ORTHOGONAL TRANSCRIPTIONAL SWITCHES DERIVED FROM TET REPRESSOR HOMOLOGS FOR SACCHAROMYCES CEREVISIAE

Abstract

The invention features compositions and methods for identifying orthogonal transcriptional switches derived from Tet repressor homologs for Saccharomyces cerevishiae regulated by 2,4-diacetylphloroglucinol (DAPG) and other ligands.

Claims

1. A system for regulating gene expression in yeast comprising: a repressible gene expression construct comprising a regulator binding sequence and a target gene sequence, wherein the regulator binding sequence comprises a phlO nucleic acid sequence; and a transcriptional activator expression construct comprising a phlF nucleic acid sequence, wherein the transcriptional activator binds to the regulator binding sequence in the absence of 2,4-diacetylphloroglucinol (DAPG) and wherein transcriptional activator binding to the regulator binding sequence is inhibited in the presence of DAPG.

2. The system of claim 1, wherein binding of the regulator binding sequence to the transcriptional activator in the absence of DAPG results in expression of the target gene sequence downstream from the regulator binding sequence.

3. The system of claim 2, wherein the repressible gene expression construct further comprises a transcription terminator sequence.

4. The system of claim 3, wherein the transcriptional terminator sequence is located upstream of the regulator binding sequence.

5. The system of claim 3, wherein the transcriptional terminator sequence comprises a nucleic acid sequence encoding alcohol dehydrogenase 1 (ADH1).

6. The system of claim 1, wherein the regulator binding sequence comprises at least one copy of a nucleic acid sequence of phlO, wherein the sequence of phlO comprises SEQ ID NO: 3.

7. The system of claim 1, wherein the repressible gene expression construct further comprises a promoter downstream from the regulator binding sequence.

8. The system of claim 7, wherein the promoter downstream from the regulator binding sequence lacks an upstream activating sequence.

9. The system of claim 8, wherein the promoter downstream from the regulator binding sequence comprises a cytochrome c isoform 1 (CYC1) promoter.

10. The system of claim 1, wherein the transcription enhancer expression construct comprises a nucleic acid sequence encoding PhlF operatively connected to a transcriptional activation domain to form phlTA.

11. The system of claim 10, wherein the transcriptional activation domain comprises at least one VP16 tandem repeat.

12. The system of claim 10, further comprising a nuclear localization signal (NLS).

13. The system of claim 10, wherein the transcription enhancer expression construct further comprises a promoter sequence comprising the human cytomegalovirus promoter (CMV).

14. The system of claim 1, wherein binding of the regulator binding sequence to the transcriptional activator in the absence of DAPG results in inhibition of expression of the target gene sequence downstream from the regulator binding sequence.

15. The system of claim 14, wherein the regulator binding sequence comprises at least one copy of a nucleic acid sequence of phlO, wherein the sequence of phlO comprises SEQ ID NO: 3.

16-19. (canceled)

20. A recombinantly engineered cell comprising: a repressible gene expression construct comprising a regulator binding sequence and a target gene sequence, wherein the regulator binding sequence comprises a phlO nucleic acid sequence; and a transcriptional activator expression construct comprising a phlF nucleic acid sequence.

21-23. (canceled)

24. A transcription enhancer expression construct comprising a nucleic acid sequence encoding a PhlF transcription regulator domain operatively linked to a transcriptional activation domain to form phlTA, or A recombinantly expressed transcriptional enhancer comprising a PhlF transcription regulator domain and a transcriptional activator domain, wherein the transcriptional activator domain comprises VP16, wherein the recombinantly expressed transcriptional enhancer comprises SEQ ID NO: 10, or A transcription enhancer expression construct comprising a nucleic acid sequence encoding a PhlF transcription regulator domain operatively linked to a nuclear localization signal (NLS) domain, wherein the transcription enhancer expression construct comprises SEQ ID NO: 11, or A recombinantly expressed transcriptional enhancer comprising a PhlF transcription regulator domain and an NLS domain.

25-31. (canceled)

32. A system for expression control of at least two peptide molecules comprising: a first peptide molecule expressed under control of a first repressible promoter; and a second peptide molecule expressed under control of a transcriptional activator comprising a PhlF transcription regulator domain and a transcriptional activation domain, wherein the at least two peptide molecules are capable of being expressed in a eukaryotic cell.

33. The system of claim 32, wherein the system is expressed in an S. cerevisiae cell.

34. A system for regulating gene expression in yeast comprising: a repressible gene expression construct comprising a regulator binding sequence and a target gene sequence, wherein the regulator binding sequence is capable of binding a CymR transcriptional regulator, repressible gene expression construct comprises SEQ ID NO: 19; and a transcriptional activator expression construct comprising a cymR nucleic acid sequence comprising SEQ ID NO: 17, wherein the transcriptional activator binds to the regulator binding sequence in the absence of p-cumate and wherein transcriptional activator binding to the regulator binding sequence is inhibited in the presence of p-cumate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIG. 1A-FIG. 1E is a series of schematics and graphs showing the characteristics of the DAPG-Off switch. FIG. 1A is a schematic diagram of DAPG (PhlF)-OFF system using GFP as a reporter. The transactivator PhlTA, consisting of PhlF and VP16 binds to the phlO (phlF operator) region of promoter phlPr (=ADH1tr-phlO-CYC1pr) in the absence of DAPG. This results in steady expression of gfp. FIG. 1B-FIG. 1D shows flow cytometric analyses in which FITC-A shows the intensity of GFP fluorescence. Strain BY4741 is wild type lacking a gfp gene, and DapG-TA is a BY4741-based strain that carries gfp gene under control of the DAPG-switch. FIG. 1B is a graph showing BY4741 in SC and Strain DapG-TA in SC (solid line). FIG. 1C is a graph showing DapG-TA in SC and with (solid line) and without 12 M DAPG (broken line). FIG. 1D is a graph showing DapG-TA in SC and with (solid line) and without 48 M of DAPG (broken line). The broken lines in FIG. 1C and FIG. 1D are the same. FIG. 1E is a line graph showing time course of GFP fluorescence. The y-axis is the mean fluorescence values out of 10,000 counts. Red circles and blue squares represent DapG-TA cells grown in the absence or presence of 48-M DAPG, respectively. Green triangles show BY4741 grown without DAPG. The plotted values are averages from four independent experiments, and the error bars reflect standard deviations that are not visible because of their small size.

[0067] FIG. 2 is a bar graph showing the effects of DAPG on plating efficiency of BY4741. Yeast strain BY4741 overnight culture in SC medium was diluted 25,000-fold, and 100 l was spread on SC, and SC containing 24-M or 48 M of DAPG. The number of colonies formed was counted after 2 days. The values were means and standard deviations calculated from three experiments.

[0068] FIG. 3A-FIG. 3D is a series of photographs showing the evaluation of the DAPG-Off system using the ADE2 gene (CDS) as a reporter. Cells were cultured for 2 days at 30 C. in SC (FIG. 3A), SC-Ade (FIG. 3B), SC with 24 M of DAPG (FIG. 3C), and SC-Ade with 24-DAPG (FIG. 3D). Cells were diluted 10-fold stepwise across each row.

[0069] FIG. 4 is a series of graphs showing evaluation of four orthogonal systems (the Tet-Off, Camphor-Off, DAPG-Off, and Cumate-Off switches). The gfp gene was used as a reporter, and fluorescence was evaluated in SC medium, SC with 25-M doxycycline, SC with 25-M D-camphor, SC with 48-M DAPG, and SC with 150-M p-cumate across each column.

[0070] FIG. 5 is a schematic diagram of the DAPG-On switch using the ADE2 gene as a reporter (Abbreviation: phlO, phlF operator sequence).

[0071] FIG. 6A-FIG. 6D is a series of photographs showing spot analysis using an ADE2 reporter under control of the DAPG-On system. Cells were cultured for 1 day at 30 C. in SC-Leu-His (FIG. 6A), SC-Leu-His-Ade (FIG. 6B), SC-Leu-His with 24-M DAPG (FIG. 6C), and SC-Leu-His-Ade with 24-M DAPG (FIG. 6D). The cells were diluted 10-fold stepwise across each row.

DETAILED DESCRIPTION

[0072] The invention is based, at least in part, on the surprising identification of a unique promoter regulated by DAPG, cumic acid, and other ligands, which are orthogonal (i.e., no cross talk) to previously described Tet and camphor regulated systems. Accordingly, described herein are orthogonal transcriptional switches derived from Tet repressor homologs for Saccharomyces cerevisiae regulated by 2,4-diacetylphloroglucinol (DAPG) and other ligands.

[0073] The yeast Saccharomyces cerevisiae is the premiere eukaryote for biotechnology, and the first to be sequenced (Goffeau et al., 1996 Science, (274)546: 563-567). More recently, the first synthetic eukaryotic chromosomes and chromosome fragments of S. cerevisiae chromosome

[0074] synIXR, semi-synVIL, synIII were built through a synthetic yeast genome project, Sc2.0 (Annaluru et al., 2014 Science, 344: 55-58; Dymond et al., 2011 Nature, 477: 471-476). The organism benefits from the powerful genetic tools available to reveal gene functions, such as various mutant libraries, including the gene knockout collection (Giaever et al., 2002 Nature, 418: 387-391) and the overexpression collection that utilizes the intrinsic GAL1 promoter (GAL1pr) as a means to individually express genes at a high level (Gelperin et al., 2005 Genes Dev, 19: 2816-2826). However, the popular GAL1pr has some potential disadvantages because it a) requires a high concentration of galactose to induce expression, b) leads to slow growth relative to glucose medium, and c) may affect fundamental metabolism in a manner unrelated to the overexpressed gene product. Furthermore, being able to regulate different pathways independently in the same cell requires a series of distinct orthogonal promoter systems, each activated or repressed by its own ligand. Thus, as described herein, it is highly desirable to have a palette of diverse chemically regulated promoters.

[0075] Another option for regulated expression of genes of interest is to use a synthetic promoter comprised of functional units from other species. The Tet-Off and -On systems are among the most popular expression switches that take advantage of the synthetic promoter. The Tet system is originally from Escherichia coli, in which the transcriptional regulator TetR binds to its operator sequence (tetO) only in the absence of its ligands, such as tetracycline and doxycycline (Ramos et al., 2005 Microbiol. Mol. Biol. Rev., 69: 326-356). Notably, the Tet system can be applied to various species, such as mammalian cells and yeast (Gari et al., 1997 Yeast, 13: 837-848; Lewandoski, M. 2001 Nat. Rev. Genet., 2: 743-755; Urlinger et al., 2000 Proceedings of the National Academy of Sciences U.S.A., 97: 7963-7968). With respect to the key component TetR, there are a vast number of TetR homologs in bacteria and metagenomic samples (Ramos et al., 2005 Microbiol. Mol. Biol. Rev., 69: 326-356; Stanton et al., 2014 Nat. Chem. Biol., 10: 99-105), some of which have well-known operators and ligands. Recently, it was reported that one

of the TetR homologs Pseudomonas putida CamR was utilized for the development of the camphor-Off switch in yeast, analogous to the Tet-Off switch (Ikushima et al., 2015 G3 (Bethesda) 5, 1983-1990). Camphor, the ligand for CamR, prevents binding between CamR and its operator (camO), resulting in camphor-dependent expression of reporter genes under the control of the camO-containing promoter. Moreover, camphor was found to have little impact on yeast growth. On the other hand, an IPTG-On switch is available, and exploits another E. coli repressor, Lad, in yeast (Grilly et al., 2007 Mol. Syst. Biol., 3: 127). Using this system, a reporter gene was activated only in the presence of the Lad ligand IPTG (Isopropyl -D-1-thiogalactopyranoside).

[0076] Similar to the research in yeast, TetR homologs have been developed as switches in heterologous organisms other than yeast. One of them is a 2,4-diacetylphloroglucinol (DAPG)-based switch using phlF gene in bacteria and mammalian cells (Stanton et al., 2014 ACS Synth. Biol., 3: 880-891). The transcriptional repressor PhlF from Pseudomonas fluorescens and related species, known to be a distant homolog of TetR, regulates expression of the DAPG biosynthetic gene phlA in a DAPG-dependent manner (Abbas et al., 2002 J Bacteriol., 184: 3008-3016; Ramette et al., 2011 Appl. Microbiol., 34: 180-188; Schnider-Keel et al., 2000 J Bacteriol, 182: 1215-1225). Besides, other TetR homologs, namely EthR and CymR, have been applied as expression switches in heterologous organisms other than yeast (Eaton, R. W. 1997 J Bacteriol, 179: 3171-3180; Mullick et al., 2006 BMC Biotechnol., 6: 43; Weber et al., 2008 Proceedings of the National Academy of Sciences U.S.A., 105: 9994-9998; Kaczmarczyk et al., 2013 Appl. Environ. Microbiol., 79: 6795-6802).

[0077] As described in detail below, TetR homologs were engineered to develop expression switches in yeast. Specifically, described herein are well-behaved switches that depend on DAPG (both off and on) and p-cumate (off). In particular, DAPG showed little effect on yeast growth at working concentrations for the system. Also described herein is the possibility and challenges of developing additional ligand-regulated TetR homolog-based expression switches in yeast.

[0078] Accordingly, described herein is the development of tightly regulated expression switches in yeast, by engineering distant homologs of Escherichia coli TetR, including the transcriptional regulator PhlF from Pseudomonas and others. Previous studies demonstrated that the PhlF protein bound its operator sequence (phlO) in the absence of 2,4-diacetylphloroglucinol (DAPG) but dissociated from phlO in the presence of DAPG. Thus, described in detail below is the development of a DAPG-Off system in which expression of a gene preceded by the phlO-embedded promoter was activated by a fusion of PhlF to a multimerized viral activator protein (VP16) domain in a DAPG-free environment but repressed when DAPG was added to growth medium. In addition, a DAPG-On system with the opposite behavior of the DAPG-Off system was constructed, i.e., DAPG triggers the expression of a reporter gene. Exposure of DAPG to yeast cells did not cause any serious deleterious effect on yeast physiology in terms of growth. Efforts to engineer additional Tet repressor homologs were partially successful and a known mammalian switch, the p-cumate switch based on CymR from Pseudomonas, was found to function in yeast. Orthogonality between the TetR (doxycycline), CamR (D-camphor), PhlF (DAPG), and CymR (p-cumate)-based Off switches was demonstrated by evaluating all 4 ligands against suitably engineered yeast strains. This study expands the toolbox of On- and Off- switches for yeast biotechnology.

