LOCUS       sequence-328832-        7661 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 7661)
  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 7661)
  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: pGK415-­phlTA2-­1E
FEATURES             Location/Qualifiers
     source          1..7661
                     /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(664..1758)
                     /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(1759..2166)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(2467..2922)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2604..2625)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2816..2835
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3048..3070
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3062..3079
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3063..3079
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3089..3108
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3089..3107
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4534..4554
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      4570..4840
                     /gene="S. cerevisae PGK1"
                     /label=PGK1 terminator
                     /note="transcription terminator for 3-phosphoglycerate 
                     kinase gene"
     primer_bind     complement(4887..4907)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4887..4905)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4926..4942)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4926..4942)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4939..4961)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4950..4966
                     /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(4974..4980,4981..4998,4999..5004))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5019..5040
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5157..5174)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5328..5916)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5408..5427)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6087..6878,6879..6947))
                     /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     6710..6729
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6948..7052)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    7089..7592
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     7634..7652
                     /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 accatatcga ctacgtcgtt aaggccgttt ctgacagagt aaaattcttg agggaacttt
      241 caccattatg ggaaatggtt caagaaggta ttgacttaaa ctccatcaaa tggtcaggtc
      301 attgagtgtt ttttatttgt tgtatttttt tttttttaga gaaaatcctc caatatataa
      361 attaggaatc atagtttcat gattttctgt tacacctaac tttttgtgtg gtgccctcct
      421 ccttgtcaat attaatgtta aagtgcaatt ctttttcctt atcacgttga gccattagta
      481 tcaatttgct tacctgtatt cctttacatc ctcctttttc tccttcttga taaatgtatg
      541 tagattgcgt atatagtttc gtctacccta tgaacatatt ccattttgta atttcgtgtc
      601 gtttctatta tgaatttcat ttataaagtt tatgtacaaa tatcataaaa aaagagaatc
      661 tttttaagca aggattttct taacttcttc ggcgacagca tcaccgactt cggtggtact
      721 gttggaacca cctaaatcac cagttctgat acctgcatcc aaaacctttt taactgcatc
      781 ttcaatggcc ttaccttctt caggcaagtt caatgacaat ttcaacatca ttgcagcaga
      841 caagatagtg gcgatagggt tgaccttatt ctttggcaaa tctggagcag aaccgtggca
      901 tggttcgtac aaaccaaatg cggtgttctt gtctggcaaa gaggccaagg acgcagatgg
      961 caacaaaccc aaggaacctg ggataacgga ggcttcatcg gagatgatat caccaaacat
     1021 gttgctggtg attataatac catttaggtg ggttgggttc ttaactagga tcatggcggc
     1081 agaatcaatc aattgatgtt gaaccttcaa tgtagggaat tcgttcttga tggtttcctc
     1141 cacagttttt ctccataatc ttgaagaggc caaaacatta gctttatcca aggaccaaat
     1201 aggcaatggt ggctcatgtt gtagggccat gaaagcggcc attcttgtga ttctttgcac
     1261 ttctggaacg gtgtattgtt cactatccca agcgacacca tcaccatcgt cttcctttct
     1321 cttaccaaag taaatacctc ccactaattc tctgacaaca acgaagtcag tacctttagc
     1381 aaattgtggc ttgattggag ataagtctaa aagagagtcg gatgcaaagt tacatggtct
     1441 taagttggcg tacaattgaa gttctttacg gatttttagt aaaccttgtt caggtctaac
     1501 actaccggta ccccatttag gaccacccac agcacctaac aaaacggcat cagccttctt
     1561 ggaggcttcc agcgcctcat ctggaagtgg aacacctgta gcatcgatag cagcaccacc
     1621 aattaaatga ttttcgaaat cgaacttgac attggaacga acatcagaaa tagctttaag
     1681 aaccttaatg gcttcggctg tgatttcttg accaacgtgg tcacctggca aaacgacgat
     1741 cttcttaggg gcagacatta gaatggtata tccttgaaat atatatatat atattgctga
     1801 aatgtaaaag gtaagaaaag ttagaaagta agacgattgc taaccaccta ttggaaaaaa
     1861 caataggtcc ttaaataata ttgtcaactt caagtattgt gatgcaagca tttagtcatg
     1921 aacgcttctc tattctatat gaaaagccgg ttccggcgct ctcacctttc ctttttctcc
     1981 caatttttca gttgaaaaag gtatatgcgt caggcgacct ctgaaattaa caaaaaattt
     2041 ccagtcatcg aatttgattc tgtgcgatag cgcccctgtg tgttctcgtt atgttgagga
     2101 aaaaaataat ggttgctaag agattcgaac tcttgcatct tacgatacct gagtattccc
     2161 acagttaact gcggtcaaga tatttcttga atcaggcgcc ttagaccgct cggccaaaca
     2221 accaattact tgttgagaaa tagagtataa ttatcctata aatataacgt ttttgaacac
     2281 acatgaacaa ggaagtacag gacaattgat tttgaagaga atgtggattt tgatgtaatt
     2341 gttgggattc catttttaat aaggcaataa tattaggtat gtagatatac tagaagttct
     2401 cctcgaccgg tcgatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     2461 atcaggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     2521 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     2581 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     2641 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     2701 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     2761 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     2821 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     2881 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc cattcaggct
