FUSION SINGLE-STRANDED DNA POLYMERASE BST, NUCLEIC ACID MOLECULE ENCODING FUSION DNA POLYMERASE NEQSSB-BST, METHOD OF PREPARATION AND UTILISATION THEREOF

Abstract

The subject of the invention is the fusion single-stranded DNA polymerase Bst linked with NeqSSB protein at the N-end of the polymerase using the linker consisting of six amino acids with the amino acid sequence Gly-Ser-Gly-Gly-Val-Asp, wherein the given polymerase is present in three different variants, and the preparation method thereof. Moreover, the subject of the invention is the nucleic acid molecule encoding the fusion DNA polymerase NeqSSB-Bst Full Length, Large Fragment, Short Fragment and their utilisation.

Claims

1. A fusion polymerase of single-stranded DNA polymerase Bst or another polymerase of this class of DNA polymerases connected with NeqSSB protein or a protein with a sequence similar to NeqSSB in the degree not higher than 50% at N-end of the polymerase using a linker of an exemplary amino acid sequence Gly-Ser-Gly-Gly-Val-Asp or fused directly without a linker, wherein the mentioned polymerase is present in three different variants.

2. Fusion The fusion DNA polymerase NeqSSB-Bst according to claim 1, wherein it contains one of the three variants of Bst polymerase: Full length—whole amino acid sequence of DNA polymerase|Bst with disabled 5′-3′ activity thanks to the point mutation; Large Fragment—DNA polymerase|Bst without 5′-3′ domain; Short Fragment—short version with the deletion of both exonucleolytic domains.

3. Fusion The fusion DNA polymerase NeqSSB-Bst according to claim 1, wherein it binds to all types of DNA and RNA.

4. Fusion The fusion DNA polymerase NeqSSB-Bst according to claim 1, comprising the sequence presented in SEQ1.

5. The fusion DNA polymerase NeqSSB-Bst according to claim 1, comprising the sequence presented in SEQ2.

6. The fusion DNA polymerase NeqSSB-Bst according to claim 1, comprising the sequence presented in SEQ3.

7. A nucleic acid molecule encoding the fusion DNA polymerase NeqSSB-Bst Full Length presented in SEQ 4, the fusion DNA polymerase NeqSSB-Bst Large Fragment presented in SEQ 5, or the fusion DNA polymerase NeqSSB-Bst Short Fragment presented in SEQ 6SEQ 6.

8-9. (canceled)

10. The nucleic acid molecule encoding the fusion DNA polymerase NeqSSB-Bst according to claim 7.

11. The A preparation method of the fusion DNA polymerase NeqSSB-Bst defined in claim 1 wherein: a first step includes the expression of the gene encoding the enzyme in the optimised conditions in microbiological shaker: growth temperature 28-37° C., incubation time of the medium after induction—3-20 h, inductor concentration—0.1-1 mM IPTG, obtained cell lysate is subjected to the disintegration using ultrasound and elimination of the DNA genomic contamination using dsDNase, a second purification step utilises the metal affinity chromatography with His-Trap beads, next steps cover triple dialysis (10 mM Tris-HCl pH 7.1, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, 0.1% Triton X-100), gel filtration, and concentration of the preparation, all processes were conducted in 4° C., the purity of the obtained proteins was tested using SDS-PAGE electrophoresis, and the number of units for the obtained preparation was determined using EvaEZ Fluorometric Polymerase Activity Assay Kit.

12. (canceled)

Description

DESCRIPTION OF THE SEQUENCES AND FIGURES

[0029] Seq. 1—presents the amino acid sequence of the fusion polymerase NeqSSB-Bst Full length

[0030] Seq. 2—presents the amino acid sequence of the fusion polymerase NeqSSB-Bst Large Fragment

[0031] Seq. 3—presents the amino acid sequence of the fusion polymerase NeqSSB-Bst Short Fragment

[0032] Seq. 4—presents the sequence of the gene encoding fusion DNA polymerase NeqSSB-Bst Full Length

[0033] Seq. 5—presents the sequence of the gene encoding fusion DNA polymerase NeqSSB-Bst Large Fragment

[0034] Seq. 6—presents the sequence of the gene encoding fusion DNA polymerase NeqSSB-Bst Short Fragment

[0035] FIG. 1—presents the electrophoretic 10% polyacrylamide gel separation of proteins from individual stages of the fusion DNA polymerase purification [0036] M—Protein mass marker (Thermo-Fischer Scientific) with the masses of the standard proteins: 116; 66.2; 45; 35; 25; 18.4; 14.4 kDa; [0037] 1—whole cell-free extract of the recombinant Escherichia coli TOP10F′-pETNeqSSB-Bst strain; [0038] 2—whole cell-free extract subjected to the preliminary thermal denaturation [0039] 3—fraction not bound with His-Trap column; [0040] 4—wash fraction of His-Trap beads containing 40 mM imidazole [0041] 5—wash fraction of His-Trap beads containing 100 mM imidazole [0042] 6—collected fraction containing fusion DNA polymerase after elution using 500 mM imidazole

[0043] FIG. 2—presents the charts concerning dependence of the EvaGreen dye fluorescence on time starting from the DNA amplification for the fusion DNA polymerases which enables the calculation of the number DNA polymerase units. The legend assigns the amounts of used DNA polymerase for the reaction in microliters to the curves.