Kits

[0079] The invention provides kits. In one embodiment, the kit includes one or more of the plasmids/constructs described herein, along with any of the ligands described herein, e.g., DAPG. In some embodiments, the kit comprises a sterile container; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0080] The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0081] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, 1989); Oligonucleotide Synthesis (Gait, 1984); Animal Cell Culture (Freshney, 1987); Methods in Enzymology Handbook of Experimental Immunology (Weir, 1996); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, 1987); Current Protocols in Molecular Biology (Ausubel, 1987); PCR: The Polymerase Chain Reaction, (Mullis, 1994); Current Protocols in Immunology (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0082] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

Example 1: Materials and Methods

[0083] The following materials and methods were utilized in the examples described herein.

Media

[0084] Yeast strains were cultured in YPD or SD-based medium supplemented with needed nutrients. SC is a fully supplemented medium of SD, and SC lacking three components, such as leucine, histidine, and adenine, is referred to as SC-Leu-His-Ade. The following drugs added to yeast media were purchased from Santa Cruz Biotechnology (Dallas, Tex.); 2,4-diacetylphloroglucinol (DAPG), coumesterol, and gentamicin. The drugs virginiamycin 51, quercetin, 2-benzyl acetate, D-camphor, and G418, were bought from Sigma-Aldrich (St. Louis, Mo.). Doxycycline, p-cumate (p-isopropylbenzoate), and fisetin were purchased from Clontech laboratories (Mountain View, Calif.), System Biosciences (Mountain View, Calif.), and Fisher Scientific (Pittsburgh, Pa.), respectively.

[0085] Escherichia coli cells were grown in Luria Broth (LB) medium. Carbenicillin (Sigma-Aldrich), kanamycin (Sigma-Aldrich), chloramphenicol (Sigma-Aldrich), or zeocin (Life Technologies, Carlsbad, Calif.) were used to select bacterial strains that had drug-resistant genes at final concentrations of 75 g/ml, 50 g/ml, 20 g/ml, and 25 g/ml respectively. Agar was added to 2% for petri plates.

Plasmids

[0086] The TOP10 strain of E. coli (F.sup. mcA(mrr-hsdRMS-mcrBC) 80lacZM15 lacX74 recA1 araD139 (araleu) 7697 galUgalKrpsL (StrR) endA1 nupG) was used for the construction and amplification of plasmids. In this study, plasmids were constructed with previously described methods (Ikushima et al., 2015 G3 (Bethesda) 5, 1983-1990; Agmon et al., 2015 ACS Synth. Biol., 4: 853-859; Mitchell et al., 2013 ACS Synth. Biol., 2: 473-477). The yeast GoldenGate (yGG) assembly method enabled one-pot plasmid construction because restriction enzymes and DNA ligase could be combined in a single reaction. The Type IIS restriction enzymes BsaI and BsmBI, were used to yield nonpalindromic sticky ends that ligate with one another in a predetermined order and directionality. The standard overhangs flanked by outward facing BsaI sites of the rfp gene were CAGT or TTTT in acceptor vectors unless otherwise described (Agmon et al., 2015 ACS Synth. Biol., 4: 853-859).

[0087] Plasmids used in the study are shown in Table 1. In addition to yGG plasmids of pSIB055, pSIB604, pLM270, and pSIB233 (Ikushima et al., 2015 G3 (Bethesda) 5, 1983-1990; Agmon et al., 2015 ACS Synth. Biol., 4: 853-859), three yGG acceptor vectors were newly constructed as follows. Vectors pSIB230 and pSIB270 are convertible plasmids that can be used as either a centromeric plasmid or an integrative plasmid directed into yeast chromosomes VI or XI. Each has a yeast selection marker of KlURA3 and LEU2, respectively. Plasmid pSIB024 is a centromeric acceptor vector that confers phenotypes of uracil prototrophy (URA3) and resistance to G418. In the yGG assembly, the plasmids above were used with the following yGG components: two promoters, TDH1pr and CMVpr from human cytomegalovirus; three coding sequences (CDS), gfp, ADE2, and the rfp gene that turned the host E. coli cells bright red; two terminators, STR1tr and GSH1tr. All these parts were described previously (Ikushima et al., 2015 G3 (Bethesda) 5, 1983-1990).

TABLE-US-00013 TABLE 1 Plasmids used in this study Plasmid Origin Description pSIB055.sup.a) AV cam.sup.r, HIS3, integrative (targetChVI), rfp pSIB604.sup.a) AV amp.sup.r, LEU2, integrative (YKL162c), rfp pLM270.sup.a) AV amp.sup.r, 2, LEU2, rfp pSIB233.sup.b) AV amp.sup.r, KlURA3, centromeric/integrative (YKL162c), rfp pSIB230 AV amp.sup.r, Sphis5, centromeric/integrative (targetChVI), rfp pSIB270 AV amp.sup.r, LEU2, integrative (YKL162c), rfp pSIB024 AV amp.sup.r, kanMX/URA3, centromeric, rfp pSIB918 pSIB604 amp.sup.r, LEU2, integrative (YKL162c), phlPr (=ADH1tr-phlF operator-CYC1pr)-rfp (BsmBI, overhang of AATG/TGAG)-GSH1tr, CMVpr-phlTA (=phlF-VP16)-STR1tr pSIB927 pSIB918 amp.sup.r, LEU2, integrative (YKL162c), phlPr (=ADH1tr-phlF operator-CYC1pr)-gfp-GSH1tr, CMVpr-phlTA (=phlF-VP16)-STR1tr pSIB883 pSIB230 amp.sup.r, Sphis5, centromeric/integrative (targetChVI), phlPr (=ADH1tr-phlF operator-CYC1pr)-ADE2-GSH1tr pSIB337 pSIB233 amp.sup.r, KlURA3, centromeric/integrative (YKL162c), CMVpr-phlTA (=phlF-VP16)-STR1tr pSIB833 pSIB230 amp.sup.r, Sphis5, centromeric/integrative (targetChVI), ADHphO1 (=ADH1pr-phlF operator single)-ADE2-GSH1tr pSIB924 pSIB230 amp.sup.r, Sphis5, centromeric/integrative (targetChVI), ADHphO2 (=ADH1pr-phlF operator double)-ADE2-GSH1tr pSIB921 pSIB270 amp.sup.r, LEU2, integrative (YKL162c), TDH1pr-NLS-phlF-STR1tr pSIB726 pLM270 amp.sup.r, LEU2, 2, TDH1pr-NLS-phlF-STR1tr pSIB170 pSIB055 cam.sup.r, HIS3, integrative (targetChVI), varPr (=ADH1tr-varR operator-CYC1pr)-gfp-GSH1tr pSIB145 pSIB024 amp.sup.r, kanMX/URA3, centromeric, CMVpr-varTA (=varR-VP16)-STR1tr pSIB084 pSIB055 cam.sup.r, HIS3, integrative (targetChVI), lmrPr (=ADH1tr-lmrA/yxaF operator-CYC1pr)-gfp-GSH1tr pSIB133 pSIB024 amp.sup.r, kanMX/URA3, centromeric, CMVpr-lmrTAv1 (=lmrA-VP16)-STR1tr pSIB220 pSIB024 amp.sup.r, kanMX/URA3, centromeric, CMVpr-lmrTAv2 (=NLS-lmrA-VP16)-STR1tr pSIB166 pSIB055 cam.sup.r, HIS3, integrative (targetChVI), icaPr (=ADH1tr-icaR operator-CYC1pr)-gfp-GSH1tr pSIB222 pSIB024 amp.sup.r, kanMX/URA3, centromeric, CMVpr-icaTA (=icaR-VP16)-STR1tr pSIB454 pSIB230 amp.sup.r, Sphis5, centromeric/integrative (targetChVI), ethPr (=ADH1tr-ethR operator-CYC1pr)-gfp-GSH1tr pSIB750 pSIB233 amp.sup.r, KlURA3, centromeric/integrative (YKL162c), CMVpr-ethTA(=NLS-ethR-VP16)-STR1tr pSIB531 pSIB233 amp.sup.r, KlURA3, centromeric/integrative (YKL162c), CMVpr-yxaTA (=NLS-yxaF-VP16-NLS)-STR1tr pSIB431 pSIB230 amp.sup.r, Sphis5, centromeric/integrative (targetChVI), dhaPr (=ADH1tr-dhaR operator-CYC1pr)-gfp-GSH1tr pSIB503 pSIB233 amp.sup.r, KlURA3, centromeric/integrative (YKL162c), CMVpr-dhaTA(=dhaR-VP16-NLS)-STR1tr pSIB803 pSIB230 amp.sup.r, Sphis5, centromeric/integrative (targetChVI), cymPr (=ADH1tr-cymR operator-CYC1pr)-gfp-GSH1tr pSIB470 pSIB233 amp.sup.r, KlURA3, centromeric/integrative (YKL162c), CMVpr-cymTA(=NLS-cymR-VP16)-STR1tr Notes. Asterisks show sources: .sup.a)Agmon et al. (2015); .sup.b) Ikushima et al. (2015). The other plasmids were constructed in this study (see Materials and methods). Abbreviations: AV, acceptor vector; targetChVI: an intergenic region between GAT1/YFL021w and PAU5/YFL020c; cam.sup.r, chloramphenicol resistant gene; amp.sup.r, ampicillin resistant gene; KlURA3, Kluyveromyces lactis URA3; Sphis5, Schizosaccharomyces pombe his5.

[0088] In particular, a specialized acceptor vector pSIB918, which is yGG-ready for putting any gene under the control of the DAPG-Off system in an integrated state at YKL162c gene with a single transformation, was constructed as follows. First, the rfp gene in the acceptor vector pSIB604 was replaced with the CMVpr, phlTA CDS, and the STR1tr. The resultant plasmid harbored a pair of BsmBI sites at the 5-side of the CMVpr part to accommodate a second transcription unit cassette, and three DNA fragments, phlPr, rfp, and GSH1tr were ligated in the BsmBI gap to generate pSIB918. Plasmid pSIB927 was built by replacing rfp of pSIB918 with the gfp gene.

[0089] A plasmid, pSIB883 was constructed by inserting phlPr, ADE2, and GSH1tr in yGG acceptor vector pSIB230. In order to build pSIB337, the rfp gene in pSIB233 was replaced with the CMVpr, CDS for phlTA, and the STR1tr.

[0090] The three parts, a promoter ADHphlO1, ADE2, and GSH1tr, were cloned as a transcription unit into pSIB230 to yield pSIB833. A related plasmid, pSIB924, contains the promoter ADHphlO2 instead of ADHphlO1 of pSIB833. The 2 plasmid pSIB628 was used as an acceptor vector to build pSIB726, harboring a transcriptional unit consisting of TDH1pr, a nuclear localization signal (NLS)-fused PhlF and the STR1tr.

[0091] Two other sets of plasmids were constructed. One set has a promoter corresponding to each of the tetR-homologs' operator in a minimal promoter fragment fused to gfp as a reporter. The other is a group of vectors that constitutively express a transactivator consisting of the TetR homolog and VP16. The DNA sequences for TetR homologs were codon-optimized using GeneDesign (Richardson et al., 2006 Genome Res., 16, 550-556) for expression in yeast.

Yeast Strains

[0092] Yeast strains are listed in Table 2. Strain BY-ADE was constructed by transforming ade2-deficient BY11204 with a PCR fragment containing the wild-type ADE2 amplified with primers SI-589 (5-TCCACAATCAATTGCGAGAAGC (SEQ ID NO: 1)) and SI-590 (5-CATTTGTTGGAGGAAAGTTGTCC (SEQ ID NO: 2)) using BY4741 genomic DNA as template, followed by selection of an adenine prototrophic phenotype. The other transformations to integrate the expression cassettes were conducted using DNA fragments prepared from the aforementioned pSIB-series of plasmids, previously digested with NotI.

TABLE-US-00014 TABLE 2 Yeast strains used in this study Another Strain name Genotype BY4741.sup.a) MATa his31 leu20 met150 ura30 DapG-TA SIY1001 BY4741 ykl162c::pSIB927 (LEU2, phlPr-gfp, phlTA) BY11204.sup.a) MATa leu21 his3200 lys20 ura3-167 met150 ade2::hisG BY-ADE SIY0979 BY11204 ade2::hisG::ADE2 phlA-TA SIY0987 BY11204 targetChVI::pSIB883 (Sphis5, phlPr-ADE2)ykl162c::pSIB337 (KlURA3, phlTA) phlA-EmV SIY0990 BY11204 targetChVI::pSIB883 (Sphis5, phlPr-ADE2)ykl162c::pSIB233 (KlURA3) varG-TA SIY0155 BY4741 targetChVI::pSIB170 (HIS3, varPr-gfp) pSIB145 (kanMX/URA3, CEN, varTA) lmr/yxaG- SIY0083 BY4741 targetChVI::pSIB084 (HIS3, lmr/yxaPr-gfp) pSIB133 (kanMX/URA3, CEN, lmrTAv1 lmrTAv1) lmr/yxaG- SIY0256 BY4741 targetChVI::pSIB084 (HIS3, lmr/yxaPr-gfp) pSIB220 (kanMX/URA3, CEN, lmrTAv2 lmrTAv2) icaG-TA SIY0261 BY4741 targetChVI::pSIB166 (HIS3, icaPr-gfp) pSIB222 (kanMX/URA3, CEN, icaTA) ethG-TA SIY0743 BY4741 targetChVI::pSIB454 (Sphis5, ethPr-gfp) ykl162c::pSIB750 (KlURA3, ethTA) lmr/yxaG-yxaTA SIY0050 BY4741 targetChVI::pSIB084 (HIS3, lmr/yxaPr-gfp) ykl162c::pSIB531(KlURA3+, yxaTA) dhaG-TA SIY0525 BY4741 targetChVI::pSIB431 (Sphis5, dhaPr-gfp) ykl162c::pSIB503 (KlURA3, dhaTA) cymG-TA SIY0809 BY4741 targetChVI::pSIB803 (Sphis5, cymPr-gfp) ykl162c::pSIB470 (KlURA3, cymTA) BY4742.sup.a) MATa his31 leu20 lys20 ura30 TetOffG.sup.b) SIY0555 BY4742 targetChVI::pSIB527 (Sphis5, tetPr-gfp, tTA) camG-TA.sup.b) SIY0733 BY4741 targetChVI::pSIB426 (Sphis5, camPr-gfp)ykl162c::pSIB619 (KlURA3, camTA) AphO1-2PhlF SIY1018 BY11204 targetChVI::pSIB833 (Sphis5, ADHphO1-ADE2) pSIB726 (2, LEU2, phlF) AphO1-2Emv SIY1016 BY11204 targetChVI::pSIB833 (Sphis5, ADHphO1-ADE2) pLM270 (2, LEU2) AphO2-2PhlF SIY1026 BY11204 targetChVI::pSIB924 (Sphis5, ADHphO2-ADE2) pSIB726 (2, LEU2, phlF) AphO2-2Emv SIY1024 BY11204 targetChVI::pSIB924 (Sphis5, ADHphO2-ADE2) pLM270 (2, LEU2) Notes. Footnotes show sources: .sup.a)Agmon et al. (2015); .sup.b)Ikushima et al. (2015). The other strains were constructed in this study.

Flow Cytometry

[0093] Cellular fluorescence from GFP was determined by flow-cytometric analysis with a previously described method (Ikushima et al., 2015 G3 (Bethesda) 5, 1983-1990).

Example 2: Construction of a PhlF-Based Transcriptional Regulator and a phlF Operator-Embedded Promoter for the DAPG-Off System

[0094] One of the TetR homologs, a PhlF-based transcriptional activator, named phlTA, was constructed by fusing Pseudomonas PhlF (GenBank AAF20928.1) to three tandem repeats of a VP16 transcriptional activation domain derived from herpes simplex virus Type 1 (Baron et al., 1997 Nucleic Acids Res., 25: 2723-2729). Here, the CMVpr from human cytomegalovirus was used to drive the appropriate level of phlTA expression.

[0095] The promoter used for phlF-dependent expression of a reporter gene was built by embedding seven repeats of the phlF operator sequence (phlO) between the alcohol dehydrogenase ADH1 terminator and a CYC1 (cytochrome c) promoter from which the endogenous UAS (upstream activating sequence) had been removed. The resulting promoter was named phlPr, the architecture of which was analogous to a promoter used in yeast Tet- and Camphor-Off systems (Gari et al., 1997 Yeast, 13: 837-848; Ikushima et al., 2015 G3 (Bethesda) 5, 1983-1990. The unit of the phlF operator used was 5-TATGTATGATACGAAACGTACCGTATCGTTAAGGTAGCGT (SEQ ID NO: 3; Abbas et al., 2002 J Bacteriol., 184: 3008-3016).

[0096] As described herein, the PhlF-derived transcriptional activator, phlTA, binds phlPr to activate transcription of a reporter gene only in the absence of DAPG, the ligand of PhlF, and DAPG ligand binding is predicted to eliminate reporter expression in the presence of DAPG (FIG. 1A).

Example 3: Performance of the DAPG-Off Switch with a GFP Reporter

[0097] The performance of the DAPG-Off system was examined using GFP as a reporter. A yeast transformant, DapG-TA (SIY1001), which had phlTA and the phlPr-gfp reporter, was constructed by integrating plasmid pSIB918 in BY4741 (Table 1 and Table 2). The DapG-TA strain showed significant expression of GFP in the absence of DAPG, unlike control strain BY4741 (FIG. 1B). In addition, the GFP fluorescence of the DapG-TA was higher than that of the BY4741 control strain (background) in the presence of 12-M DAPG, but decreased to the same level as BY4741 at 48-M DAPG (FIG. 1B-FIG. 1D). These data indicated that the expression of the reporter GFP was regulated in a DAPG-dependent manner, consistent with predictions. Furthermore, the DapG-TA strain was then used to evaluate the kinetics of the DAPG-Off switch. The intensity of GFP showed an increase over a 19-h period in the absence of DAPG, whereas the fluorescence reduced to background levels over 14-h of culture in the presence of 48 M DAPG (FIG. 1E). This duration of GFP expression is consistent with a half-life of GFP protein in yeast (around 7 h) and suggests transcriptional shutoff is rapid. Moreover, in terms of overall promoter strength, the GFP fluorescence of DapG-TA at 19 h in SC is approximately 13% of the intensity a TDH1pr-gfp and 8-fold higher than KEX2pr-gfp. Thus the DAPG-Off switch should be useful to regulate gene expression for practical, middle-strength expression.

[0098] With regards to effects of DAPG on growth, the results presented herein showed that there was little difference in the number of colonies formed in three media types: (a) SC with 24-M (5-g/ml) DAPG; (b) SC with 48-M (10-g/ml) DAPG; (c) SC without DAPG (FIG. 2). However, previous work suggests that 10-g/ml DAPG did not inhibit growth of BY4741 (Kwak et al., 2011 Appl. Environ. Microbiol., 77: 1770-1776), whereas cells of this background did show sensitivity to higher concentrations (Troppens et al., 2013 FEMS Yeast Res., 13: 322-334).

Example 4: DAPG-Off System with an ADE2 Reporter

[0099] A second reporter gene was used to further assess the performance of the DAPG-Off switch. Here, it was examined whether the ADE2 gene under the control of phlPr complements the adenine-auxotrophy of strain BY11204 in a DAPG-dependent manner (FIG. 3A-FIG. 3D). The BY11204-based yeast transformant phlA-EmV (phlA Empty vector), which had the phlPr-ADE2 reporter gene but not phlTA, showed solid growth on SC medium, but failed to grow in SC-Ade medium, irrespective of DAPG concentration. On the other hand, the phlA-TA strain, harboring both phlPr-ADE2 and phlTA showed proliferation in a DAPG-free SC-Ade medium. In contrast, the strain did not grow on SC-Ade medium containing 24 M DAPG. Similarly to the GFP reporter (FIG. 1B-FIG. 1E), the DAPG-Off switch enabled very tight regulation of the reporter gene phlPr-ADE2. Moreover, no revertants appeared on SC-Ade agar plates on plating of 2.310.sup.5 cells, consistent with a genetically stable DAPG-Off system.

Example 5: Construction of Off-Switches Using Other TetR Homologs

[0100] This study assessed other TetR homologs' ligands and operators previously reported (Table 3): varR (Namwat et al., 2001 J. Bacteriol., 183: 2025-2031), lmrA (Yoshida et al., 2004 J. Bacteriol., 186: 5640-5648; Hirooka et al., 2007 J. Bacteriol., 189: 5170-5182), icaR (Jeng et al., Nucleic Acids Res., 36: 1567-1577), yxaF (Yoshida et al., 2004 J. Bacteriol., 186: 5640-5648; Hirooka et al., 2007 J. Bacteriol., 189: 5170-5182), dhaR (Poelarends et al., 2000 J. Bacteriol., 182: 2191-2199), ethR (Weber et al., 2008 Proceedings of the National Academy of Sciences U.S.A., 105: 9994-9998), and cymR (Eaton, R. W. 1997 J Bacteriol, 179: 3171-3180; Mullick et al., 2006 BMC Biotechnol., 6: 43; Kaczmarczyk et al., 2013 Appl. Environ. Microbiol., 79: 6795-6802). The scheme to evaluate the switch candidates was similar to the DAPG-Off switch. A transcriptional activation domain of VP16 was added to the TetR homologs (transactivator), and operator sequences of them were interposed between the ADH1 terminator and UAS-less CYC1 promoter, followed by the gfp reporter. First, it was observed that all strains which had only the gfp reporter did not express GFP; the strains were then transformed with plasmids to express the corresponding transactivators and expression was evaluated.

TABLE-US-00015 TABLE3 Yeastoff-switchcandidatesevaluatedinthisstudy Expressionof Switch TetR GFP candidate homolog Drug w/o with (Strain) (Accession) NLS Operator Species (Concentration) drug drug Var-Off VarR w/o varR Streptomyces VirginiamycinS1 On On (varG-TA) (BAB32408) operator.sup.a) virginae (2mM) Lmr- LmrA w/o lmrA/yxaF Bacillussubtilis Quercetin,Fisetin, Off ND Offv1 (NP_388150) operator.sup.b) Coumesterol (lmr/yxaG- lmrTAv1) Lmr- LmrA N-end lmrA/yxaF Bacillussubtilis Quercetin(0.13M), On On Offv2 (NP_388150) operator.sup.b) Fisetin(0.19M), (lmr/yxaG- Coumesterol(0.22M) lmrTAv2) lcaR-Off IcaR N-end icaR Staphylococcus Gentamicin(10mM) On On (icaG-TA) (AAN17770.1) operator.sup.c) epidermidis Eth-Off EthR N-end EthR Mycobacterium 2-Benzylacetate On On (ethG-TA) (NP_218372) operator.sup.d) tuberculosis (0.01%) Yxa-Off YxaF N,C- lmrA/yxaF Bacillussubtilis Quercetin,Fisetin, Off ND (lmr/yxaG- (BAA21585) ends operator.sup.b) Coumesterol) yxaTA) Dha-Off DhaR N-end dhaR Mycobacterium 1-Chlorobutane Off ND (dhaG-TA) (CAB65288) operator.sup.e) sp.GP1 Cumate- CymR N-end cymR Pseudomonas p-Cumate On Off Off (AAB62296) operator.sup.f) putida (150M) (cymG-TA) Notes. .sup.a)TGTCACTTGTACATCGTATAACTCTCATATACGTTGTAGAACAGTTC (SEQ ID NO: 4) (4 repeats) .sup.b)CTTTCTCCTACAATTATATAGAACGGTCTAGACAAATGAATGATAATATATAGACTGGTCTAAATTGGAGGAAGCGATA (SEQ ID NO: 5)(3 repeats) .sup.c)ACAACCTAACTAACGAAAGGTAGGTGAA (SEQ ID NO: 6) (6 repeats) .sup.d)GTGTCGATAGTGTCGACATCTCGTTGACGGCCTCGACATTACGTTGATAGCGTGG (SEQ ID NO: 7) (5 repeats) .sup.e)AAGATGACCGGTCACCTT (7 repeats) .sup.f)AAGAAAGAAACAAACCAACCTGTCTGTATTATCTC (SEQ ID NO: 8)(6 repeats) Abbreviations: ND, Not determined.

[0101] Streptomyces virginiae antibiotic resistance regulator VarR tagged with VP16 induced strong GFP expression both in the absence and presence of the ligand virginiamycin S. This result suggested the hybrid transactivator is active, but virginiamycin may not be stably taken up in yeast cells. On the other hand, expressing a transactivator based on LmrA and VP16 gave rise to no gfp activity in the presence or absence of ligand. Since the fusion protein must enter the nucleus for expression in yeast, a nuclear localization signal (NLS) from SV40 (Kalderon et al., 1984 Cell, 39: 499-509) was added to LmrTA, resulting in strong expression of GFP; however, as in the case of VarR-TA, it was not ligand (quercetin)-responsive. Differently from TetR and PhlF in the Tet- and DAPG-Off systems, these results show that some TetR homologs require an NLS in order to activate transcription while others do not. The TetR homologs IcaR and EthR also showed solid ligand-independent GFP fluorescence with gentamicin and 2-benzyl acetate respectively. EthR was previously reported as a useful switch in mammalian cells (Weber et al., 2008 Proceedings of the National Academy of Sciences U.S.A., 105: 9994-9998), but tight regulation of the switch could not be achieved in yeast. The reason behind residual gfp expression in the presence of the ligands remains unclear, but the amount of ligand able to penetrate yeast cell envelopes may not be sufficient to thoroughly prevent transactivator from binding to the operators. Potentially, the use of pdr5 mutants or other drug-sensitized strains might confer responsiveness to such switches. By contrast, YxaF and DhaR did not confer detectable GFP expression even in the absence of their ligands, even though a NLS was appended. Possible reasons are that either amount of the transactivator expression, or alternatively, the affinity between the transactivators and the operator sequences chosen might be insufficient to induce reporter expression. Finally, CymR, a repressor involved in the p-cymene catabolic pathway in Pseudomonas putida did result in the expected expression profile of the relevant GFP reporter with p-cumate. Thus, one additional off-switch was obtained for yeast; the strain with the appropriate reporter and activator is called CymG-TA.

Example 6: Orthogonality of the Yeast Off-Switches

[0102] A series of switches anticipated for use together must be evaluated for orthogonality to maximize their utility. Thereby, four switches of the Tet-Off, Camphor-Off, DAPG-Off, and Cumate-Off systems were evaluated. When four strains that contained the switches independently were cultured in the absence and presence of the four ligands (doxycycline, camphor, DAPG, cumate), the intensity of reporter GFP fluorescence decreased to the background level only in the presence of the appropriate ligands, whereas in the presence of the inappropriate ligands, a strong GFP signal was observed, similar to that observed in the absence of any ligands (FIG. 4). An exception to this trend was that cells with the Tet or Cam system showed lowered GFP fluorescence in the DAPG medium. Thus, DAPG may have slightly reduced orthogonality, but the GFP signal can also certainly be affected by other factors, such as growth phase (FIG. 1E). In short, the results suggest that the four switches are useful to construct complex biological circuits in which multiple reporter genes are regulated separately in a single strain. Indeed, this is the case for the Tet and Cam systems independently regulated in the same strain (Ikushima et al., 2015 G3 (Bethesda) 5, 1983-1990).

Example 7: Construction of Two Components for the DAPG-on System

[0103] Next, On-switches that make it possible to induce expression of a gene of interest by ligand treatment were developed. In particular, a DAPG-On system based on the native transcriptional regulator and a phlO-containing promoter was developed. Here, a single and double repeat of phlO sequences were embedded downstream of the ADH1 promoter (ADH1pr) to build ADHphlO1 and ADHphlO2, leading to constitutive reporter expression in the absence of the transcriptional regulator. The two promoters were analogous in design to the ADHi promoter, which carries a single copy of the lac operator downstream of the ADH1pr (Grilly et al., 2007 Mol. Syst. Biol., 3: 127). Next, a transcriptional regulator consisting of NLS and PhlF was constructed. As shown in FIG. 5, this binds to the ADH1phlO promoter and sterically blocks transcription. The reporter gene expresses only in the presence of DAPG and the NLS-PhlF protein (FIG. 5).

Example 8: DAPG-on System with an ADE2 Reporter

[0104] The performance of the DAPG-On system was assessed using ADE2 as a reporter in the ade2 BY11204 strain. When PhlF was not expressed in a strain that had the ADHphlOx-ADE2 cassette, the strain, named AphOx-2EmV, grew in adenine-deficient medium irrespective of DAPG addition as well as in adenine-containing medium (FIG. 6A-FIG. 6D). On the other hand, strain AphOx-2PhlF, which carried multiple copies of PhlF in addition to the ADHphlOx-ADE2 cassette, hardly grew at all on DAPG-free SC-Ade medium, consistent with binding of PhlF to phlO (phlF operator sequence) preventing expression of the ADHphlOx-driven ADE2 gene. However, both strains AphOx-2PhlF showed vigorous growth on a SC-Ade medium in the presence of 5 M DAPG. These results suggest that DAPG released PhlF from the phlO region and led to expression of ADE2. With regard to the genetic stability of the DAPG-On system, the reversion frequencies to adenine-prototrophic (Ade.sup.+) clones were 9.410.sup.6 and 1.210.sup.6 for strains AphO1-2PhlF and AphO2-2PhlF respectively. The stability is enhanced further by increasing the copy number of phlO downstream of ADH1pr, since there was an 8-fold difference in reversion frequency between the two strains. These results suggest that the DAPG-On system is useful for regulating expression of a gene of interest with the opposite logic of the DAPG-Off system.

