LOCUS       sequence-328862-        7670 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses phlTA(2-­1E) mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7670)
  AUTHORS   Tominaga M, Nozaki K, Umeno D, Ishii J, Kondo A
  TITLE     Robust and flexible platform for directed evolution of yeast genetic
            switches.
  JOURNAL   Nat Commun. 2021 Mar 23;12(1):1846. doi: 10.1038/s41467-021-22134-y.
  PUBMED    33758180
REFERENCE   2  (bases 1 to 7670)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2021-03-23"; volume: "12(1):1846. doi"; pages: " 
            10.1038/s41467-021-22134-y"
COMMENT     Sequence Label: pRS405red-­phlTA2-­1E
FEATURES             Location/Qualifiers
     source          1..7670
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(1187..2281)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(2282..2689)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(2990..3445)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3127..3148)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3339..3358
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3571..3593
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3585..3602
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3586..3602
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3612..3631
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3612..3630
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5057..5077
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      5093..5363
                     /gene="S. cerevisae PGK1"
                     /label=PGK1 terminator
                     /note="transcription terminator for 3-phosphoglycerate 
                     kinase gene"
     primer_bind     complement(5410..5430)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5410..5428)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5449..5465)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5449..5465)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5462..5484)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5473..5489
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5497..5503,5504..5521,5522..5527))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5542..5563
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5680..5697)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5851..6439)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5931..5950)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6610..7401,7402..7470))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7233..7252
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7471..7575)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7643..7661
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatagtca atgaaatgaa cattgattta ctatcaattt cttctcacaa gatttacggt
      241 ccaaagggaa taggtgccat ctatgtaaga aggagaccaa gagttagatt agaaccttta
      301 ctatccggtg gtggccaaga gagaggttta agatctggta ctttggcccc cccattggta
      361 gcgggatttg gtgaagctgc gagattgatg aagaaagaat ttgacaacga tcaagctcac
      421 atcaaaagac tatccgataa attagtcaaa ggtctattat ccgctgaaca taccacgttg
      481 aacggatctc cagatcatcg ttatccaggg tgtgttaacg tttctttcgc ctacgtggaa
      541 ggagaatctt tattgatggc actaagggat atcgcattat cctcgggttc agcctgtaca
      601 tctgcttccc tagaaccttc ttatgtttta catgcgctgg gtaaggatga tgcattagcc
      661 cattcttcca tcagatttgg tattggtaga tttagtactg aagaggagga cgactacgtc
      721 gttaaggccg tttctgacag agtaaaattc ttgagggaac tttcaccatt atgggaaatg
      781 gttcaagaag gtattgactt aaactccatc aaatggtcag gtcattgagt gttttttatt
      841 tgttgtattt tttttttttt agagaaaatc ctccaatata taaattagga atcatagttt
      901 catgattttc tgttacacct aactttttgt gtggtgccct cctccttgtc aatattaatg
      961 ttaaagtgca attctttttc cttatcacgt tgagccatta gtatcaattt gcttacctgt
     1021 attcctttac atcctccttt ttctccttct tgataaatgt atgtagattg cgtatatagt
     1081 ttcgtctacc ctatgaacat attccatttt gtaatttcgt gtcgtttcta ttatgaattt
     1141 catttataaa gtttatgtac aaatatcata aaaaaagaga atctttttaa gcaaggattt
     1201 tcttaacttc ttcggcgaca gcatcaccga cttcggtggt actgttggaa ccacctaaat
     1261 caccagttct gatacctgca tccaaaacct ttttaactgc atcttcaatg gccttacctt
     1321 cttcaggcaa gttcaatgac aatttcaaca tcattgcagc agacaagata gtggcgatag
     1381 ggttgacctt attctttggc aaatctggag cagaaccgtg gcatggttcg tacaaaccaa
     1441 atgcggtgtt cttgtctggc aaagaggcca aggacgcaga tggcaacaaa cccaaggaac
     1501 ctgggataac ggaggcttca tcggagatga tatcaccaaa catgttgctg gtgattataa
     1561 taccatttag gtgggttggg ttcttaacta ggatcatggc ggcagaatca atcaattgat
     1621 gttgaacctt caatgtaggg aattcgttct tgatggtttc ctccacagtt tttctccata
     1681 atcttgaaga ggccaaaaca ttagctttat ccaaggacca aataggcaat ggtggctcat
     1741 gttgtagggc catgaaagcg gccattcttg tgattctttg cacttctgga acggtgtatt
     1801 gttcactatc ccaagcgaca ccatcaccat cgtcttcctt tctcttacca aagtaaatac
     1861 ctcccactaa ttctctgaca acaacgaagt cagtaccttt agcaaattgt ggcttgattg
     1921 gagataagtc taaaagagag tcggatgcaa agttacatgg tcttaagttg gcgtacaatt
     1981 gaagttcttt acggattttt agtaaacctt gttcaggtct aacactaccg gtaccccatt
     2041 taggaccacc cacagcacct aacaaaacgg catcagcctt cttggaggct tccagcgcct
     2101 catctggaag tggaacacct gtagcatcga tagcagcacc accaattaaa tgattttcga
     2161 aatcgaactt gacattggaa cgaacatcag aaatagcttt aagaacctta atggcttcgg
     2221 ctgtgatttc ttgaccaacg tggtcacctg gcaaaacgac gatcttctta ggggcagaca
     2281 ttagaatggt atatccttga aatatatata tatatattgc tgaaatgtaa aaggtaagaa
     2341 aagttagaaa gtaagacgat tgctaaccac ctattggaaa aaacaatagg tccttaaata
     2401 atattgtcaa cttcaagtat tgtgatgcaa gcatttagtc atgaacgctt ctctattcta
     2461 tatgaaaagc cggttccggc gctctcacct ttcctttttc tcccaatttt tcagttgaaa
     2521 aaggtatatg cgtcaggcga cctctgaaat taacaaaaaa tttccagtca tcgaatttga
     2581 ttctgtgcga tagcgcccct gtgtgttctc gttatgttga ggaaaaaaat aatggttgct
     2641 aagagattcg aactcttgca tcttacgata cctgagtatt cccacagtta actgcggtca
     2701 agatatttct tgaatcaggc gccttagacc gctcggccaa acaaccaatt acttgttgag
     2761 aaatagagta taattatcct ataaatataa cgtttttgaa cacacatgaa caaggaagta
     2821 caggacaatt gattttgaag agaatgtgga ttttgatgta attgttggga ttccattttt
     2881 aataaggcaa taatattagg tatgtagata tactagaagt tctcctcgac cggtcgatat
     2941 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc
     3001 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     3061 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     3121 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     3181 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     3241 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     3301 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     3361 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     3421 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag
     3481 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     3541 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     3601 gtgagcgcgc gtaatacgac tcactatagg gcgaattggg taccgggccc cccctcgaga
     3661 aagatgccga tttgggcgcg aatcctttat tttggcttca ccctcatact attatcaggg
     3721 ccagaaaaag gaagtgtttc cctccttctt gaattgatgt taccctcata aagcacgtgg
     3781 cctcttatcg agaaagaaat taccgccgct cgtgatttgt ttgcaaaaag aacaaaactg
     3841 aaaaaaccca gacgcgctcg acttcctgtc ttcctattga ttgcagcttc caatttcgtc
     3901 acacaacaag gtcctagcga cggctcacag gttttgtaac aagcaatcga tggttctgga
     3961 atggcgggaa agggtttagt acacatgcta tgatgcccac tgtgatctcc agagcaaagt
     4021 tcgttcgatc gtactgttac tctctctctt tcaaacagaa ttgtccgaat cgtgtgacaa
     4081 caacagcctg ttctcacaca ctcttttctt ccaaccaagg gggtggttta gtttagtaga
     4141 acctcgtgaa acttacattt acatatatat aaacttgcat aaattggtca atgcaagtaa
     4201 tacatatttg gtcttttcta attcgtagtt tatcaagttc ttagatgctt tctttttctc
     4261 ttttttacag atcatcaagg aagtaattat ctacttttta caacaaatat aaaacgctag
     4321 cgtcgacatg gctagaactt cacccagatc atctattggt tctctgagat caccacatac
     4381 tcataaggct attttgacct ccaccatcga aatcttgaaa gaatgtggtt actccggttt
     4441 gtccattgaa tctgttgcta gacgtgctgg tgcttctaaa cctactatct acagatggtg
     4501 gactaacaag gctgctttga ttgctgaagt ttacgaaaac gaatccgaac aggttagaaa
     4561 gtttccagat ttgggttcct ttacggccga tttggatttc ttgttgagaa acttgtggaa
     4621 ggtttggaga gaaactattt gtggtgaagc cctcagatgc gttattgcag aagctcgatt
     4681 agatccagct actttgactc aattgaagga ccaattcatg gaaagacgta gagaaatgcc
     4741 aaaaaagttg gttaaaaacg ccatctctaa cggtgaattg ccaaaggata caaaccgtga
     4801 gttgttgttg gatatgattt tcggtttttg ctggtacagg ttgttgactg aacaattgac
     4861 tgttgaacaa gacatcgaag agttcacgtt cttgttaatc aatggtgttt gtccaggtac
     4921 tcaaagaggt ccagcagacg ctttggatga ttttgatttg gatatgttgc ctgctgatgc
     4981 cttggatgac ttcgacttag acatgttacc agccgatgca ttagacgatt tcgaccttga
     5041 tatgttacct ggtccaccaa aaaagaagag aaaggtctaa gatctgaaat aaattgaatt
     5101 gaattgaaat cgatagatca atttttttct tttctctttc cccatccttt acgctaaaat
     5161 aatagtttat tttatttttt gaatattttt tatttatata cgtatatata gactattatt
     5221 tatcttttaa tgattattaa gatttttatt aaaaaaaaat tcgctcctct tttaatgcct
     5281 ttatgcagtt tttttttccc attcgatatt tctatgttcg ggttcagcgt attttaagtt
     5341 taataactcg aaaattctgc gttcgttaaa gctgcggccg ccaccgcggt ggagctccag
     5401 cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt
     5461 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5521 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5581 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5641 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5701 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5761 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5821 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5881 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5941 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6001 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6061 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6121 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6181 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6241 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6301 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6361 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6421 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6481 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6541 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6601 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6661 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6721 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6781 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6841 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6901 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6961 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7021 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7081 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7141 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7201 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7261 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7321 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7381 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7441 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7501 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7561 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     7621 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