[0044] FIG. 3—presents the electrophoretic 10% polyacrylamide gel separation of lysates for various expression conditions [0045] M—Protein mass marker (Thermo-Fischer Scientific) with the masses of the standard proteins: 116; 66.2; 45; 35; 25; 18.4; 14.4 kDa; [0046] 1—whole cell-free extract of the recombinant Escherichia coli TOP10F′-pETNeqSSB-Bst strain before induction; [0047] 2—whole cell-free extract 3 h after induction with 1 mM IPTG, expression conducted in 28° C.; [0048] 3—whole cell-free extract 4 h after induction with 1 mM IPTG, expression conducted in 28° C. [0049] 4—whole cell-free extract 5 h after induction with 1 mM IPTG, expression conducted in 28° C. [0050] 5—whole cell-free extract 6 h after induction with 1 mM IPTG, expression conducted in 28° C. [0051] 6—whole cell-free extract 20 h after induction with 1 mM IPTG, expression conducted in 28° C. [0052] 7—whole cell-free extract 3 h after induction with 0.1 mM IPTG, expression conducted in 28° C.; [0053] 8—whole cell-free extract 4 h after induction with 0.1 mM IPTG, expression conducted in 28° C. [0054] 9—whole cell-free extract 5 h after induction with 0.1 mM IPTG, expression conducted in 28° C. [0055] 10—whole cell-free extract 6 h after induction with 0.1 mM IPTG, expression conducted in 28° C. [0056] 11—whole cell-free extract 20 h after induction with 0.1 mM IPTG, expression conducted in 28° C. [0057] 12—whole cell-free extract of the recombinant Escherichia coli TOP10F′-pETNeqSSB-Bst strain before induction; [0058] 13—whole cell-free extract 3 h after induction with 1 mM IPTG, expression conducted in 37° C.; [0059] 14—whole cell-free extract 4 h after induction with 1 mM IPTG, expression conducted in 37° C. [0060] 15—whole cell-free extract 5 h after induction with 1 mM IPTG, expression conducted in 37° C. [0061] 16—whole cell-free extract 6 h after induction with 1 mM IPTG, expression conducted in 37° C. [0062] 17—whole cell-free extract 20 h after induction with 1 mM IPTG, expression conducted in 37° C. [0063] 18—whole cell-free extract of the recombinant Escherichia coli TOP10F′-pETNeqSSB-Bst strain before induction; [0064] 19—whole cell-free extract 3 h after induction with 0.1 mM IPTG, expression conducted in 37° C.; [0065] 20—whole cell-free extract 4 h after induction with 0.1 mM IPTG, expression conducted in 37° C. [0066] 21—whole cell-free extract 5 h after induction with 0.1 mM IPTG, expression conducted in 37° C. [0067] 22—whole cell-free extract 6 h after induction with 0.1 mM IPTG, expression conducted in 37° C. [0068] 23—whole cell-free extract 20 h after induction with 0.1 mM IPTG, expression conducted in 37° C.

[0069] FIG. 4—shows the graphs presenting the change in the activity of the fusion DNA polymerases with the increase in temperature comparing with the reference DNA polymerase|Bst. The blue line presents the result of DNA polymerase|Bst, the red line for fusion DNA polymerase Bst Full length, violet line for fusion DNA polymerase Bst Large Fragment, and the green line for the fusion DNA polymerase Bst Short Fragment. The activity is described using the GelAnalyzer program based on the intensity of the obtained PCR products in an agarose gel.

[0070] FIG. 5—shows the electrophoretic separation in 1.5% agarose gel with ethidium bromide presenting the comparison of the processivity of DNA polymerases defined as the amplification rate during isothermal PCR. The reactions were conducted in various periods of time what is indicated over the lines.

[0071] FIG. 6—shows the electrophoretic separation in 1.5% agarose gel presenting the comparison of the DNA polymerase resistance to the inhibitors: blood lactoferrin (A), soil polyphenols (B).

A:

[0072] A: 1—the reaction product generated as the result of the DNA amplification with added 6 μg of lactoferrin

[0073] 2—the reaction product generated as the result of the DNA amplification with added 0.6 μg of lactoferrin

[0074] 3—the reaction product generated as the result of the DNA amplification with added 0.06 μg of lactoferrin

[0075] 4—the reaction product generated as the result of the DNA amplification with added 6 ng of lactoferrin

[0076] K+ reaction product generated during DNA amplification without the addition of an inhibitor.