Perspectives

[0105] Expression switches that can be regulated with small compounds are widely used in biology-related studies. The Tet-Off and -On systems are among the most popular switches because of their advantages, e.g., tight regulation and ease of handling, but the number of such ligand regulated switches is very limited. Thus, more and better switches would enable more options to control gene expression.

[0106] Described herein is the assessment of seven TetR homologs to develop transcription switches in yeast, resulting in switches based on two TetR homologs, PhlF and CymR. They were named DAPG-Off, DAPG-On, and Cumate-Off switches in which DAPG or p-cumate prevented or triggered the expression of a reporter gene such as gfp and ADE2 at a concentration. As described herein, the Cumate-Off switch is further evaluated for reversion and performance with an auxotrophic reporter. However, most importantly, the two switches showed robust orthogonality to other useful switches such as the Tet- and camphor switches. These switches expand the repertoire of regulated gene expression in yeast (Table 4). On the other hand, this study did not yield controllable switches in the case of five other TetR homologs including EthR, which has been shown to work as a switch in mammalian cells. Thus, not all TetR homologs are directly applicable for use as a high performing expression switch in yeast.

TABLE-US-00016 TABLE 4 Useful On and Off switch reagents for end users Restriction enzyme site Plasmid yGG-use Description (overhang of top/end-sides) <DAPG-Off system> pSIB918 AV amp.sup.r, LEU2, integrative (YKL162c), phlPr (=ADH1tr-phlF operator- BsmBI (AATG/TGAG) CYC1pr)-rfp-GSH1tr, CMVpr-phlTA (=phlF-VP16)-STR1tr pSIB289 Transactivator kan.sup.r, phlTA (=phlF-VP16) BsmBI (AATG/TGAG) pSIB153 Promoter with kan.sup.r, phlPr (=ADH1tr-phlF operator-CYC1pr) BsaI (CAGT/AATG) operator <Tet-Off system> pSIB498 AV amp.sup.r, Sphis5, integrative (targetChVI), CMVpr-tTA(=tetR-VP16)- BsmBI (AATG/TGAG) STR1tr, tetPr (=ADH1tr-tetR operator-CYC1pr)-rfp-GSH1tr pSIB009 Transactivator amp.sup.r, tTA (=tetR-VP16) BsaI (AATG/TGAG) pSIB022 Promoter with kan.sup.r, tetOPr (=ADH1tr-tetO operator-CYC1pr) BsaI (CAGT/AATG) operator <Camphor-Off system> pSIB859 AV amp.sup.r, KlURA3, integrative (HO), TDH1pr-camTA (=camR-VP16- BsmBI (AATG/TGAG) NLS)-STR1tr, camPr (=ADH1tr-camR operator-CYC1pr)-rfp-SOL3tr pSIB477 Transactivator kan.sup.r, camTA (=camR-VP16-NLS) BsmBI (AATG/TGAG) pSIB396 Promoter with kan.sup.r, camPr (=ADH1tr-camR operator-CYC1pr) BsaI (CAGT/AATG) operator <Cumate-Off system> pSIB665 Transactivator kan.sup.r, cymTA (=NLS-cymR-VP16) BsaI (AATG/TGAG) pSIB795 Promoter with kan.sup.r, cymPr (=ADH1tr-cymR operator-CYC1pr) BsaI (CAGT/AATG) operator <DAPG-On system> pSIB199 Transactivator kan.sup.r, NLS-phlF BsaI (AATG/TGAG) pSIB916 Promoter with kan.sup.r, ADHphO2 (=ADH1pr-phlF operator double) BsaI (CAGT/AATG) operator <Tet-On system> pSIB499 AV amp.sup.r, Sphis5, integrative (targetChVI), CMVpr-rtTA(=mutated BsmBI (AATG/TGAG) tetR-VP16)-STR1tr, tetPr (=ADH1tr-tetR operator-CYC1pr)-rfp-GSH1tr pSIB010 Transactivator amp.sup.r, rtTA (=muated tetR-VP16) BsaI (AATG/TGAG) pSIB022 See above (Tet-Off) [Common parts for yGG] pSIB027 Promoter kan.sup.r, TDH1 promoter (=TDH1pr) BsaI (CAGT/AATG) pSIB237 Promoter kan.sup.r, CMV promoter (mutated to be BsmBI-free) (=CMVpr) BsaI (CAGT/AATG) pSIB031 Terminator kan.sup.r, GSH1 terminator (=GSH1pr) BsaI (TGAG/TTTT) pSIB206 Terminator kan.sup.r, STR1 terminator (mutated to be BsmBI-free) (=STR1tr) BsaI (TGAG/TTTT) pSIB639 Terminator kan.sup.r, SOL3 terminator (=SOL3tr) BsaI (TGAG/TTTT) Notes. Plasmids except for pSIB918 were not listed in Table 1, but details on the plasmids are described in Supporting information. Abbreviations: AV, acceptor vector; targetChVI: an intergenic region between GAT1/YFL021w and PAU5/YFL020c; amp.sup.r, ampicillin resistant gene; kanr.sup.r, kanamycin resistant gene.

[0107] In the development of the DAPG-On switch, it was speculated that a DAPG-On switch could be constructed by mutating phlTA, fusion of PhlF and triple repeats of VP16, because the transactivators of the Tet-On and Cumate-On switches (named rev-tTA) could be isolated from multiple missense mutants of the transactivators used in the Tet-Off and Cumate-Off systems, VP16-tethered TetR or CymR, respectively. In fact, it was confirmed that the Tet-On switch a transactivator based on the 5-amino acid residue TetR variant named rtTA-M2 (Urlinger et al., 2000 Proceedings of the National Academy of Sciences U.S.A., 97: 7963-7968) showed doxycycline-dependent GFP expression in the opposite manner to the Tet-Off system in yeast. However, such a mutant with opposite behavior to the DAPG-Off system was not isolated. In this study, another way to develop the DAPG-On system was developed in which the binding of PhlF to the operator could sterically prevent the transcription of a reporter gene, resulting in relatively tight regulation of the ADE2 reporter (FIG. 6A-FIG. 6D). This strategy is effective for developing additional On-switches in yeast.

[0108] As an alternative strategy, other sources are identified for constructing a Compound-On switch. Notably, in natural environments, there is another type of TetR homolog that binds to an operator preferentially in the presence of a specific ligand. As described herein, those proteins are made use of as a kind of natural rev-tTA. Many quorum-sensing (QS) molecules that function as transcriptional regulators are known (Safari et al., 2014 Appl. Microbiol. Biotechnol., 98: 3401-3412). Interestingly, the binding of a TetR homolog, such as LuxR and TraR, to an operator sequence is triggered with a QS molecule. Prior to the invention described herein, a functional switch has not been obtained using the QS-related transactivators. However, a QS molecule-On switch is constructed from a number of QS-related TetR homologs. Additional transcriptional switches are developed using TetR homologs as a great tool in biotechnology.

[0109] An additional description of plasmids listed in Table 4 is provided below.

DAPG-Off System: pSIB918, pSIB289, and pSIB153

[0110] All three plasmids were constructed in this study: pSIB918 was referred to in Table 1. pSIB289 and pSIB153 were used to build pSIB918. The sequences for the DAPG-Off system are provided above.

Tet-Off System: pSIB498, pSIB009, and pSIB022

[0111] Plasmids pSIB009 and pSIB022 provided elements of the transactivator and operator-embedded promoter to construct pSIB498, and all three plasmids were prepared previously but the names were not mentioned as such (Ikushima et al., 2015 G3 (Bethesda), Vol. 5 (10), 1983-1990, incorporated herein by reference). In particular, pSIB498 is the most generally useful yeast GoldenGate-ready acceptor vector carrying the Tet-Off switch. Plasmid pSIB527 shown in the article was constructed by replacing rfp gene of pSIB498 with gfp gene.

[0112] An exemplary nucleic acid sequence for tTA (tetR-VP16) in pSIB498 for the Tet-Off system is provided below (1-618, label=TetR; 619-744, VP16; 745-747, Stop codon; SEQ ID NO: 22):

TABLE-US-00017 atgagtagattggacaagtctaaggttatcaactctgctttggaattgag aacgaagttggtatcgaaggtttgaccaccagaaagttggctcaaaagtt gggtgttgaacaaccaaccttgtactggcacgttaagaacaagagagctt tgaggacgctttggctatcgaaatgttggacagacaccacacccacttct gtccattggaaggtgaatcttggcaagacttcttgagaaacaacgctaag tctttcagatgtgctttgctctctcaccgcgacggtgctaaggttcactt gggaaccagaccaaccgaaaagcaatacgaaaccttggaaaaccaattgg ctttcttgtgtcaacaaggtttctctttggaaaacgctttgtacgctttg tctgctgaggtcacttcaccttgggttgtgttttggaagaccaagaacac caagttgctaaggaagaaagagaaaccccaaccaccgactctatgccacc attgctcagacaagctatcgaattgttcgaccaccaaggtgctgaaccag ctttcttgttcggtttggaattgatcatctgtggtttggaaaagcaattg aagtgtgaatctggtgggccggccgacgctttggacgacttcgacttgga catgttgccagctgacgctttggacgacttcgacttggacatgttgccag ctgacgctttggacgacttcgacttggacatgttgccaggttga

[0113] An exemplary amino acid sequence for tTA (tetR-VP16) in pSIB498 for the Tet-Off system is provided below (SEQ ID NO: 23):

TABLE-US-00018 MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRA LLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVH LGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQE HQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQ LKCESGGPADALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG

[0114] An exemplary nucleic acid sequence for tetOpr (ADH1tr-tetO operator-CYC1pr) in pSIB498 for the Tet-Off system is provided below (1-203, ADH1 transcriptional terminator; 216-508, tetR operator; 521-667, CYC1 promoter; SEQ ID NO: 24):

TABLE-US-00019 cacttctaaataagcgaatttcttatgatttatgatttttattattaaat aagttataaaaaaaataagtgtatacaaattttaaagtgactcttaggtt ttaaaacgaaaattcttgttcttgagtaactctttcctgtaggtcaggtt gctttctcaggtatagcatgaggtcgctcttattgaccacacctctaccg gcaggccggcccgggtcgagtttaccactccctatcagtgatagagaaaa gtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagt cgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagttta ccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactccc tatcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtg atagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaa aagtgaaacccgggccggcctatggcatgcatgtgctctgtatgtatata aaactcttgttttcttcttttctctaaatattctttccttatacattagg tcctttgtagcataaattactatacttctatagacacgcaaacacaaata cacacactaaattaata
Camphor-Off System: pSIB859, pSIB477, and pSIB396

[0115] As was the case with the Tet-Off switch plasmids above, pSIB477 and pSIB396 provided elements of transactivator and operator-embedded promoter parts for pSIB859, and all the three plasmids were prepared previously (Ikushima et al., 2015 G3 (Bethesda), Vol. 5 (10), 1983-1990, incorporated herein by reference). Particularly, pSIB859 is the most generally useful yeast GoldenGate-ready acceptor vector carrying the Camphor-Off switch. Accordingly, this plasmid was used to construct pSIB872 described herein.

[0116] An exemplary nucleic acid sequence for camTA (camR-VP16-NLS) in pSIB859 for the Camphor-Off system is provided below (1-558, CamR (Pseudomonas putida); 559-684, VP16; 685-708, NLS (SV40); 709-711, Stop codon; SEQ ID NO: 25):

TABLE-US-00020 atggacatcaagcaatctttgttgcacgctgctatgagattgttgtctgc taagggtcgcgacggtgctaccatgcgaccaatctgtgctgaagttggtg ttaccccaccaaccttgtaccaccactacggtgacttgcaaggtttgcac aaggctgctatcgacgaaacctacagacaagttgctgaagcttaccacgg tggtaccgaagaaagaggtccattgaagggtatccgcgacggttgggcta ccttcttgcaattcgcttactctgaaccaaacatgtgtagaatgttggtt caacacatcatggctggtgaaccaccatctatggttgctgacaccttgag aggtgttgctgacgacttggctcaattccacgctcaaggtagattgacct tcccaccaagagaagctgctcaattgttgtggatgggtgctttgggtgct ttgacctacgctttgtctagagaaggtgctggttacacccaagacttggc tttgcaaaaggctaagttggacatcaccttggttgctttgttcaacatcg aagaagaagggccggccgacgctttggacgacttcgacttggacatgttg cctgcagatgcacttgatgattttgatcttgatatgcttccagcagacgc attggatgactttgaccttgacatgcttcctggtatgccaaagaagaaga gaaaggtatga

[0117] An exemplary amino acid sequence for camTA (camR-VP16-NLS) in pSIB859 for the Camphor-Off system is provided below (SEQ ID NO: 26):

TABLE-US-00021 MDIKQSLLHAAMRLLSAKGRDGATMRPICAEVGVTPPTLYHHYGDLQGLH KAAIDETYRQVAEAYHGGTEERGPLKGIRDGWATFLQFAYSEPNMCRMLV QHIMAGEPPSMVADTLRGVADDLAQFHAQGRLTFPPREAAQLLWMGALGA LTYALSREGAGYTQDLALQKAKLDITLVALFNIEEEGPADALDDFDLDML PADALDDFDLDMLPADALDDFDLDMLPGMPKKKRKV

[0118] An exemplary nucleic acid sequence for camPR (ADH1tr-camR operator-CYC1pr) for the Camphor-Off system is provided below (1-203, ADH1 transcriptional terminator; 208-430, camR operator; 432-574, CYC1 promoter; SEQ ID NO: 27):

TABLE-US-00022 cacttctaaataagcgaatttcttatgatttatgatttttattattaaat aagttataaaaaaaataagtgtatacaaattttaaagtgactcttaggtt ttaaaacgaaaattcttgttcttgagtaactctttcctgtaggtcaggtt gctttctcaggtatagcatgaggtcgctcttattgaccacacctctaccg gcaaggtcaggctctatatctgcgatatactgagcatatcccccccaggc tctatatctgcgatatactgagcatatcccccccaggctctatatctgcg atatactgagcatatcccccccaggctctatatctgcgatatactgagca tatccccccaggctctatatctgcgatatactgagcatatccccccaggc tctatatctgcgatatactgagcatatcccaatggcatgcatgtgctctg tatgtatataaaactcttgttttcttcttttctctaaatattctttcctt atacattaggtcctttgtagcataaattactatacttctatagacacgca aacacaaatacacacactaaattaata
Cumate-Off System: pSIB665 and pSIB795

[0119] Plasmids pSIB665 and pSIB795 were constructed herein. They provided the transactivator and operator-embedded promoter parts for pSIB470 and pSIB803, respectively.

DAPG-on System: pSIB199 and pSIB916

[0120] Plasmids pSIB199 and pSIB916 were constructed herein. They provided transactivator and operator-embedded promoter parts for pSIB470 and pSIB803, respectively.

Tet-on System: pSIB499, pSIB010, and pSIB022

[0121] Plasmids pSIB010 and pSIB022 provided transactivator and operator-embedded promoter parts for pSIB499. Specifically, pSIB499 is the most generally useful yeast GoldenGate-ready acceptor vector specialized for the Tet-On system in which the transactivator is the TetR variant with alteration of 5-amino acids (S12G E19G A56P D148E H179R), known as rtTA-M2 (Urlinger et al., 2000 Proc. Natl. Acad. Sci. USA., Vol. 97 (14), 7963-7968, incorporated herein by reference).

[0122] The rtTA acts in the opposite manner against tTA of the Tet-Off system. The present study yielded BY4741-derived transformants with a plasmid that harbored gfp gene in the position of rfp of pSIB499. Only background GFP fluorescence was observed in the absence of doxycycline, while GFP significantly expressed in the presence of more than 10-M doxycycline.

[0123] An exemplary nucleic acid sequence for rtTA (tetR mutant-VP16) in pSIB499 for the Tet-On system is provided below (1-618, mutated TetR; 619-744, VP16; 745-747, Stop codon; SEQ ID NO: 28):

TABLE-US-00023 atgagtagattggacaagtctaaggttatcaacggtgctttggaattgtt gaacggtgttggtatcgaaggtttgaccaccagaaagttggctcaaaagt tgggtgttgaacaaccaaccttgtactggcacgttaagaacaagagagct ttgttggacgctttgccaatcgaaatgttggacagacaccacacccactt ctgtccattggaaggtgaatcttggcaagacttcttgagaaacaacgcta agtctttcagatgtgctttgctctctcaccgcgacggtgctaaggttcac ttgggaaccagaccaaccgaaaagcaatacgaaaccttggaaaaccaatt ggctttcttgtgtcaacaaggtttctctttggaaaacgctttgtacgctt tgtctgctgttggtcacttcaccttgggttgtgttttggaagaacaagaa caccaagttgctaaggaagaaagagaaaccccaaccaccgactctatgcc accattgctcagacaagctatcgaattgttcgacagacaaggtgctgaac cagctttcttgttcggtttggaattgatcatctgtggtttggaaaagcaa ttgaagtgtgaatctggtgggccggccgacgctttggacgacttcgactt ggacatgttgccagctgacgctttggacgacttcgacttggacatgagcc agctgacgctttggacgacttcgacttggacatgttgccaggttga

[0124] An exemplary amino acid sequence for rtTA (tetR-VP16) in pSIB499 for the Tet-On system is provided below (SEQ ID NO: 29):

TABLE-US-00024 MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRA LLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVH LGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQE HQVAKEERETPTTDSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQ LKCESGGPADALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG

[0125] The tetOpr (ADH1tr-teto operator CYC1pr) in pSI499 is identical to tetOpr (ADH1tr-tetO operator-CYC1pr) in pSIB498 above.

[0126] Common parts for yGG include the following. An exemplary nucleic acid sequence for yGG-TDH1pr in pSIB027 is provided below (12-871, TDH1 promoter; SEQ ID NO: 30):

TABLE-US-00025 ggtctcacagtctcgatggattagtttctcacaggtaacataacaaaaac caagaaaagcccgcttctgaaaactacagttgacttgtatgctaaagggc cagactaatgggaggagaaaaagaaacgaatgtatatgctcatttacact ctatatcaccatatggaggataagttgggctgagcttctgatccaattta ttctatccattagttgctgatatgtcccaccagccaacacttgatagtat ctactcgccattcacttccagcagcgccagtagggttgttgagcttagta aaaatgtgcgcaccacaagcctacatgactccacgtcacatgaaaccaca ccgtggggccttgttgcgctaggaataggatatgcgacgaagacgcttct gcttagtaaccacaccacattttcagggggtcgatctgcttgcttccttt actgtcacgagcggcccataatcgcgclattattaaaaggcgcgagacag caaacaggaagctcgggittcaaccttcggagtggtcgcagatctggaga ctggatctttacaatacagtaaggcaagccaccatctgcttcttaggtgc atgcgacggtatccacgtgcagaacaacatagtctgaagaagggggggag gagcatgttcattctctgtagcagtaagagcttggtgataatgaccaaaa ctggagtctcgaaatcatataaatagacaatatattttcacacaatgaga tttgtagtacagttctattctctctcttgcataaataagaaattcatcaa gaacttggtttgatatttcaccaacacacacaaaaaacagtacttcacta aatttacacacaaaacaaaatgagagacc

[0127] An exemplary nucleic acid sequence for yGG-mutated CMVpr in pSIB237 is provided below (12-781, mutated CMV promoter; SEQ ID NO: 31):

TABLE-US-00026 ggtctcacagtgagcttggcccattgcatacgttgtatccatatcataat atgtacatttatattggctcatgtccaacattaccgccatgttgacattg attattgactagttattaatagtaatcaattacggggtcattagttcata gcccatatatggagttccgcgttacataacttacggtaaatggcccgcct ggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgt tcccatagtaacgccaatagggactttccattgacgtcaatgggtggagt atttacggtaaactgcccacttggcagtacatcaagtgtatcatatgcca agtacgccccctattgacgtcaatgacggtaaatggcccgcctggcatta tgcccagtacatgaccttatgggactacctacttggcagtacatctacgt attagtcatcgctattaccatggtgatgcggttaggcagtacatcaatgg gcgtggatagcggtttgactcacggggatttccaagtctccaccccattg acgtcaatgggagtttgttaggcaccaaaatcaacgggactttccaaaat gtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacgg tgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctg tagacgccatccacgctgttttgacctccatagaagacaccgggaccgat ccagcctccgcggcccgaattaattcataatgagagacc

[0128] An exemplary nucleic acid sequence for yGG-ScGSH1tr in pSIB031 is provided below (12-718, GSH1 terminator; SEQ ID NO: 32):

TABLE-US-00027 ggtctcgtgagactccttttacttcggttgtgaaagaaagttgacattat cgatttgggtgacacggtgattgaaaaagcaacgaccagtattatacctc ttttttttattattcagtttatatttttgcaagtgatcttaagcatttct acacaaacttatgccaacgtgaccatttattattttatatagcaaaaaaa aatgaggggccttgcagaacaattgttgcgagtttctaataacaagcacg tagaatattggccatttaatttttctcttcaatttatagaatggttgtgt tagtgacaaaaagaatattcttccccgccaggactcgaacctggaatctc ctggttcgtagccagacgccgtgaccattgggccacgaggaacaagaata taaagatctctgagggcaaggtatgcctatgtcgcaataaaatgtttgtt cctgcgcaaaagtaaagttctattaatatacaactacacagttatcggtt cacactattcgatagttgtaaaaaccattttgataaagatataacaaggc gtttattaaggacatttttgctacaagtcgtgaagtattgattgtaggcg atcgttggtaactttctccatatcggaatattcaatattgaactcacccc tcccttgcgataagctccttagcttattggtgtaggtggtaatttccctt agtggcactttcgcttttcgagacc

[0129] An exemplary nucleic acid sequence for yGG-mutaed STR1tr in pSIB206 is provided below (12-741, mutated STR1; SEQ ID NO: 33):

TABLE-US-00028 ggtctcgtgagtcgccagtgccatgtttctgccttcgaccggaccattta agtacgataaatatccattataaatatatagtctaaaatatccattaata ctgtgctcaatcaatcgtgttagatgatttagttattccaaatcgttatt atagtgcagaagtagtatacataaaggcatatgcatgcgatttggaagta acgctcgccgtagacaagtaagaatgcctgctgtcttgagaaccaggtcc aaagaatcctctatagagcagaagcctgcttccagaactagaacgagatc aagaaggggcaagcgtggtcgtgacgatgatgatgatgacgacgatgagg aaagcgatgatgcatacgatgaagtaggtaatgactatgacgagtatgct tcaagagcgaagctggccaccaataggcccttcgaaatagtcgcgggact gcctgctagtgtggagctgcccaactataactcttcgcttactcatccgc aatcaattaaaaattctggggtgctttacgactctctggtcagttccaga agaacctgggttcagggtgagatgtttgaactgtattggcgaagacctaa gaaaattgttagtgaatctaccccagcagcgacggagagtccaacatctg gaacgattcctttgattcgagataagatgcagaaaatgtgcgattgtgta atgagtggaggtcctcacacgttcaaagttagacttttggagacc

[0130] An exemplary nucleic acid sequence for yGG-SOL3tr in pSIB639 is provided below (12-562, SOL3 terminator; SEQ ID NO: 34):

TABLE-US-00029 ggtctcatgagaaaagacacacatgcgagctttcgaacctcagatgctaa tattacgtgttatatataccaaactttataaaatgacatagatattttat gctgtgatagctacctgttatggagaagctcttcttattccccctgtcaa ctttcatactcttgtagaatttcctttatgataggtttatcgcttacgaa tttagactttgatgtgatgggtttggcacctgttctttaccacaaccttt gcgtgcctcatcaatagcgtttgatctgtcgggaaatttgtatttgtaga gtgcatccttgcacattgtatagacccaattacgctcttctaacaggttc acgaacgattttatttcaggaacagagccgattgtactttttgaacctat aatgatcagcttggatttggcccttgtcatggcaacattgactcttctta gctattcagcagcgctcctccatttaattgagaatttcttctaaccatgg aaataataatgcactttagtcacgaccttgaaactgatcagcagtcaaga tctctagcttttagagacc
Common Parts for yGG: pSIB027, pSIB237, pSIB031, pSIB206, and pSIB639

[0131] These parts were described previously Ikushima et al., 2015 G3 (Bethesda), Vol. 5 (10), 1983-1990, incorporated herein by reference) and represent promoter or terminator parts for acceptor vectors in yeast GoldenGate assembly.

Example 9: Select Annotated Sequences

[0132] Described below are select annotated sequences for the constructs provided herein.

TABLE-US-00030 LOCUSNLS24bpds-DNAlinear03-DEC-2016 DEFINITION. ACCESSION VERSION SOURCE. ORGANISM. COMMENT COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers misc_feature 1..24 /label=NLS(SV40) /ApEinfo_fwdcolor=#36ff21 /ApEinfo_revcolor=#36ff21 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 ORIGIN 1ATGCCAAAGAAGAAGAGAAAGGTT(SEQIDNO:35) //

TABLE-US-00031 LOCUSSEQ_ID_NO_9_PhIF729bpds-DNAlinear03-DEC-2016 DEFINITION. ACCESSION VERSION SOURCE. ORGANISM. COMMENT COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers misc_feature 1..600 /label=PhIF /ApEinfo_fwdcolor=cyan /ApEinfo_revcolor=cyan /ApEinfo_graphicformat=arrowdata{{01200-1}{}0} width5offset0 misc_feature 601..726 /label=VP16 /ApEinfo_fwdcolor=#2109ff /ApEinfo_revcolor=#2109ff /ApEinfo_graphicformat=arrowdata{{01200-1}{}0} width5offset0 misc_feature 727..729 /label=Stopcodon /ApEinfo_fwdcolor=#ff8080 /ApEinfo_revcolor=#ff8080 /ApEinfo_graphicformat=arrowdata{{01200-1}{}0} width5offset0 ORIGIN 1 ATGGCTAGAACCCCATCTCGATCTTCTATCGGTTCTTTGCGATCTCCACACACCCACAAG 61 GCTATCTTGACCTCTACCATCGAAATCTTGAAGGAATGTGGTTACTCTGGTTTGTCTATC 121 GAATCTGTTGCTAGAAGAGCTGGTGCTTCTAAGCCAACCATCTACAGATGGTGGACCAAC 181 AAGGCTGCTTTGATCGCTGAAGTTTACGAAAACGAATCTGAACAAGTTAGAAAGTTCCCA 241 GACTTGGGTTCTTTCAAGGCTGACTTGGACTTCTTGTTGAGAAACTTGTGGAAGGTTTGG 301 AGAGAAACCATCTGTGGTGAGGCTTTCAGATGTGTTATCGCTGAAGCTCAATTGGACCCA 361 GCTACCTTGACCCAATTGAAGGACCAATTCATGGAAAGAAGAAGAGAAATGCCAAAGAAG 421 TTGGTTGAAAACGCTATCTCTAACGGTGAATTGCCAAAGGACACCAACAGAGAATTGTTG 481 TTGGACATGATCTTCGGTTTCTGTTGGTACAGATTGTTGACCGAACAATTGACCGTTGAA 541 CAAGACATCGAAGAGTTCACCTTCTTGTTGATCAACGGTGTTTGTCCAGGAACCCAAAGA 601 gggccggccGACGCTTTGGACGACTTCGACTTGGACATGTTGCCTGCAGATGCACTTGAT 661 GATTTTGATCTTGATATGCTTCCAGCAGACGCATTGGATGACTTTGACCTTGACATGCTT 721 CCTGGTTGA(SEQIDNO:9;PhIF-VP16inSIB337) //

TABLE-US-00032 LOCUSSIB921_G339_A63L624bpds-DNAlinear02-DEC-2016 DEFINITION. ACCESSION VERSION SOURCE. ORGANISM. COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers misc_feature 25..621 /label=PhIF /ApEinfo_fwdcolor=cyan /ApEinfo_revcolor=cyan /ApEinfo_graphicformat=arrowdata{{01200-1}{}0} width5offset0 misc_feature 622..624 /label=STOPcodon /ApEinfo_fwdcolor=#fflfdd /ApEinfo_revcolor=#fflfdd /ApEinfo_graphicformat=arrowdata{{01200-1}{}0} width5offset0 misc_feature 1..24 /label=NLS(SV40) /ApEinfo_fwdcolor=#36ff21 /ApEinfo_revcolor=#36ff21 /ApEinfo_graphicformat=arrowdata{{01200-1}{}0} width5offset0 ORIGIN 1 ATGCCAAAGAAGAAGAGAAAGGTTGCTAGAACCCCATCTCGATCTTCTATCGGTTCTTTG 61 CGATCTCCACACACCCACAAGGCTATCTTGACCTCTACCATCGAAATCTTGAAGGAATGT 121 GGTTACTCTGGTTTGTCTATCGAATCTGTTGCTAGAAGAGCTGGTGCTTCTAAGCCAACC 181 ATCTACAGATGGTGGACCAACAAGGCTGCTTTGATCGCTGAAGTTTACGAAAACGAATCT 241 GAACAAGTTAGAAAGTTCCCAGACTTGGGTTCTTTCAAGGCTGACTTGGACTTCTTGTTG 301 AGAAACTTGTGGAAGGTTTGGAGAGAAACCATCTGTGGTGAGGCTTTCAGATGTGTTATC 361 GCTGAAGCTCAATTGGACCCAGCTACCTTGACCCAATTGAAGGACCAATTCATGGAAAGA 421 AGAAGAGAAATGCCAAAGAAGTTGGTTGAAAACGCTATCTCTAACGGTGAATTGCCAAAG 481 GACACCAACAGAGAATTGTTGTTGGACATGATCTTCGGTTTCTGTTGGTACAGATTGTTG 541 ACCGAACAATTGACCGTTGAACAAGACATCGAAGAGTTCACCTTCTTGTTGATCAACGGT 601 GTTTGTCCAGGAACCCAAAGATGA(SEQIDNO:11;NLS-PhIFinSIB921) //

TABLE-US-00033 LOCUSphlPr_in_pSIB918653bpds-DNAcircular03-DEC-2016 DEFINITION. ACCESSION VERSION SOURCE. ORGANISM. COMMENT COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers misc_feature 220..249 /label=coreofPhIFoperator /ApEinfo_fwdcolor=cyan /ApEinfo_revcolor=cyan /ApEinfographicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 335..374 /label=1unit(PhIFop.core+flank) /ApEinfo_fwdcolor=#ffd580 /ApEinfo_revcolor=#ffd580 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 204..214 /label=Adapter_FseI-XmaI /ApEinfo_fwdcolor=#fffbf4 /ApEinfo_revcolor=#fffbf4 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 255..294 /label=1unit(PhIFop.core+flank)(1) /ApEinfo_label=1unit(PhIFop.core+flank) /ApEinfo_fwdcolor=#ffd580 /ApEinfo_revcolor=#ffd580 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 380..409 /label=coreofPhIFoperator(1) /ApEinfo_label=coreofPhIFoperator /ApEinfo_fwdcolor=cyan /ApEinfo_revcolor=cyan /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 375..414 /label=1unit(PhIFop.core+flank)(2) /ApEinfo_label=1unit(PhIFop.core+flank) /ApEinfo_fwdcolor=#ffd580 /ApEinfo_revcolor=#ffd580 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 215..254 /label=1unit(PhIFop.core+flank)(3) /ApEinfo_label=1unit(PhIFop.core+flank) /ApEinfo_fwdcolor=#ffd580 /ApEinfo_revcolor=#ffd580 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 420..449 /label=coreofPhIFoperator(2) /ApEinfo_label=coreofPhIFoperator /ApEinfo_fwdcolor=cyan /ApEinfo_revcolor=cyan /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 507..653 /label=ScCYC1promoter /ApEinfo_fwdcolor=#1dff0f /ApEinfo_revcolor=#1dff0f /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 260..289 /label=coreofPhIFoperator(3) /ApEinfo_label=coreofPhIFoperator /ApEinfo_fwdcolor=cyan /ApEinfo_revcolor=cyan /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 415..454 /label=1unit(PhIFop.core+flank)(4) /ApEinfo_label=1unit(Ph1Fop.core+flank) /ApEinfo_fwdcolor=#ffd580 /ApEinfo_revcolor=#ffd580 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 300..329 /label=coreofPhIFoperator(4) /ApEinfo_label=coreofPhIFoperator /ApEinfo_fwdcolor=cyan /ApEinfo_revcolor=cyan /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 295..334 /label=1unit(PhIFop.core+flank)(5) /ApEinfo_label=1unit(PhIFop.core+flank) /ApEinfo_fwdcolor=#ffd580 /ApEinfo_revcolor=#ffd580 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 1..203 /label=ADH1transcriptionalterminator /ApEinfo_fwdcolor=#ffec1f /ApEinfo_revcolor=#ffec1f /ApEinfo_graphicformat=arrow_data {{01200-1}{}0} width5offset0 misc_feature 455..494 /label=1unit(PhIFop.core+flank)(6) /ApEinfo_label=1unit(PhIFop.core+flank) /ApEinfo_fwdcolor=#ffd580 /ApEinfo_revcolor=#ffd580 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 340..369 /label=coreofPhIFoperator(5) /ApEinfo_label=coreofPhIFoperator /ApEinfo_fwdcolor=cyan /ApEinfo_revcolor=cyan /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 460..489 /label=coreofPhIFoperator(6) /ApEinfo_label=coreofPhIFoperator /ApEinfo_fwdcolor=cyan /ApEinfo_revcolor=cyan /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 495..506 /label=Adapter_XmaI-FseI /ApEinfo_fwdcolor=#fffefb /ApEinfo_revcolor=#fffefb /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 215..494 /label=phIFoperator /ApEinfo_fwdcolor=#ff8080 /ApEinfo_revcolor=#ff8080 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 ORIGIN 1 cacttctaaataagcgaatttcttatgatttatgatttttattattaaataagttataaa 61 aaaaataagtgtatacaaattttaaagtgactcttaggttttaaaacgaaaattcttgtt 121 cttgagtaactctttcctgtaggtcaggttgctttctcaggtatagcatgaggtcgctct 181 tattgaccacacctctaccggcaggccggcccggTATGTATGATACGAAACGTACCGTAT 241 CGTTAAGGTAGCGTTATGTATGATACGAAACGTACCGTATCGTTAAGGTAGCGTTATGTA 301 TGATACGAAACGTACCGTATCGTTAAGGTAGCGTTATGTATGATACGAAACGTACCGTAT 361 CGTTAAGGTAGCGTTATGTATGATACGAAACGTACCGTATCGTTAAGGTAGCGTTATGTA 421 TGATACGAAACGTACCGTATCGTTAAGGTAGCGTTATGTATGATACGAAACGTACCGTAT 481 CGTTAAGGTAGCGTcccgggccggcctatggcatgcatgtgctctgtatgtatataaaac 541 tcttgttttcttcttttctctaaatattctttccttatacattaggtcctttgtagcata 601 aattactatacttctatagacacgcaaacacaaatacacacactaaattaATA(SEQIDNO:13;phlPrinpSIB918) //

TABLE-US-00034 LOCUSSEQ_ID_NO_15_ADH823bpds-DNAcircular03-DEC-2016 DEFINITION. ACCESSION VERSION SOURCE. ORGANISM. COMMENT COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers misc_feature 693..732 /label=1unit(PhIFop.core+flank) /ApEinfo_fwdcolor=#ffd580 /ApEinfo_revcolor=green /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 733..762 /label=coreofPhIFoperator /ApEinfo_fwdcolor=cyan /ApEinfo_revcolor=green /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 733..767 /label=1unit(PhIFop.core+flank)(1) /ApEinfo_label=1unit(PhIFop.core+flank) /ApEinfo_fwdcolor=#ffd580 /ApEinfo_revcolor=green /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 698..727 /label=coreofPhIFoperator(1) /ApEinfo_label=coreofPhIFoperator /ApEinfo_fwdcolor=cyan /ApEinfo_revcolor=green /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 768..793 /label=ADHli(down) /ApEinfo_fwdcolor=#fff449 /ApEinfo_revcolor=#fff449 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 promoter 794..823 /label=syntheticsequencefromADHipr /ApEinfo_fwdcolor=#b0ffa8 /ApEinfo_revcolor=#b0ffa8 /ApEinfographicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 1..692 /label=ADHli(up) /ApEinfo_fwdcolor=#c7ff70 /ApEinfo_revcolor=#c7ff70 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 179..179 /label=T->A(mutation) /ApEinfo_fwdcolor=cyan /ApEinfo_revcolor=cyan /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 693..767 /label=PhIFoperator /ApEinfo_fwdcolor=#ff8080 /ApEinfo_revcolor=#ff8080 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 ORIGIN 1 TAAAGTCCAATGCTAGTAGAGAAGGGGGGTAACACCCCTCCGCGCTCTTTTCCGATTTTT 61 TTCTAAACCGTGGAATATTTCGGATATCCTTTTGTTGTTTCCGGGTGTACAATATGGACT 121 TCCTCTTTTCTGGCAACCAAACCCATACATCGGGATTCCTATAATACCTTCGTTGGTCaC 181 CCTAACATGTAGGTGGCGGAGGGGAGATATACAATAGAACAGATACCAGACAAGACATAA 241 TGGGCTAAACAAGACTACACCAATTACACTGCCTCATTGATGGTGGTACATAACGAACTA 301 ATACTGTAGCCCTAGACTTGATAGCCATCATCATATCGAAGTTTCACTACCCTTTTTCCA 361 TTTGCCATCTATTGAAGTAATAATAGGCGCATGCAACTTCTTTTCTTTTTTTTTCTTTTC 421 TCTCTCCCCCGTTGTTGTCTCACCATATCCGCAATGACAAAAAAATGATGGAAGACACTA 481 AAGGAAAAAATTAACGACAAAGACAGCACCAACAGATGTCGTTGTTCCAGAGCTGATGAG 541 GGGTATCTCGAAGCACACGAAACTTTTTCCTTCCTTCATTCACGCACACTACTCTCTAAT 601 GAGCAACGGTATACGGCCTTCCTTCCAGTTACTTGAATTTGAAATAAAAAAAAGTTTGCT 661 GTCTTGCTATCAAGTATAAATAGACCTGCAATTATGTATGATACGAAACGTACCGTATCG 721 TTAAGGTAGCGTATGATACGAAACGTACCGTATCGTTAAGGTAGCGTCTTTCTTCCTTGT 781 TTCTTTTTCTGCAggtcgactctagaggatccccgggtattaa(SEQIDNO:15;ADHph02inpS1B924) //

TABLE-US-00035 LOCUSSEQ_ID_NO_17_cym762bpds-DNAlinear03-DEC-2016 DEFINITION. ACCESSION VERSION SOURCE. ORGANISM. COMMENT COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers misc_feature 634..759 /label=VP16 /ApEinfo_fwdcolor=#2109ff /ApEinfo_reycolor=#2109ff' /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 25..633 /label=CymR /ApEinfo_fwdcolor=cyan /ApEinfo_revcolor=cyan /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 1..24 /label=NLS(SV40) /ApEinfo_fwdcolor=#ff7b78 /ApEinfo_revcolor=#ff7b78 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 760..762 /label=Stopcodon /ApEinfo_fwdcolor=#ff8080 /ApEinfo_revcolor=#ff8080 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 ORIGIN 1 ATGCCAAAGAAGAAGAGAAAGGTAATGTCTCCAAAGAGAAGAACTCAAGCTGAAAGAGCT 61 ATGGAAACTCAAGGTAAGTTGATCGCTGCTGCTTTGGGTGTTTTGAGAGAAAAGGGTTAC 121 GCTGGTTTCAGAATCGCTGACGTTCCAGGTGCTGCTGGTGTTTCTAGAGGTGCTCAATCT 181 CACCACTTCCCAACCAAGTTGGAATTGTTGTTGGCTACCTTCGAATGGTTGTACGAACAA 241 ATCACCGAAAGATCTAGAGCTAGATTGGCTAAGTTGAAGCCAGAAGACGACGTTATCCAA 301 CAAATGTTGGACGACGCTGCTGAATTCTTCTTGGACGACGACTTCTCTATCTCTTTGGAC 361 TTGATCGTTGCTGCTGACCGCGATCCTGCTTTGAGAGAAGGTATCCAAAGAACCGTTGAA 421 AGAAACAGATTCGTTGTTGAAGACATGTGGTTGGGTGTTTTGGTTTCTAGAGGTTTGTCT 481 CGCGACGACGCTGAAGACATCTTGTGGTTGATCTTCAACTCTGTTAGAGGTTTGGCTGTT 541 AGATCTTTGTGGCAAAAGGACAAGGAAAGATTCGAAAGAGTTAGAAACTCTACCTTGGAA 601 ATCGCTAGAGAAAGATACGCTAAGTTCAAGAGGgggccggccGACGCTTTGGACGACTTC 661 GACTTGGACATGTTGCCTGCAGATGCACTTGATGATTTTGATCTTGATATGCTTCCAGCA 721 GACGCATTGGATGACTTTGACCTTGACATGCTTCCTGGTTGA(SEQIDNO:17;cymTAinpSIB470) //

TABLE-US-00036 LOCUSSEQ_ID_NO_19_Cym564bpds-DNAcircular04-DEC-2016 DEFINITION. ACCESSION VERSION SOURCE. ORGANISM. COMMENT COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers misc_feature 383..417 /label=CymRoperatorP1 /ApEinfo_fwdcolor=#83ff70 /ApEinfo_revcolor=#83ff70 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 204..207 /label=acceptor /ApEinfo_fwdcolor=cyan /ApEinfo_revcolor=green /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 348..382 /label=CymRoperatorP1(1) /ApEinfo_label=CymRoperatorP1 /ApEinfo_fwdcolor=#83ff70 /ApEinfo_revcolor=#83ff70 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 388..413 /label=bindingsite /ApEinfo_fwdcolor=#ff95f8 /ApEinfo_revcolor=#ff95f8 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 313..347 /label=CymRoperatorP1(2) /ApEinfo_label=CymRoperatorPl /ApEinfo_fwdcolor=#83ff70 /ApEinfo_revcolor=#83ff70 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 278..312 /label=CymRoperatorP1(3) /ApEinfo_label=CymRoperatorPl /ApEinfo_fwdcolor=#83ff70 /ApEinfo_revcolor=#83ff70 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 418..421 /label=acceptor(1) /ApEinfo_label=acceptor /ApEinfo_fwdcolor=#00ffff /ApEinfo_revcolor=green /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 243..277 /label=CymRoperatorP1(4) /ApEinfo_label=CymRoperatorPl /ApEinfo_fwdcolor=#83ff70 /ApEinfo_revcolor=#83ff70 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 208..242 /label=CymRoperatorP1(5) /ApEinfo_label=CymRoperatorP1 /ApEinfo_fwdcolor=#83ff70 /ApEinfo_revcolor=#83ff70 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 1..203 /label=ADH1transcriptionalterminator /ApEinfo_fwdcolor=#ffec1f /ApEinfo_revcolor=green /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 353..378 /label=bindingsite(1) /ApEinfo_label=bindingsite /ApEinfo_fwdcolor=#ff95f8 /ApEinfo_revcolor=#ff95f8 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 318..343 /label=bindingsite(2) /ApEinfo_label=bindingsite /ApEinfo_fwdcolor=#ff95f8 /ApEinfo_revcolor=#ff95f8 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 283..308 /label=bindingsite(3) /ApEinfo_label=bindingsite /ApEinfo_fwdcolor=#ff95f8 /ApEinfo_revcolor=#ff95f8 /ApEinfo_graphicformat=arrow_data width5offset0 misc_feature 419..564 /label=ScCYC1promoter /ApEinfo_fwdcolor=#1dff0f /ApEinfo_revcolor=green /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 248..273 /label=bindingsite(4) /ApEinfo_label=bindingsite /ApEinfo_fwdcolor=#ff95f8 /ApEinfo_revcolor=#ff95f8 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 213..238 /label=bindingsite(5) /ApEinfo_label=bindingsite /ApEinfo_fwdcolor=#ff95f8 /ApEinfo_revcolor=#ff95f8 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 208..417 /label=cymRoperator /ApEinfo_fwdcolor=#ff0000 /ApEinfo_revcolor=#ff0000 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 ORIGIN 1 cacttctaaataagcgaatttcttatgatttatgattntattattaaataagttataaa 61 aaaaataagtgtatacaaattttaaagtgactcttaggttttaaaacgaaaattchgtt 121 cttgagtaactctttcctgtaggtcaggttgctttctcaggtatagcatgaggtcgctct 181 tattgaccacacctctaccggcaaggtAAGAAAGAAACAAACCAACCTGTCTGTATTATC 241 TCAAGAAAGAAACAAACCAACCTGTCTGTATTATCTCAAGAAAGAAACAAACCAACCTGT 301 CTGTATTATCTCAAGAAAGAAACAAACCAACCTGTCTGTATTATCTCAAGAAAGAAACAA 361 ACCAACCTGTCTGTATTATCTCAAGAAAGAAACAAACCAACCTGTCTGTATTATCTCaat 421 ggcatgcatgtgctctgtatgtatataaaactcttgattcttcttnctctaaatattc 481 tttccttatacattaggtcctttgtagcataaattactatacttctatagacacgcaaac 541 acaaatacacacactaaattaATA(SEQIDNO:19;CymPrinpSIB803) //

TABLE-US-00037 LOCUSSEQ_ID_NO_22_Tab747bpds-DNAcircular04-DEC-2016 DEFINITION. ACCESSION VERSION SOURCE. ORGANISM. COMMENT COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers misc_feature 1..618 /label=TetR /ApEinfo_fwdcolor=cyan /ApEinfo_revcolor=cyan /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 619..744 /label=VP16 /ApEinfo_fwdcolor=#ff1fdd /ApEinfo_revcolor=#ff1fdd /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 misc_feature 745..747 /label=Stopcodon /ApEinfo_fwdcolor=#ff8080 /ApEinfo_revcolor=#ff8080 /ApEinfo_graphicformat=arrow_data{{01200-1}{}0} width5offset0 ORIGIN 1 ATGAGTAGATTGGACAAGTCTAAGGTTATCAACTCTGCTTTGGAATTGTTGAACGAAGTT 61 GGTATCGAAGGTTTGACCACCAGAAAGTTGGCTCAAAAGTTGGGTGTTGAACAACCAACC 121 TTGTACTGGCACGTTAAGAACAAGAGAGCTTTGTTGGACGCTTTGGCTATCGAAATGTTG 181 GACAGACACCACACCCACTTCTGTCCATTGGAAGGTGAATCTTGGCAAGACTTCTTGAGA 241 AACAACGCTAAGTCTTTCAGATGTGCTTTGCTCTCTCACCGCGACGGTGCTAAGGTTCAC 301 TTGGGAACCAGACCAACCGAAAAGCAATACGAAACCTTGGAAAACCAATTGGCTTTCTTG 361 TGTCAACAAGGTTTCTCTTTGGAAAACGCTTTGTACGCTTTGTCTGCTGTTGGTCACTTC 421 ACCTTGGGTTGTGTTTTGGAAGACCAAGAACACCAAGTTGCTAAGGAAGAAAGAGAAACC 481 CCAACCACCGACTCTATGCCACCATTGCTCAGACAAGCTATCGAATTGTTCGACCACCAA 541 GGTGCTGAACCAGCTTTCTTGTTCGGTTTGGAATTGATCATCTGTGGTTTGGAAAAGCAA 601 TTGAAGTGTGAATCTGGTGGGCCGGCCGACGCTTTGGACGACTTCGACTTGGACATGTTG 661 CCAGCTGACGCTTTGGACGACTTCGACTTGGACATGTTGCCAGCTGACGCTTTGGACGAC 721 TTCGACTTGGACATGTTGCCAGGTTGA(SEQIDNO:22;Table4tTA(=tetR-VP16)inpSIB498) //

TABLE-US-00038 LOCUSSEQ_ID_NO_24_Tab667bpds-DNAcircular04-DEC-2016 DEFINITION. ACCESSION VERSION SOURCE. ORGANISM. COMMENT COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers misc_feature 521...667 /label= ScCYC1promoter /ApEinfo_fwdcolor= #1dff0f /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 1...203 /label= ADH1transcriptionalterminator /ApEinfo_fwdcolor= #ffec1f /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 216...508 /label= tetO*7 /ApEinfo_fwdcolor= cyan /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 356...373 /label= tetOmin /ApEinfo_fwdcolor= #ff051a /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 216...228 /label= fw /ApEinfo_fwdcolor= #8b7aff /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 384...396 /label= fw(1) /ApEinfo_label= fw /ApEinfo_fwdcolor= #9480ff /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 509...520 /label= Adapter_XmaI-FseI /ApEinfo_fwdcolor= #fffefb /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 229...247 /label= tetOmin(1) /ApEinfo_label= tetOmin /ApEinfo_fwdcolor= #ff2222 /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 300...312 /label= fw(2) /ApEinfo_label= fw /ApEinfo_fwdcolor= #6e79ff /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 426...438 /label= fw(3) /ApEinfo_label= fw /ApEinfo_fwdcolor= #9990ff /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 272...289 /label= tetOmin(2) /ApEinfo_label= tetOmin /ApEinfo_fwdcolor= #ff1527 /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 342...355 /label= fw(4) /ApEinfo_label= fw /ApEinfo_fwdcolor= #7f67ff /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 468...480 /label= fw(5) /ApEinfo_label= fw /ApEinfo_fwdcolor= #9568ff /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 204...215 /label= Adapter_FseI-XmaI /ApEinfo_fwdcolor= #fffbf4 /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 313...331 /label= tetOmin(3) /ApEinfo_label= tetOmin /ApEinfo_fwdcolor= #ff0e26 /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 481...499 /label= tetOmin(4) /ApEinfo_label= tetOmin /ApEinfo_fwdcolor= #ff0f1e /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 258...271 /label= fw(6) /ApEinfo_label= fw /ApEinfo_fwdcolor= #8172ff /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 221...257 /label= NativeTet0ofE.coli /ApEinfo_fwdcolor= #f1beff /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 397...415 /label= tetOmin(5) /ApEinfo_label= tetOmin /ApEinfo_fwdcolor= #ff020e /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 439...457 /label= tetOmin(6) /ApEinfo_label= tetOmin /ApEinfo_fwdcolor= #ff1919 /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 216...508 /label= tetRoperator /ApEinfo_fwdcolor= #ff0000 /ApEinfo_revcolor= #ff0000 /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 ORIGIN 1 cacttctaaataagcgaatttcttatgatttatgatttttattattaaataagttataaa 61 aaaaataagtgtatacaaattttaaagtgactcttaggttttaaaacgaaaattcttgtt 121 cttgagtaactctttcctgtaggtcaggttgctttctcaggtatagcatgaggtcgctct 181 tattgaccacacctctaccggcaggccggcccgggtcgagtttaccactccctatcagtg 241 atagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagt 301 cgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccactcccta 361 tcagtgatagagaaaagtgaaagtcgagtttaccactccctatcagtgatagagaaaagt 421 gaaagtcgagtttaccactccctatcagtgatagagaaaagtgaaagtcgagtttaccac 481 tccctatcagtgatagagaaaagtgaaacccgggccggcctatggcatgcatgtgctctg 541 tatgtatataaaactcttgttttcttcttttctctaaatattctttccttatacattagg 601 tcctttgtagcataaattactatacttctatagacacgcaaacacaaatacacacactaa 661 attaATA(SEQIDNO:24;Table4;tetOpr(=ADH1tr-tetOoperator- CYC1pr)inpSIB498) //

TABLE-US-00039 LOCUSSEQ_ID_NO_25_Tab711bpds-DNAcircular04-DEC-2016 DEFINITION. ACCESSION VERSION SOURCE. ORGANISM. COMMENT COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers misc_feature 559...684 /label= VP16 /ApEinfo_fwdcolor= #2109ff /ApEinfo_revcolor= #2109ff /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 685...708 /label= NLS(SV40) /ApEinfo_fwdcolor= #ff7b78 /ApEinfo_revcolor= #ff7b78 /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 1...558 /label= CamR_PseudomonasputidaBAA03510 /ApEinfo_fwdcolor= cyan /ApEinfo_revcolor= cyan /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 709...711 /label= Stopcodon /ApEinfo_fwdcolor= #ff0000 /ApEinfo_revcolor= #ff0000 /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 ORIGIN 1 ATGGACATCAAGCAATCTTTGTTGCACGCTGCTATGAGATTGTTGTCTGCTAAGGGTCGC 61 GACGGTGCTACCATGCGACCAATCTGTGCTGAAGTTGGTGTTACCCCACCAACCTTGTAC 121 CACCACTACGGTGACTTGCAAGGTTTGCACAAGGCTGCTATCGACGAAACCTACAGACAA 181 GTTGCTGAAGCTTACCACGGTGGTACCGAAGAAAGAGGTCCATTGAAGGGTATCCGCGAC 241 GGTTGGGCTACCTTCTTGCAATTCGCTTACTCTGAACCAAACATGTGTAGAATGTTGGTT 301 CAACACATCATGGCTGGTGAACCACCATCTATGGTTGCTGACACCTTGAGAGGTGTTGCT 361 GACGACTTGGCTCAATTCCACGCTCAAGGTAGATTGACCTTCCCACCAAGAGAAGCTGCT 421 CAATTGTTGTGGATGGGTGCTTTGGGTGCTTTGACCTACGCTTTGTCTAGAGAAGGTGCT 481 GGTTACACCCAAGACTTGGCTTTGCAAAAGGCTAAGTTGGACATCACCTTGGTTGCTTTG 541 TTCAACATCGAAGAAGAAgggccggccGACGCTTTGGACGACTTCGACTTGGACATGTTG 601 CCTGCAGATGCACTTGATGATTTTGATCTTGATATGCTTCCAGCAGACGCATTGGATGAC 661 TTTGACCTTGACATGCTTCCTGGTATGCCAAAGAAGAAGAGAAAGGTATGA (SEQIDNO:25;Table4camTA(=camR-VP16-NLS)inpSIB859) //

TABLE-US-00040 LOCUSSEQ_ID_NO_27_Tab577bpds-DNAcircular04-DEC-2016 DEFINITION. ACCESSION VERSION SOURCE. ORGANISM. COMMENT COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers misc_feature 204...207 /label= acceptor /ApEinfo_fwdcolor= cyan /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{0121} { } 0} width5offset0 misc_feature 431...434 /label= acceptor(1) /ApEinfo_label= acceptor /ApEinfo_fwdcolor= #00ffff /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{0121} { } 0} width5offset0 misc_feature 396...427 /label= CamOcore /ApEinfo_fwdcolor= #ff2f14 /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{0121} { } 0} width5offset0 misc_feature 208...239 /label= CamOcore(1) /ApEinfo_label= CamOcore /ApEinfo_fwdcolor= #ff2f14 /ApEinfo_revcolor= green /ApEinfographicformat= arrow_data{{0121} { } 0} width5offset0 misc_feature 284...315 /label= CamOcore(2) /ApEinfo_label= CamOcore /ApEinfo_fwdcolor= #ff2f14 /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{0121} { } 0} width5offset0 misc_feature 1...203 /label= ADH1transcriptionalterminator /ApEinfo_fwdcolor= #ffec1f /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{0121} { } 0} width5offset0 misc_feature 432...574 /gene= pCYC1 /product= CYC1TATAregion /label= CYC1TATAregion /ApEinfo_fwdcolor= pink /ApEinfo_revcolor= pink /ApEinfo_graphicformat= arrow_data{{0121} { } 0} width5offset0 misc_feature 359...390 /label= CamOcore(3) /ApEinfo_label= CamOcore /ApEinfo_fwdcolor= #ff2f14 /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{0121} { } 0} width5offset0 misc_feature 246...277 /label= CamOcore(4) /ApEinfo_label= CamOcore /ApEinfo_fwdcolor= #ff2f14 /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{0121} { } 0} width5offset0 misc_feature 322...353 /label= CamOcore(5) /ApEinfo_label= CamOcore /ApEinfo_fwdcolor= #ff2f14 /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{0121} { } 0} width5offset0 misc_feature 432...577 /label= ScCYC1promoter /ApEinfo_fwdcolor= #1dff0f /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{0121} { } 0} width5offset0 misc_feature 208...430 /label= camRoperator /ApEinfo_fwdcolor= #ff8040 /ApEinfo_revcolor= #ff8040 /ApEinfo_graphicformat= arrow_data{{0121} { } 0} width5offset0 ORIGIN 1 cacttctaaataagcgaatttcttatgatttatgatttttattattaaataagttataaa 61 aaaaataagtgtatacaaattttaaagtgactcttaggttttaaaacgaaaattcttgtt 121 cttgagtaactctttcctgtaggtcaggttgctttctcaggtatagcatgaggtcgctct 181 tattgaccacacctctaccggcaaggtCAGGCTCTATATCTGCGATATACTGAGCATATC 241 CCCCCCAGGCTCTATATCTGCGATATACTGAGCATATCCCCCCCAGGCTCTATATCTGCG 301 ATATACTGAGCATATCCCCCCCAGGCTCTATATCTGCGATATACTGAGCATATCCCCCCA 361 GGCTCTATATCTGCGATATACTGAGCATATCCCCCCAGGCTCTATATCTGCGATATACTG 421 AGCATATCCCaatggcatgcatgtgctctgtatgtatataaaactcttgttttcttcttt 481 tctctaaatattctttccttatacattaggtcctttgtagcataaattactatacttcta 541 tagacacgcaaacacaaatacacacactaaattaATA(SEQIDNO:27; Table4camPr(=ADH1tr-camRoperator-CYC1pr)inpSIB859) //

