LOCUS       sequence-328820-        7664 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses rTetTA (K8N, L131L) mutant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7664)
  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 7664)
  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-­rtetTAK8N, L131L
FEATURES             Location/Qualifiers
     source          1..7664
                     /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             join(3806..4423,4424..4429,4430..4465,4466..4468,4469..4504,
                     4505..4507,4508..4543,4544..4552)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSNVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     terminator      4573..4843
                     /gene="S. cerevisae PGK1"
                     /label=PGK1 terminator
                     /note="transcription terminator for 3-phosphoglycerate 
                     kinase gene"
     primer_bind     complement(4890..4910)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4890..4908)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4929..4945)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4929..4945)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4942..4964)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4953..4969
                     /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(4977..4983,4984..5001,5002..5007))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5022..5043
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5160..5177)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5331..5919)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5411..5430)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6090..6881,6882..6950))
                     /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     6713..6732
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6951..7055)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    7092..7595
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     7637..7655
                     /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 catcaaggaa gtaattatct actttttaca
     3781 acaaatataa aacgctagcg tcgacatgtc tagactggac aagagcaatg tcataaacgg
     3841 cgctctggaa ttactcaatg gagtcggtat cgaaggcctg acgacaagga aactcgctca
     3901 aaagctggga gttgagcagc ctaccctgta ctggcacgtg aagaacaagc gggccctgct
     3961 cgatgccctg ccaatcgaga tgctggacag gcatcatacc cacttctgcc ccctggaagg
     4021 cgagtcatgg caagactttc tgcggaacaa cgccaagtca ttccgctgtg ctctcctctc
     4081 acatcgcgac ggggctaaag tgcatctcgg cacccgccca acagagaaac agtacgaaac
     4141 cctggaaaat cagctcgcgt tcctgtgtca gcaaggcttc tccctggaga acgcactata
     4201 cgctctgtcc gccgtgggcc actttacact gggctgcgta ttggaggaac aggagcatca
     4261 agtagcaaaa gaggaaagag agacacctac caccgattct atgcccccac ttctgagaca
     4321 agcaattgag ctgttcgacc ggcagggagc cgaacctgcc ttccttttcg gcctggaact
     4381 aatcatatgt ggcctggaga aacagctaaa gtgcgaaagc ggcgggccgg ccgacgccct
     4441 tgacgatttt gacttagaca tgctcccagc cgatgccctt gacgactttg accttgatat
     4501 gctgcctgct gacgctcttg acgattttga ccttgacatg ctccccgggt aactaagtaa
     4561 gatctgaaat aaattgaatt gaattgaaat cgatagatca atttttttct tttctctttc
     4621 cccatccttt acgctaaaat aatagtttat tttatttttt gaatattttt tatttatata
     4681 cgtatatata gactattatt tatcttttaa tgattattaa gatttttatt aaaaaaaaat
     4741 tcgctcctct tttaatgcct ttatgcagtt tttttttccc attcgatatt tctatgttcg
     4801 ggttcagcgt attttaagtt taataactcg aaaattctgc gttcgttaaa gctgcggccg
     4861 ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg
     4921 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4981 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     5041 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5101 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5161 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5221 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5281 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5341 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5401 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5461 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5521 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5581 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5641 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5701 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5761 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5821 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5881 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5941 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6001 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6061 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6121 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6181 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6241 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6301 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6361 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6421 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6481 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6541 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6601 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6661 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6721 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6781 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6841 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6901 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6961 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7021 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7081 gggtcctttt catcacgtgc tataaaaata attataattt aaatttttta atataaatat
     7141 ataaattaaa aatagaaagt aaaaaaagaa attaaagaaa aaatagtttt tgttttccga
     7201 agatgtaaaa gactctaggg ggatcgccaa caaatactac cttttatctt gctcttcctg
     7261 ctctcaggta ttaatgccga attgtttcat cttgtctgtg tagaagacca cacacgaaaa
     7321 tcctgtgatt ttacatttta cttatcgtta atcgaatgta tatctattta atctgctttt
     7381 cttgtctaat aaatatatat gtaaagtacg ctttttgttg aaatttttta aacctttgtt
     7441 tatttttttt tcttcattcc gtaactcttc taccttcttt atttactttc taaaatccaa
     7501 atacaaaaca taaaaataaa taaacacaga gtaaattccc aaattattcc atcattaaaa
     7561 gatacgaggc gcgtgtaagt tacaggcaag cgatccgtcc taagaaacca ttattatcat
     7621 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtc
//