B:

[0077] 1—the reaction product generated as the result of the DNA amplification with added 100 μg of polyphenols

[0078] 2—the reaction product generated as the result of the DNA amplification with added 10 μg of polyphenols

[0079] 3—the reaction product generated as the result of the DNA amplification with added 1 μg of polyphenols

[0080] 4—the reaction product generated as the result of the DNA amplification with added 0.1 μg of polyphenols

[0081] 5—the reaction product generated as the result of the DNA amplification with added 0.01 μg of polyphenols

[0082] K+ reaction product generated during DNA amplification without the addition of an inhibitor.

[0083] FIG. 7—shows the electrophoretic separation in 2% agarose gel with ethidium bromide presenting the results of the DNA electrophoretic mobility shift assay in the presence of the fusion DNA polymerases. The reaction mixture contained 10 pmol (dT.sub.76) of fluorescein-labelled (green) and 2.5 pmol PCR product of 100 bp (orange)

1—d(T).sub.76
2—100 bp
3—d(T).sub.76+100 bp+3.3 pmol of the fusion DNA polymerase
4—d(T).sub.76+100 bp+6.6 pmol of the fusion DNA polymerase
5—d(T).sub.76+100 bp+13.2 pmol of the fusion DNA polymerase
6—d(T).sub.76+100 bp+26.4 pmol of the fusion DNA polymerase
7—d(T).sub.76+100 bp+52.8 pmol of the fusion DNA polymerase
8—d(T).sub.76+100 bp+105.6 pmol of the fusion DNA polymerase
9—d(T).sub.76+100 bp+211.2 pmol of the fusion DNA polymerase

[0084] The invention is illustrated by the embodiment, including but not limited to.

EXAMPLE

Fusion DNA Polymerase NeqSSB-Bst

[0085] Fusion DNA polymerases NeqSSB-Bst were obtained by fusion of three various Bst polymerases with NeqSSB protein at polymerase N-end using the linker consisting of six amino acids of the following sequence: Gly-Ser-Gly-Gly-Val-Asp. The sequences of the three variants of fusion DNA polymerase are presented in the figure SEQ. 1-3 (amino acid sequences) and SEQ. 4-6 (nucleotide sequences). DNA polymerases were obtained in the laboratory scale in the prokaryotic system based on Escherichia coli bacteria.

Preparation—Example 1

[0086] First step of the DNA polymerase preparation includes the expression of the gene encoding the enzyme in the optimised conditions in microbiological shaker: growth temperature—30° C., incubation time of the medium after induction—3 to 20 h, inductor concentration—0.1 to 1 mM IPTG. In the protein purification process the obtained cell lysate is subjected to the disintegration using ultrasound and removal of the DNA genomic contamination using dsDNase. Thanks to the presence of the oligohistidine domain, the second purification step utilises the metal affinity chromatography with His-Trap beads (FIG. 1). The next steps cover triple dialysis until obtaining the conditions which provide the stability for DNA polymerase (10 mM Tris-HCl pH 7.1, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, 0.1% Triton X-100), gel filtration, and densification of the preparation. All the processes were conducted in 4° C. The purity of the obtained proteins was tested using SDS-PAGE electrophoresis, and the number of units for the obtained preparation was determined using EvaEZ Fluorometric Polymerase Activity Assay Kit Biotium (USA) according to the definition of unit: 1 activity unit [1 U] is the amount of DNA polymerase which can incorporate 10 nmol of nucleotides in 30 min in its optimum operation temperature 65° C. (FIG. 2). 1 litre of the laboratory scale culture provides approx. 5 mg of purified preparation with the activity of approx. 10 000 U, what enables the respective number of amplification reactions.

Preparation—Example 2

[0087] Expression of the gene encoding the fusion DNA polymerase was conducted in the conditions providing appropriate oxygenation of the liquid culture in the temperature of 28° C. Logarithmic-phase cultures were induced using IPTG with the amount providing the protein expression—IPTG in the range of 1 mM to 0.1 mM and incubation of 3 to 20 hours (FIG. 3). After that the cell lysate was mechanically disintegrated and purified using metal affinity chromatography and ion-exchange chromatography. The obtained fusion DNA polymerases were subjected to the dialysis for the storage conditions (10 mM Tris-HCl pH 7.1, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, 0.1% Triton X-100) and provided in the concentration of 1 U/μL based on the commercial EvaEZ Fluorometric Polymerase Activity Assay Kit of Biotium (USA) according to the definition of unit.

Preparation—Example 3

[0088] An efficient expression of the gene encoding the polymerase Bst fused with NeqSSB protein was obtained in culture in 37° C. and induction of IPTG in range of 1 mM to 0.1 mM for 3 to 20 hours (FIG. 3). Centrifuged and mechanically disrupted cell lysate was purified using chromatographic techniques (metal affinity chromatography and ion-exchange chromatography), suspended in the formulation buffer (10 mM Tris-HCl pH 7.1, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, 0.1% Triton X-100) and provided in the concentration of 1 U/μL. The amount of DNA units was defined based on the definition of unit using the EvaEZ Fluorometric Polymerase Activity Assay Kit of Biotium (USA).