TABLE-US-00041 LOCUSSEQ_ID_NO_28_Tab747bpds-DNAcircular04-DEC-2016 DEFINITION. ACCESSION VERSION SOURCE. ORGANISM. COMMENT COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers misc_feature 1...618 /label= mutatedTetR /ApEinfo_fwdcolor= cyan /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 619...744 /label= VP16 /ApEinfo_fwdcolor= #ff1fdd /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 745...747 /label= Stopcodon /ApEinfo_fwdcolor= #ff8080 /ApEinfo_revcolor= #ff8080 /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 ORIGIN 1 ATGAGTAGATTGGACAAGTCTAAGGTTATCAACGGTGCTTTGGAATTGTTGAACGGTGTT 61 GGTATCGAAGGTTTGACCACCAGAAAGTTGGCTCAAAAGTTGGGTGTTGAACAACCAACC 121 TTGTACTGGCACGTTAAGAACAAGAGAGCTTTGTTGGACGCTTTGCCAATCGAAATGTTG 181 GACAGACACCACACCCACTTCTGTCCATTGGAAGGTGAATCTTGGCAAGACTTCTTGAGA 241 AACAACGCTAAGTCTTTCAGATGTGCTTTGCTCTCTCACCGCGACGGTGCTAAGGTTCAC 301 TTGGGAACCAGACCAACCGAAAAGCAATACGAAACCTTGGAAAACCAATTGGCTTTCTTG 361 TGTCAACAAGGTTTCTCTTTGGAAAACGCTTTGTACGCTTTGTCTGCTGTTGGTCACTTC 421 ACCTTGGGTTGTGTTTTGGAAGAACAAGAACACCAAGTTGCTAAGGAAGAAAGAGAAACC 481 CCAACCACCGACTCTATGCCACCATTGCTCAGACAAGCTATCGAATTGTTCGACAGACAA 541 GGTGCTGAACCAGCTTTCTTGTTCGGTTTGGAATTGATCATCTGTGGTTTGGAAAAGCAA 601 TTGAAGTGTGAATCTGGTGGGCCGGCCGACGCTTTGGACGACTTCGACTTGGACATGTTG 661 CCAGCTGACGCTTTGGACGACTTCGACTTGGACATGTTGCCAGCTGACGCTTTGGACGAC 721 TTCGACTTGGACATGTTGCCAGGTTGA(SEQIDNO:28;Table4rtTA (=mutatedtetR-VP16)inpSIB499) //

TABLE-US-00042 LOCUSSIB027partial881bpds-DNAlinear30-SEP-2013 DEFINITION. ACCESSION VERSION SOURCE. ORGANISM. COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers misc_feature 12...871 /label= ScTDHpr /ApEinfo_fwdcolor= #baff87 /ApEinfo_revcolor= #baff87 /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 8...11 /label= Adapter(CAGT) /ApEinfo_fwdcolor= #ff1916 /ApEinfo_revcolor= #ff1916 /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 871...874 /label= Adapter(AATG) /ApEinfo_fwdcolor= ff1638 /ApEinfo_revcolor= ff1638 /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 ORIGIN 1 GGTCTCacagtCTCGATGGATTAGTTTCTCACAGGTAACATAACAAAAACCAAGAAAAGC 61 CCGCTTCTGAAAACTACAGTTGACTTGTATGCTAAAGGGCCAGACTAATGGGAGGAGAAA 121 AAGAAACGAATGTATATGCTCATTTACACTCTATATCACCATATGGAGGATAAGTTGGGC 181 TGAGCTTCTGATCCAATTTATTCTATCCATTAGTTGCTGATATGTCCCACCAGCCAACAC 241 TTGATAGTATCTACTCGCCATTCACTTCCAGCAGCGCCAGTAGGGTTGTTGAGCTTAGTA 301 AAAATGTGCGCACCACAAGCCTACATGACTCCACGTCACATGAAACCACACCGTGGGGCC 361 TTGTTGCGCTAGGAATAGGATATGCGACGAAGACGCTTCTGCTTAGTAACCACACCACAT 421 TTTCAGGGGGTCGATCTGCTTGCTTCCTTTACTGTCACGAGCGGCCCATAATCGCGCTTT 481 TTTTTTAAAAGGCGCGAGACAGCAAACAGGAAGCTCGGGTTTCAACCTTCGGAGTGGTCG 541 CAGATCTGGAGACTGGATCTTTACAATACAGTAAGGCAAGCCACCATCTGCTTCTTAGGT 601 GCATGCGACGGTATCCACGTGCAGAACAACATAGTCTGAAGAAGGGGGGGAGGAGCATGT 661 TCATTCTCTGTAGCAGTAAGAGCTTGGTGATAATGACCAAAACTGGAGTCTCGAAATCAT 721 ATAAATAGACAATATATTTTCACACAATGAGATTTGTAGTACAGTTCTATTCTCTCTCTT 781 GCATAAATAAGAAATTCATCAAGAACTTGGTTTGATATTTCACCAACACACACAAAAAAC 841 AGTACTTCACTAAATTTACACACAAAACAAAatgaGAGACC(SEQIDNO:30; Table4yGG-TDH1prinpSIB027) //

TABLE-US-00043 LOCUSSEQ_ID_NO_31_Tab792bpds-DNAlinear04-DEC-2016 DEFINITION. ACCESSION VERSION SOURCE. ORGANISM. COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers misc_feature 12...781 /gene= CMV-P /product= CMVpromotor /label= CMVpromoter /ApEinfo_fwdcolor= #fffea3 /ApEinfo_revcolor= #fffea3 /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 704...704 /label= G->T(todisruptBsmBI) /ApEinfo_fwdcolor= cyan /ApEinfo_revcolor= cyan /ApEinfo_graphicformatarrow_data{{012001} { } 0} width5offset0 misc_feature 8...11 /label= Acceptor(CAGT) /ApEinfo_fwdcolor= cyan /ApEinfo_revcolor= cyan /ApEinfo_graphicformatarrow_data{{012001} { } 0} width5offset0 misc_feature 782...785 /label= Acceptor(AATG) /ApEinfo_fwdcolor= cyan /ApEinfo_revcolor= cyan /ApEinfo_graphicformatarrow_data{{012001} { } 0} width5offset0 ORIGIN 1 GGTCTCacagtgagcttggcccattgcatacgttgtatccatatcataatatgtacattt 61 atattggctcatgtccaacattaccgccatgttgacattgattattgactagttattaat 121 agtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataac 181 ttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataa 241 tgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagt 301 atttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgcccc 361 ctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttat 421 gggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgc 481 ggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtc 541 tccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaa 601 aatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggagg 661 tctatataagcagagctcgtttagtgaaccgtcagatcgcctgTagacgccatccacgct 721 gttttgacctccatagaagacaccgggaccgatccagcctccgcggcccgaattaattca 781 tAatgaGAGACC(SEQIDNO:31;Table4yGG-mutatedCMVprin pSIB237) //

TABLE-US-00044 LOCUSSEQ_ID_NO_32_Tab725bpds-DNAlinear04-DEC-2016 DEFINITION. ACCESSION VERSION SOURCE. ORGANISM. COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers misc_feature 12...718 /label= ScGSH1tr /ApEinfo_fwdcolor= #96ff9b /ApEinfo_revcolor= green /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 715...718 /label= adapter /ApEinfo_fwdcolor= #ff304b /ApEinfo_revcolor= #ff304b /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0/ misc_feature 8...11 /label= adapter(1) /ApEinfo_label= adapter /ApEinfo_fwdcolor= #ff0000 /ApEinfo_revcolor= #ff0000 /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 ORIGIN 1 GGTCTCgtgagACTCCTTTTACTTCGGTTGTGAAAGAAAGTTGACATTATCGATTTGGGT 61 GACACGGTGATTGAAAAAGCAACGACCAGTATTATACCTCTTTTTTTTATTATTCAGTTT 121 ATATTTTTGCAAGTGATCTTAAGCATTTCTACACAAACTTATGCCAACGTGACCATTTAT 181 TATTTTATATAGCAAAAAAAAATGAGGGGCCTTGCAGAACAATTGTTGCGAGTTTCTAAT 241 AACAAGCACGTAGAATATTGGCCATTTAATTTTTCTCTTCAATTTATAGAATGGTTGTGT 301 TAGTGACAAAAAGAATATTCTTCCCCGCCAGGACTCGAACCTGGAATCTCCTGGTTCGTA 361 GCCAGACGCCGTGACCATTGGGCCACGAGGAACAAGAATATAAAGATCTCTGAGGGCAAG 421 GTATGCCTATGTCGCAATAAAATGTTTGTTCCTGCGCAAAAGTAAAGTTCTATTAATATA 481 CAACTACACAGTTATCGGTTCACACTATTCGATAGTTGTAAAAACCATTTTGATAAAGAT 541 ATAACAAGGCGTTTATTAAGGACATTTTTGCTACAAGTCGTGAAGTATTGATTGTAGGCG 601 ATCGTTGGTAACTTTCTCCATATCGGAATATTCAATATTGAACTCACCCCTCCCTTGCGA 661 TAAGCTCCTTAGCTTATTGGTGTAGGTGGTAATTTCCCTTAGTGGCACTTTCGCTTTTcG 721 AGACC(SEQIDNO:32;Table4yGG-ScGSH1trinpSIB031) //

TABLE-US-00045 LOCUSSEQ_ID_NO_33_Tab748bpds-DNAlinear04-DEC-2016 DEFINITION. ACCESSION VERSION SOURCE. ORGANISM. COMMENT COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers misc_feature 8...11 /label= Adapter /ApEinfo_fwdcolor= #ff0000 /ApEinfo_revcolor= #ff0000 /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 738...741 /label= adapter /ApEinfo_fwdcolor= #ff0000 /ApEinfo_revcolor= #ff0000 /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 12...741 /label= mutatedScSTR1tr(BsmBI-free) /ApEinfo_fwdcolor= #afffdd /ApEinfo_revcolor= #afffdd /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 ORIGIN 1 GGTCTCgtgagTCGCCAGTGCCAtGTtTCTGCCTTCGACCGGACCTTTTTAAGTACGATA 61 AATATCCTTTTATAAATATATAGTCTAAAATATCCATTAATACTGTGCTCAATCAATCGT 121 GTTAGATGATTTAGTTTTTTCCAAATCGTTATTATAGTGCAGAAGTAGTATACATAAAGG 181 CATATGCATGCGATTTGGAAGTAACGCTCGCCGTAGACAAGTAAGAATGCCTGCTGTCTT 241 GAGAACCAGGTCCAAAGAATCCTCTATAGAGCAGAAGCCTGCTTCCAGAACTAGAACGAG 301 ATCAAGAAGGGGCAAGCGTGGTCGTGACGATGATGATGATGACGACGATGAGGAAAGCGA 361 TGATGCATACGATGAAGTAGGTAATGACTATGACGAGTATGCTTCAAGAGCGAAGCTGGC 421 CACCAATAGGCCCTTCGAAATAGTCGCGGGACTGCCTGCTAGTGTGGAGCTGCCCAACTA 481 TAACTCTTCGCTTACTCATCCGCAATCAATTAAAAATTCTGGGGTGCTTTACGACTCTCT 541 GGTCAGTTCCAGAAGAACCTGGGTTCAGGGTGAGATGTTTGAACTGTATTGGCGAAGACC 601 TAAGAAAATTGTTAGTGAATCTACCCCAGCAGCGACGGAGAGTCCAACATCTGGAACGAT 661 TCCTTTGATTCGAGATAAGATGCAGAAAATGTGCGATTGTGTAATGAGTGGAGGTCCTCA 721 CACGTTCAAAGTTAGACTTTTgGAGACC(SEQIDNO:33;Table4 yGG-mutatedSTR1trinpSIB206) //

TABLE-US-00046 LOCUSSEQ_ID_NO_34_Tab573bpds-DNAlinear04-DEC-2016 DEFINITION. ACCESSION VERSION SOURCE. ORGANISM. COMMENT COMMENT COMMENT ApEinfo:methylated:1 FEATURES Location/Qualifiers misc_feature 1...6 /label= BsaI /ApEinfo_fwdcolor= cyan /ApEinfo_revcolor= cyan /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 568...573 /label= BsaI(1) /ApEinfo_label= BsaI /ApEinfo_fwdcolor= cyan /ApEinfo_revcolor= cyan /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 8...11 /label= Adapter /ApEinfo_fwdcolor= #ff0000 /ApEinfo_revcolor= #ff0000 /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 563...566 /label= Adapter(1) /ApEinfo_label= Adapter /ApEinfo_fwdcolor= #ff0000 /ApEinfo_revcolor= #ff0000 /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 misc_feature 12...562 /label= SOL3tr /ApEinfo_fwdcolor= #84ff89 /ApEinfo_revcolor= #84ff89 /ApEinfo_graphicformat= arrow_data{{012001} { } 0} width5offset0 ORIGIN 1 GGTCTCaTGAGAAAAGACACACATGCGAGCTTTCGAACCTCAGATGCTAATATTACGTGT 61 TATATATACCAAACTTTATAAAATGACATAGATATTTTATGCTGTGATAGCTTTCCTGTT 121 ATGGAGAAGCTCTTCTTATTCCCCCTGTCAACTTTCATACTCTTGTAGAATTTCCTTTAT 181 GATAGGTTTATCGCTTACGAATTTAGACTTTGATGTGATGGGTTTGGCACCTGTTCTTTT 241 TCCACAACCTTTGCGTGCCTCATCAATAGCGTTTGATCTGTCGGGAAATTTGTATTTGTA 301 GAGTGCATCCTTGCACATTGTATAGACCCAATTACGCTCTTCTAACAGGTTCACGAACGA 361 TTTTATTTCAGGAACAGAGCCGATTGTACTTTTTGAACCTATAATGATCAGCTTGGATTT 421 GGCCCTTGTCATGGCAACATTGACTCTTCTTAGCTCTTTCAGCAGCGCTCCTCCATTTAA 481 TTGAGAATTTCTTCTAACCATGGAAATAATAATGCACTTTTTGTCACGACCTTGAAACTG 541 ATCAGCAGTCAAGATCTCTAGCTTTTaGAGACC(SEQIDNO:34;Table4 yGG-SOL3trinpSIB639) //

OTHER EMBODIMENTS

[0133] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0134] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

[0135] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.