     2941 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     3001 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     3061 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac
     3121 cgggcccccc ctcgagaaag atgccgattt gggcgcgaat cctttatttt ggcttcaccc
     3181 tcatactatt atcagggcca gaaaaaggaa gtgtttccct ccttcttgaa ttgatgttac
     3241 cctcataaag cacgtggcct cttatcgaga aagaaattac cgccgctcgt gatttgtttg
     3301 caaaaagaac aaaactgaaa aaacccagac gcgctcgact tcctgtcttc ctattgattg
     3361 cagcttccaa tttcgtcaca caacaaggtc ctagcgacgg ctcacaggtt ttgtaacaag
     3421 caatcgatgg ttctggaatg gcgggaaagg gtttagtaca catgctatga tgcccactgt
     3481 gatctccaga gcaaagttcg ttcgatcgta ctgttactct ctctctttca aacagaattg
     3541 tccgaatcgt gtgacaacaa cagcctgttc tcacacactc ttttcttcca accaaggggg
     3601 tggtttagtt tagtagaacc tcgtgaaact tacatttaca tatatataaa cttgcataaa
     3661 ttggtcaatg caagtaatac atatttggtc ttttctaatt cgtagtttat caagttctta
     3721 gatgctttct ttttctcttt tttacagatc atcaaggaag taattatcta ctttttacaa
     3781 caaatataaa acgctagcgt cgacatggct agaacttcac ccagatcatc tattggttct
     3841 ctgagatcac cacatactca taaggctatt ttgacctcca ccatcgaaat cttgaaagaa
     3901 tgtggttact ccggtttgtc cattgaatct gttgctagac gtgctggtgc ttctaaacct
     3961 actatctaca gatggtggac taacaaggct gctttgattg ctgaagttta cgaaaacgaa
     4021 tccgaacagg ttagaaagtt tccagatttg ggttccttta cggccgattt ggatttcttg
     4081 ttgagaaact tgtggaaggt ttggagagaa actatttgtg gtgaagccct cagatgcgtt
     4141 attgcagaag ctcgattaga tccagctact ttgactcaat tgaaggacca attcatggaa
     4201 agacgtagag aaatgccaaa aaagttggtt aaaaacgcca tctctaacgg tgaattgcca
     4261 aaggatacaa accgtgagtt gttgttggat atgattttcg gtttttgctg gtacaggttg
     4321 ttgactgaac aattgactgt tgaacaagac atcgaagagt tcacgttctt gttaatcaat
     4381 ggtgtttgtc caggtactca aagaggtcca gcagacgctt tggatgattt tgatttggat
     4441 atgttgcctg ctgatgcctt ggatgacttc gacttagaca tgttaccagc cgatgcatta
     4501 gacgatttcg accttgatat gttacctggt ccaccaaaaa agaagagaaa ggtctaagat
     4561 ctgaaataaa ttgaattgaa ttgaaatcga tagatcaatt tttttctttt ctctttcccc
     4621 atcctttacg ctaaaataat agtttatttt attttttgaa tattttttat ttatatacgt
     4681 atatatagac tattatttat cttttaatga ttattaagat ttttattaaa aaaaaattcg
     4741 ctcctctttt aatgccttta tgcagttttt ttttcccatt cgatatttct atgttcgggt
     4801 tcagcgtatt ttaagtttaa taactcgaaa attctgcgtt cgttaaagct gcggccgcca
     4861 ccgcggtgga gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
     4921 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4981 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     5041 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5101 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     5161 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     5221 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     5281 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     5341 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     5401 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     5461 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     5521 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     5581 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5641 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5701 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5761 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5821 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5881 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5941 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6001 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6061 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     6121 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     6181 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     6241 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     6301 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     6361 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     6421 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     6481 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     6541 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     6601 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6661 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     6721 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6781 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6841 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6901 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6961 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7021 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctggg
     7081 tccttttcat cacgtgctat aaaaataatt ataatttaaa ttttttaata taaatatata
     7141 aattaaaaat agaaagtaaa aaaagaaatt aaagaaaaaa tagtttttgt tttccgaaga
     7201 tgtaaaagac tctaggggga tcgccaacaa atactacctt ttatcttgct cttcctgctc
     7261 tcaggtatta atgccgaatt gtttcatctt gtctgtgtag aagaccacac acgaaaatcc
     7321 tgtgatttta cattttactt atcgttaatc gaatgtatat ctatttaatc tgcttttctt
     7381 gtctaataaa tatatatgta aagtacgctt tttgttgaaa ttttttaaac ctttgtttat
     7441 ttttttttct tcattccgta actcttctac cttctttatt tactttctaa aatccaaata
     7501 caaaacataa aaataaataa acacagagta aattcccaaa ttattccatc attaaaagat
     7561 acgaggcgcg tgtaagttac aggcaagcga tccgtcctaa gaaaccatta ttatcatgac
     7621 attaacctat aaaaataggc gtatcacgag gccctttcgt c
//