[0089] The comparative analysis of the properties of the enzymes being the subject of the invention with the reference DNA polymerase Bst have shown that the presence of an additional DNA-binding NeqSSB protein has a positive impact on the DNA polymerase properties. The thermostability of the all obtained fusion variants of the DNA polymerases in comparison with the reference DNA polymerase Bst was increased by approx. 20% (FIG. 4). Moreover, the DNA polymerases in fusion with NeqSSB protein shown the threefold increase of processivity (FIG. 5). The fusion DNA polymerases tolerate the concentration of clinical (lactoferrin, heparin) and environmental (humic acid, soil, polyphenols) inhibitors in the reaction mixtures increased even by several dozen of times comparing with the reference polymerase (FIG. 6). The fusion DNA polymerases exhibited increased sensitivity by several times and thus increased affinity to the DNA matrix comparing with the reference DNA polymerase Bst.

BIBLIOGRAPHY

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TABLE-US-00001 Nucleotide and amino acid sequence SEQ. 1 MDEEELIQLIIEKTGKSREEIEKMVEEKIKAFNNLISRRGALLLVAKKLGVLYKNTPKEKKIGELESWEYVKVKG KILKSFGLISYSKGKFQPIILGDETGTIKAIIWNTDKELPENTVIEAIGKTKINKKTGNLELHIDSYKILESDLE IKPQKQEFVGICIVKYPKKQTQKGTIVSKAILTSLDRELPVVYFNDFDWEIGHIYKVYGKLKKNIKTGKIEFFAD KVEEATLKDLKAFKGEADGSGGVDLKNKLVLIDGNSVAYRAFFALPLLHNDKGIHTNAVYGFTMMLNKILAEEQPT HILVAFDAGKTTFRHETFQDAKGGRQQTPPELSEQFPLVRELLKAYRIPAYELDHYEADDIIGTMAARAEREG FAVKVISGDRDLTQLASPQVIVEITKKGITDIESYTPETVVEKYGLIPEQIVDLKGLMGDKSDNIPGVPGIGKK TAVKLLKQFGTVENVIASIDEIKGEKLKENLRQYRDLALLSKCILAAICRDAPVELTLDDIVYKGEDREKVVALF QELGFQSFLDKMAVQTDEGEKPLAGMDFAIADSVTMEMLADKAALVVEVVGDNYHHAPIVGIALANERGR FFLRPETAVADPKFLAWLGDETKKKTMFDSKRAAVALNGKGIELAGVGVVFDLLLAAYLLDPAQAAGDVAA VAKMHQYEAVRSDEAVYGKGAKRTVPDEPTLAEQLVRKAAAIWALEEPLMDELRRNEQDRLLTELEHALA GILANMEFTGVKVDTKRLEQMGAELTEQLQAVERRIYELAGQEFNINSPKQLGTVLFDKLQLPVLKKTKTGY STSADVLEKLAPHHEIVEHILHYRQLGKLQSTYIEGLLKVVHPVTGKVHTMFNQALTQTGRLSSVEPNLQNIPI RLEEGRKIRQAFVPSEPDWLIFAADYSQIELRVLAHIAEDDNLIEAFRRWLDIHTKTAMDIFHVSEEDVTANM RRQAKAVNFGIVYGISDYGLAQNLNITRKEAAEFIERYFASFPGVKQYMDNIVQEAKQKGYVTILLHRRRYLP DITSRNFNVRTFAERTAMNTPIQGSAADIIKKAMIDLSVSVREERLQARLLLQGHDELILEAPKEEIGRLCRLVP EVMECIAVTLRVPLKVDYHYGPTVVYDAK. LENGTH: 1127 aa TYPE: amino acid sequence TYPE OF THE MOLECULAR: Protein SEQ. 2 MDEEELIQLIIEKTGKSREEIEKMVEEKIKAFNNLISRRGALLLVAKKLGVLYKNTPKEKKIGELESWEYVKVKG KILKSFGLISYSKGKFQPIILGDETGTIKAIIWNTDKELPENTVIEAIGKTKINKKTGNLELHIDSYKILESDLE IKPQKQEFVGICIVKYPKKQTQKGTIVSKAILTSLDRELPVVYFNDFDWEIGHIYKVYGKLKKNIKTGKIEFFAD KVEEATLKDLKAFKGEADGSGGVDLADKAALVVEVVGDNYHHAPIVGIALANERGRFFLRPETAVADPKFLAWLG DETKKKTMFDSKRAAVALNGKGIELAGVGVVFDLLLAAYLLDPAQAAGDVAAVAKMHQYEAVRSDEAVYG KGAKRTVPDEPTLAEQLVRKAAAIWALEEPLMDELRRNEQDRLLTELEHALAGILANMEFTGVKVDTKRLE QMGAELTEQLQAVERRIYELAGQEFNINSPKQLGTVLFDKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVEHIL HYRQLGKLQSTYIEGLLKVVHPVTGKVHTMFNQALTQTGRLSSVEPNLQNIPIRLEEGRKIRQAFVPSEPDW LIFAADYSQIELRVLAHIAEDDNLIEAFRRWLDIHTKTAMDIFHVSEEDVTANMRRQAKAVNFGIVYGISDYG LAQNLNITRKEAAEFIERYFASFPGVKQYMDNIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRTFAERTAM NTPIQGSAADIIKKAMIDLSVSVREERLQARLLLQGHDELILEAPKEEIGRLCRLVPEVMEQAVTLRVPLKVDY HYGPTVVYDAK. LENGTH: 816 aa TYPE: amino acid sequence TYPE OF THE MOLECULAR: Protein SEQ. 3 MDEEELIQLIIEKTGKSREEIEKMVEEKIKAFNNLISRRGALLLVAKKLGVLYKNTPKEKKIGELESWEYVKVKG KILKSFGLISYSKGKFQPIILGDETGTIKAIIWNTDKELPENTVIEAIGKTKINKKTGNLELHIDSYKILESDLE IKPQKQEFVGICIVKYPKKQTQKGTIVSKAILTSLDRELPVVYFNDFDWEIGHIYKVYGKLKKNIKTGKIEFFAD KVEEATLKDLKAFKGEADGSGGVDLELRRNEQDRLLTELEHALAGILANMEFTGVKVDTKRLEQMGAELTEQLQA VERRIYELAGQEFNINSPKOLGTVLFDKLQLPVLKKTKTGYSTSADVLEKLAPHHEIVEHILHYRQLGKLQSTY1 EGLLKVVHPVTGKVHTMFNQALTQTGRLSSVEPNLQNIPIRLEEGRKIRQAFVPSEPDWLIFAADYSQIELRV LAHIAEDDNLIEAFRRWLDIHTKTAMDIFHVSEEDVTANMRRQAKAVNFGIVYGISDYGLAQNLNITRKEAA EFIERYFASFPGVKQYMDNIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRTFAERTAMNTPIQGSAADIIKK AMIDLSVSVREERLQARLLLQGHDELILEAPKEEIGRLCRLVPEVMEQAVTLRVPLKVDYHYGPTWYDAK. LENGTH: 663 aa TYPE: amino acid sequence TYPE OF THE MOLECULAR: Protein SEQ. 4 ATGGATGAAGAGGAACTAATACAACTAATAATAGAAAAAACTGGCAAATCTCGAGAGGAAATAGAAA AAATGGTGGAAGAAAAAATTAAAGCTTTTAACAATTTAATATCTCGTAGGGGGGCTTTACTATTAGTAG CAAAAAAACTTGGTGTTTTGTATAAAAACACTCCGAAAGAGAAAAAAATTGGCGAATTAGAAAGCTGG GAATATGTAAAAGTAAAGGGCAAAATTCTCAAATCTTTTGGATTAATTAGTTATTCGAAAGGGAAATTC CAACCTATTATTTTAGGAGACGAAACCGGTACTATTAAAGCTATTATTTGGAATACCGATAAAGAATTA CCTGAAAACACTGTAATAGAAGCTATTGGGAAAACCAAAATTAATAAGAAAACTGGCAATTTAGAATTA CATATAGACAGTTATAAAATTTTAGAAAGCGATTTAGAGATAAAACCCCAAAAGCAAGAATTTGTTGGG ATTTGCATAGTTAAATATCCAAAAAAACAAACCCAAAAAGGCACAATAGTATCGAAAGCAATTTTAACT AGCTTAGATAGGGAATTGCCTGTAGTATATTTCAACGATTTTGATTGGGAAATAGGCCATATATATAAA GTATATGGAAAGCTTAAGAAAAACATAAAAACTGGTAAAATAGAATTTTTCGCTGACAAAGTTGAGGA AGCAACATTAAAAGATCTAAAAGTTTAAAGGAGAGGCCGATGGAAGCGGAGGGGTCGACTTGAAA AACAAGCTCGTCTTAATTGACGGCAACAGCGTGGCGTACCGCGCCTTTTTTGCGTTGCCGCTTTTGCATA ACGATAAAGGGATTCATACGAACGCAGTCTACGGGTTTACGATGATGTTAAACAAAATTTTGGCGGAA GAGCAGCCGACCCACATTCTCGTTGCGTTTGACGCCGGGAAAACGACGTTCCGCCATGAAACGTTCCAA GACGCCAAAGGCGGGCGGCAGCAGACGCCGCCGGAACTGTCGGAACAGTTTCCGCTCGTGCGCGAAT TGCTCAAAGCGTACCGCATCCCCGCCTATGAGCTCGACCATTATGAAGCGGATGACATCATCGGAACGA TGGCGGCGCGGGCTGAGCGAGAAGGGTTTGCAGTGAAAGTCATTTCCGGCGACCGCGATTTAACCCA GCTTGCTTCCCCGCAAGTGACGGTGGAGATTACGAAAAAAGGGATTACCGACATCGAGTCGTACACGC CGGAGACGGTCGTGGAAAAATACGGCCTCACCCCGGAGCAAATTGTCGACTTGAAAGGATTGATGGG CGACAAATCCGACAACATCCCTGGCGTGCCCGGCATCGGGAAAAAAACAGCCGTCAAGCTGCTCAAGC AATTCGGCACGGTCGAAAACGTACTGGCATCGATCGATGAGATCAAAGGGGAGAAGCTGAAAGAAAA TTTGCGCCAATACCGGGATTTGGCGCTTTTAAGCAAACAGCTGGCCGCTATTTGCCGCGACGCCCCGGT TGAGCTGACGCTCGATGACATTGTCTACAAAGGAGAAGACCGGGAAAAAGTGGTCGCCTTGTTTCAGG AGCTCGGATTCCAGTCGTTTCTCGACAAGATGGCCGTCCAAACGGATGAAGGCGAAAAGCCGCTCGCC GGGATGGATTTTGCGATCGCCGACAGCGTCACGGACGAAATGCTCGCCGACAAAGCGGCCCTCGTCGT GGAGGTGGTGGGCGACAACTATCACCATGCCCCGATTGTCGGGATCGCCTTGGCCAACGAACGCGGG CGGTTTTTCCTGCGCCCGGAGACGGCCGTCGCCGATCCGAAATTTCTCGCTTGGCTTGGCGATGAGACG AAGAAAAAAACGATGTTTGATTCAAAGCGGGCGGCCGTCGCGCTAAATGGGAAAGGAATCGAACTGG CTGGCGTCGGCGTCGTGTTCGATCTGTTGCTGGCCGCTTACTTGCTCGATCCGGCGCAGGCGGCGGGC GACGTTGCCG CGGTGGCGAAAATGCATCAGTACGAGGCGGTGCGATCGGATGAGGCGGTCTATGGAA AAGGAGCGAAGCGGACGGTTCCTGATGAACCGACGCTTGCCGAGCAGCTCGTCCGCAAGGCGGCGGC CATTTGGGCGCTTGAAGAGCCGTTGATGGACGAACTGCGCCGCAACGAACAAGATCGGCTGCTGACCG AGCTCGAACACGCGCTGGCTGGCATTTTGGCCAATATGGAATTTACTGGAGTGAAAGTGGACACGAAG CGGCTTGAACAGATGGGGGCGGAGCTCACCGAGCAGCTGCAGGCGGTCGAGCGGCGCATTTACGAAC TCGCCGGCCAAGAGTTCAACATTAACTCGCCGAAACAGCTCGGGACGGTTTTATTTGACAAGCTGCAGC TCCCGGTGTTGAAAAAGACAAAAACCGGCTATTCGACTTCAGCCGATGTGCTAGAAAAGCTTGCACCG CACCATGAAATCGTCGAACATATTTTGCATTACCGCCAACTCGGCAAGCTGCAGTCAACGTATATTGAA GGGCTGCTGAAAGTGGTGCACCCCGTGACGGGCAAAGTGCACACGATGTTCAATCAGGCGTTGACGC AAACCGGGCGCCTCAGCTCCGTCGAACCGAATTTGCAAAACATTCCGATTCGGCTTGAGGAAGGGCGG AAAATCCGCCAGGCGTTCGTGCCGTCGGAGCCGGACTGGCTCATCTTTGCGGCCGACTATTCGCAAATC GAGCTGCGCGTCCTCGCCCATATCGCGGAAGATGACAATTTGATTGAAGCGTTCCGGCGCTGGTTGGA CATCCATACGAAAACAGCCATGGACATTTTCCATGTGAGCGAAGAAGACGTGACAGCCAACATGCGCC GCCAAGCGAAGGCCGTCAATTTTGGCATCGTGTACGGCATTAGTGATTACGGTCTGGCGCAAAACTTG AACATTACGCGCAAAGAAGCGGCTGAATTTATTGAGCGATATTTTGCCAGTTTTCCAGGTGTAAAGCAA TATATGGACAACATTGTGCAAGAAGCGAAACAAAAAGGGTATGTGACGACGCTGCTGCATCGGCGCC GCTATTTGCCCGATATTACAAGCCGCAACTTCAACGTCCGCACGTTCGCCGAGCGGACGGCGATGAACA CACCGATCCAGGGATCCGCTGCCGACATCATTAAGAAAGCGATGATCGATCTAAGCGTGAGCGTGCGC GAAGAACGGCTGCAGGCGCGCCTGTTGCTGCAAGGTCATGACGAACTCATTTTGGAGGCGCCGAAAG AGGAAATCGGACGGCTGTGCCGCCTCGTTCCGGAAGTGATGGAGCAAGCCGTGACACTTCGCGTGCCG CTGAAAGTCGATTACCATTACGGTCCGACGTGGTACGACGCCAAATAA. LENGTH: 3384 nucleotides TYPE: nucleic acid TOPOLOGY: plasmid NUBER OF STRANDS: one TYPE OF THE MOLECULAR: DNA SEQ. 5 ATGGATGAAGAGGAACTAATACAACTAATAATAGAAAAAACTGGCAAATCTCGAGAGGAAATAGAAA AAATGGTGGAAGAAAAAATTAAAGOTTTAACAATTTAATATCTCGTAGGGGGGCTTTACTATTAGTAG CAAAAAAACTTGGTGTTTTGTATAAAAACACTCCGAAAGAGAAAAAAATTGGCGAATTAGAAAGCTGG GAATATGTAAAAGTAAAGGGCAAAATTCTCAAATCTTTTGGATTAATTAGTTATTCGAAAGGGAAATTC CAACCTATTATTTTAGGAGACGAAACCGGTACTATTAAAGCTATTATTTGGAATACCGATAAAGAATTA CCTGAAAACACTGTAATAGAAGCTATTGGGAAAACCAAAATTAATAAGAAAACTGGCAATTTAGAATTA CATATAGACAGTTATAAAATTTTAGAAAGCGATTTAGAGATAAAACCCCAAAAGCAAGAATTTGTTGGG ATTTGCATAGTTAAATATCCAAAAAAACAAACCCAAAAAGGCACAATAGTATCGAAAGCAATTTTAACT AGCTTAGATAGGGAATTGCCTGTAGTATATTTCAACGATTTTGATTGGGAAATAGGCCATATATATAAA GTATATGGAAAGUTAAGAAAAACATAAAAACTGGTAAAATAGAATTTTTCGCTGACAAAGTTGAGGA AGCAACATTAAAAGATCTAAAAGCTTTTAAAGGAGAGGCCGATGGAAGCGGAGGGGTCGACTTGGCC GACAAAGCGGCCCTCGTCGTGGAGGTGGTGGGCGACAACTATCACCATGCCCCGATTGTCGGGATCGC CTTGGCCAACGAACGCGGGCGGTTTTTCCTGCGCCCGGAGACGGCCGTCGCCGATCCGAAATTTCTCG CTTGGCTTGGCGATGAGACGAAGAAAAAAACGATGTTTGATTCAAAGCGGGCGGCCGTCGCGCTAAAT GGGAAAGGAATCGAACTGGCTGGCGTCGGCGTCGTGTTCGATCTGTTGCTGGCCGCTTACTTGCTCGA TCCGGCGCAGGCGGCGGGCGACGTTGCCGCGGTGGCGAAAATGCATCAGTACGAGGCGGTGCGATCG GATGAGGCGGTCTATGGAAAAGGAGCGAAGCGGACGGTTCCTGATGAACCGACGCTTGCCGAGCAGC TCGTCCGCAAGGCGGCGGCCATTTGGGCGCTTGAAGAGCCGTTGATGGACGAACTGCGCCGCAACGA ACAAGATCGGCTGCTGACCGAGCTCGAACACGCGCTGGCTGGCATTTTGGCCAATATGGAATTTACTG GAGTGAAAGTGGACACGAAGCGGCTTGAACAGATGGGGGCGGAGCTCACCGAGCAGCTGCAGGCGG TCGAGCGGCGCATTTACGAACTCGCCGGCCAAGAGTTCAACATTAACTCGCCGAAACAGCTCGGGACG GTTTTATTTGACAAGCTGCAGCTCCCGGTGTTGAAAAAGACAAAAACCGGCTATTCGACTTCAGCCGAT GTGCTAGAAAAGCTTGCACCGCACCATGAAATCGTCGAACATATTTTGCATTACCGCCAACTCGGCAAG CTGCAGTCAACGTATATTGAAGGGCTGCTGAAAGTGGTGCACCCCGTGACGGGCAAAGTGCACACGAT GTTCAATCAGGCGTTGACGCAAACCGGGCGCCTCAGCTCCGTCGAACCGAATTTGCAAAACATTCCGAT TCGGCTTGAGGAAGGGCGGAAAATCCGCCAGGCGTT*CGTGCCGTCGGAGCCGGACTGGCTCATCTTTG CGGCCGACTATTCGCAAATCGAGCTGCGCGTCCTCGCCCATATCGCGGAAGATGACAATTTGATTGAAG CGTTCCGGCGCTGGTTGGACATCCATACGAAAACAGCCATGGACATTTTCCATGTGAGCGAAGAAGAC GTGACAGCCAACATGCGCCGCCAAGCGAAGGCCGTCAATTTTGGCATCGTGTACGGCATTAGTGATTA CGGTCTGGCGCAAAACTTGAACATTACGCGCAAAGAAGCGGCTGAATTTATTGAGCGATATTTTGCCA GTTTTCCAGGTGTAAAGCAATATATGGACAACATTGTGCAAGAAGCGAAACAAAAAGGGTATGTGACG ACGCTGCTGCATCGGCGCCGCTATTTGCCCGATATTACAAGCCGCAACTTCAACGTCCGCACGT1CGCC GAGCGGACGGCGATGAACACACCGATCCAGGGATCCGCTGCCGACATCATTAAGAAAGCGATGATCG