCCGCGACGAAAA GGTCAGTGAGACTATCATTCAAAAGTGCCTGATCGCAAAGAAGTCGAACC CGGACCGTCCATTGTTTGTTCTTGGGGGACCACCCTGCCAAGGGTTTTCG ACTGCAGGAAAAAAACGTTCAATCGTGGACGAGCGCAACTGGCTGTTCGA AAGTTACGTGAGTATCCTGAAAGAAGTCAAGCCAGACGGTTTTATTTTTG AAAACGTGACTGGCCTGTTGTCGATGGAGAAGGGAGCATTCTTTGAGATG GTTAAATCGGAATTGTCTAAGACGGTGTCGAATCTTTTCGTATATAAACT TAATTCGGTGGATTACGGTGTGCCCCAACGCCGCAACCGCGTAGTCATCA TCGGGGACTCCACCGGAACGAAGAACAGCGAGCCCCCTATCCCAATTACG TCTCTTAAAGGAGAAAAGACCCTGTTTGACGCCTTAAGCAGTGCCATTTC CGTAAAGGAGGCCCTTTCAGACTTACCCCTTTTGTCGCCTAATGAGGACG GCTCTTGGAAGAACTACGTTTGTGAGCCACAAAATATTTATCAGTCATTC ATGCGCAAGAAGATCACAGCCCAGCAGTATATCGAGATGCTGTCCTCCTT AGCTATCATTAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACT GAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCC ACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTT GAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT