LOCUS       sequence-328836-        7809 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses luxTA(2-­4F) mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7809)
  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 7809)
  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-­luxTA2-­4F
FEATURES             Location/Qualifiers
     source          1..7809
                     /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             3935..3955
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      4718..4988
                     /gene="S. cerevisae PGK1"
                     /label=PGK1 terminator
                     /note="transcription terminator for 3-phosphoglycerate 
                     kinase gene"
     primer_bind     complement(5035..5055)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5035..5053)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5074..5090)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5074..5090)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5087..5109)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5098..5114
                     /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(5122..5128,5129..5146,5147..5152))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(5305..5322)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5476..6064)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5556..5575)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6235..7026,7027..7095))
                     /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     6858..6877
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7096..7200)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    7237..7740
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     7782..7800
                     /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 cgtcgctcgt gatttgtttg
     3301 caaaaagaac aaaactgaaa aaacccagac acgctcgact tcctgtcttc ctattgattg
     3361 cagcttccaa tttcgtcaca caacaaggtc ctagcgacgg ctcacaggtt ttgtaacaag
     3421 caatcgaagg ttctggaatg gcgggaaagg gtttagtacc acatgctatg atgcccactg
     3481 tgatctccag agcaaagttc gttcgatcgt actgttactc tctctctttc aaacagaatt
     3541 gtccgaatcg tgtgacaaca acagcctgtt ctcacacact cttttcttct aaccaagggg
     3601 gtggtttagt ttagtagaac ctcgtgaaac ttacatttac atatatataa acttgcataa
     3661 attggtcaat gcaagaaata catatttggt cttttctaat tcgtagtttt tcaagttctt
     3721 agatgctttc tttttctctt ttttacagat caccaaggaa gtaattatct actttttaca
     3781 acaaatataa aacgctagcg tcgacatgcc agccgatgct ttggatgatt tcgatttgga
     3841 tatgttgcct gctgatgcat tggacgattt tgacttagac atgttaccag cagacgcatt
     3901 ggatgacttc gaccttgata tgctaccagg tccaccaaaa aagaagagaa aggttgaaaa
     3961 catcaacgct gatgacacct acagaatcat caacaagatt aaggcttgca ggtctaacaa
     4021 cgatattaac cagtgtttgt ccgacatgac caagatggtt cattgcgaat attacttgtt
     4081 ggccatcatc tacccacact ctatggttaa gtccgacatt tccattttgg acaactaccc
     4141 aaaaaaatgg cgtcagtatt acgatgatgc caacttgatt aagtacgacc caatcgttga
     4201 ttactccaac tctaatcatt ctcccatcaa ctggaacatc ttcgaaaaca atgccgtcaa
     4261 caaaaagtcc ccaaacgtta tcaaagaagc taagacctat ggtttgatta ccggtttttc
     4321 tttcccaatt cataccgcta acaatggttt cggtatgttg tcttttgccc actctgaaaa
     4381 ggataactac atcgactcat tattcttgca tgcctgcatg aacattccat tgatagttcc
     4441 atctctggtc gataactata ggaagattaa cattgccaac aacaagtcca acaacgattt
     4501 gaccaaaagg gaaaaagaat gtttggcttg ggcttgcgaa ggtaaatctt ctagggatat
     4561 ttccaagatt ttgggttgct ctgaaagaac cgttactttc catttgacta acgcccaaat
     4621 gaagttgaac actaccaaca gatgccagtc tatttctaag gctattttga ccggtgctat
     4681 tgattgtcca tactttaaga attaagatct gaaataaatt gaattgaatt gaaatcgata
     4741 gatcaatttt tttcttttct ctttccccat cctttacgct aaaataatag tttattttat
     4801 tttttgaata ttttttattt atatacgtat atatagacta ttatttatct tttaatgatt
     4861 attaagattt ttattaaaaa aaaattcgct cctcttttaa tgcctttatg cagttttttt
     4921 ttcccattcg atatttctat gttcgggttc agcgtatttt aagcttaata actcgaaaat
     4981 tctgcgttcg ttaaagctgc ggccgccacc gcggtggagc tccagctttt gttcccttta
     5041 gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     5101 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     5161 tgcctaaaga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     5221 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5281 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5341 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5401 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5461 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5521 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5581 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5641 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5701 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5761 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5821 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5881 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     5941 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     6001 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     6061 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6121 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     6181 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6241 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6301 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6361 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6421 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6481 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6541 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6601 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6661 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6721 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6781 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6841 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6901 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6961 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     7021 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     7081 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     7141 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     7201 cacatttccc cgaaaagtgc cacctgggtc cttttcatca cgtgctataa aaataattat
     7261 aatttaaatt ttttaatata aatatataaa ttaaaaatag aaagtaaaaa aagaaattaa
     7321 agaaaaaata gtttttgttt tccgaagatg taaaagactc tagggggatc gccaacaaat
     7381 actacctttt atcttgctct tcctgctctc aggtattaat gccgaattgt ttcatcttgt
     7441 ctgtgtagaa gaccacacac gaaaatcctg tgattttaca ttttacttat cgttaatcga
     7501 atgtatatct atttaatctg cttttcttgt ctaataaata tatatgtaaa gtacgctttt
     7561 tgttgaaatt ttttaaacct ttgtttattt ttttttcttc attccgtaac tcttctacct
     7621 tctttattta ctttctaaaa tccaaataca aaacataaaa ataaataaac acagagtaaa
     7681 ttcccaaatt attccatcat taaaagatac gaggcgcgtg taagttacag gcaagcgatc
     7741 cgtcctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     7801 cctttcgtc
//