ATCTAAGCGTGAGCGTGCGCGAAGAACGGCTGCAGGCGCGCCTGTTGCTGCAAGGTCATGACGAACTC ATTTTGGAGGCGCCGAAAGAGGAAATCGGACGGCTGTGCCGCCTCGTTCCGGAAGTGATGGAGCAAG CCGTGACACTTCGCGTGCCGCTGAAAGTCGATTACCATTACGGTCCGACGTGGTACGACGCCAAATAA. LENGTH: 2451 nucleotides TYPE: nucleic acid TOPOLOGY: plasmid NUMBER OF STRANDS: one TYPE OF THE MOLECULAR: DNA SEQ. 6 ATGGATGAAGAGGAACTAATACAACTAATAATAGAAAAAACTGGCAAATCTCGAGAGGAAATAGAAA AAATGGTGGAAGAAAAAATTAAAGCTTTTAACAATTTAATATCTCGTAGGGGGGCTTTACTATTAGTAG CAAAAAAACTTGGTGTTTTGTATAAAAACACTCCGAAAGAGAAAAAAATTGGCGAATTAGAAAGCTGG GAATATGTAAAAGTAAAGGGCAAAATTCTCAAATCTTTTGGATTAATTAGTTATTCGAAAGGGAAATTC CAACCTATTATTTTAGGAGACGAAACCGGTACTATTAAAGCTATTATTTGGAATACCGATAAAGAATTA CCTGAAAACACTGTAATAGAAGCTATTGGGAAAACCAAAATTAATAAGAAAACTGGCAATTTAGAATTA CATATAGACAGTTATAAAATTTTAGAAAGCGATTTAGAGATAAAACCCCAAAAGCAAGAATTTGTTGGG ATTTGCATAGTTAAATATCCAAAAAAACAAACCCAAAAAGGCACAATAGTATCGAAAGCAATTTTAACT AGCTTAGATAGGGAATTGCCTGTAGTATATTTCAACGATTTTGATTGGGAAATAGGCCATATATATAAA GTATATGGAAAGCTTAAGAAAAACATAAAAACTGGTAAAATAGAATTTTTCGCTGACAAAGTTGAGGA AGCAACATTAAAAGATCTAAAAGCTTTTAAAGGAGAGGCCGATGGAAGCGGAGGGGTCGACTTGGAA CTGCGCCGCAACGAACAAGATCGGCTGCTGACCGAGCTCGAACACGCGCTGGCTGGCATTTTGGCCAA TATGGAATTTACTGGAGTGAAAGTGGACACGAAGCGGCTTGAACAGATGGGGGCGGAGCTCACCGAG CAGCTGCAGGCGGTCGAGCGGCGCATTTACGAACTCGCCGGCCAAGAGTTCAACATTAACTCGCCGAA ACAGCTCGGGACGGTTTTATTTGACAAGCTGCAGCTCCCGGTGTTGAAAAAGACAAAAACCGGCTATTC GACTTCAGCCGATGTGCTAGAAAAGCTTGCACCGCACCATGAAATCGTCGAACATATTTTGCATTACCG CCAACTCGGCAAGCTGCAGTCAACGTATATTGAAGGGCTGCTGAAAGTGGTGCACCCCGTGACGGGCA AAGTGCACACGATGTTCAATCAGGCGTTGACGCAAACCGGGCGCCTCAGCTCCGTCGAACCGAATTTG CAAAACATTCCGATTCGGCTTGAGGAAGGGCGGAAAATCCGCCAGGCGTTCGTGCCGTCGGAGCCGG ACTGGCTCATCTTTGCGGCCGACTATTCGCAAATCGAGCTGCGCGTCCTCGCCCATATCGCGGAAGATG ACAATTTGATTGAAGCGTTCCGGCGCTGGTTGGACATCCATACGAAAACAGCCATGGACATTTTCCATG TGAGCGAAGAAGACGTGACAGCCAACATGCGCCGCCAAGCGAAGGCCGTCAATTTTGGCATCGTGTAC GGCATTAGTGATTACGGTCTGGCGCAAAACTTGAACATTACGCGCAAAGAAGCGGCTGAATTTATTGA GCGATATTTTGCCAGTTTTCCAGGTGTAAAGCAATATATGGACAACATTGTGCAAGAAGCGAAACAAA AAGGGTATGTGACGACGCTGCTGCATCGGCGCCGCTATTFGCCCGATATTACAAGCCGCAACTTCAACG TCCGCACGTTCGCCGAGCGGACGGCGATGAACACACCGATCCAGGGATCCGCTGCCGACATCATTAAG AAAGCGATGATCGATCTAAGCGTGAGCGTGCGCGAAGAACGGCTGCAGGCGCGCCTGTTGCTGCAAG GTCATGACGAACTCATTTTGGAGGCGCCGAAAGAGGAAATCGGACGGCTGTGCCGCCTCGTTCCGGAA GTGATGGAGCAAGCCGTGACACTTCGCGTGCCGCTGAAAGTCGATTACCATTACGGTCCGACGTGGTA CGACGCCAAATAA. LENGTH: 1992 nucleotides TYPE: nucleic acid TOPOLOGY: plasmid NUMBER OF STRANDS: one TYPE OF THE MOLECULAR: DNA