LOCUS       sequence-328819-        6713 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 6713)
  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 6713)
  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: pGK401red-­rtetTAK8N, L131L
FEATURES             Location/Qualifiers
     source          1..6713
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        897..1247
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     CDS             1248..2582
                     /codon_start=1
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /product="O-acetylserine and O-acetylhomoserine 
                     sulfhydrylase, required for sulfur amino acid synthesis"
                     /label=MET17
                     /translation="MPSHFDTVQLHAGQENPGDNAHRSRAVPIYATTSYVFENSKHGSQ
                     LFGLEVPGYVYSRFQNPTSNVLEERIAALEGGAAALAVSSGQAAQTLAIQGLAHTGDNI
                     VSTSYLYGGTYNQFKISFKRFGIEARFVEGDNPEEFEKVFDERTKAVYLETIGNPKYNV
                     PDFEKIVAIAHKHGIPVVVDNTFGAGGYFCQPIKYGADIVTHSATKWIGGHGTTIGGII
                     VDSGKFPWKDYPEKFPQFSQPAEGYHGTIYNEAYGNLAYIVHVRTELLRDLGPLMNPFA
                     SFLLLQGVETLSLRAERHGENALKLAKWLEQSPYVSWVSYPGLASHSHHENAKKYLSNG
                     FGGVLSFGVKDLPNADKETDPFKLSGAQVVDNLKLASNLANVGDAKTLVIAPYFTTHKQ
                     LNDKEKLASGVTKDLIRVSVGIEFIDDIIADFQQSFETVFAGQKP"
     CDS             join(3438..4055,4056..4061,4062..4097,4098..4100,4101..4136,
                     4137..4139,4140..4175,4176..4184)
                     /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      4205..4475
                     /gene="S. cerevisae PGK1"
                     /label=PGK1 terminator
                     /note="transcription terminator for 3-phosphoglycerate 
                     kinase gene"
     primer_bind     complement(4522..4542)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4522..4540)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4561..4577)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4561..4577)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4574..4596)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4585..4601
                     /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(4609..4615,4616..4633,4634..4639))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4654..4675
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4792..4809)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4963..5551)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5043..5062)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5722..6513,6514..6582))
                     /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     6345..6364
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6583..6687)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cctcaaagtt actgacgaag cttatgatgg tttggttatt gttattggta gatggattca
       61 agggtttttg tcccaaaaga ataataacac tattgaaggc ttgaacttca tcagattact
      121 tgaaaaagat tcaggtaaat ctgagttctt attaagctac ggtaaggaag taaacaaaat
      181 tccacaaagc tacgaaaatt tgaagaaagg ttccactgta accagcaatg ggttgaactg
      241 ggaagttatt gaatatcacg cttaataaag gagaataaat cgttttctac tttcttctgc
      301 tgctataata agcacctatg ggatctatat agtattttta taacgataga ctttataaaa
      361 gaaaatacct aagtgaaaat ttggtgaatt ttgagataat tgttgggatt ccatttttaa
      421 taaggcaata atattaggta tgtagaatat actagaagtt ctcctcgagg atttaggaat
      481 ccataaaagg gaatctgcaa ttctacacaa ttctataaat attattatca tcgttttata
      541 tgttaatatt cattgatcct attacattat caatccttgc gtttcagctt ccactaattt
      601 agatgactat ttctcatcat ttgcgtcatc ttctaacacc gtatatgata atatactagt
      661 aacgtaaata ctagttagta gatgatagtt gatttttatt ccaacactaa gaaataattt
      721 cgccatttct tgaatgtatt taaagatatt taatgctata atagacattt aaatccaatt
      781 cttccaacat acaatgggag tttggccgag tggtttaagg cgtcagattt aggtggattt
      841 aacctctaaa atctctgata tcttcggatg caagggttcg aatcccttag ctctcattat
      901 tttttgcttt ttctcttgag gtcacatgat cgcaaaatgg caaatggcac gtgaagctgt
      961 cgatattggg gaactgtggt ggttggcaaa tgactaatta agttagtcaa ggcgccatcc
     1021 tcatgaaaac tgtgtaacat aataaccgaa gtgtcgaaaa ggtggcacct tgtccaattg
     1081 aacacgctcg atgaaaaaaa taagatatat ataaggttaa gtaaagcgtc tgttagaaag
     1141 gaagtttttc ctttttcttg ctctcttgtc ttttcatcta ctatttcctt cgtgtaatac
     1201 agggtcgtca gatacataga tacaattcta ttacccccat ccatacaatg ccatctcatt
     1261 tcgatactgt tcaactacac gccggccaag agaaccctgg tgacaatgct cacagatcca
     1321 gagctgtacc aatttacgcc accacttctt atgttttcga aaactctaag catggttcgc
     1381 aattgtttgg tctagaagtt ccaggttacg tctattcccg tttccaaaac ccaaccagta
     1441 atgttttgga agaaagaatt gctgctttag aaggtggtgc tgctgctttg gctgtttcct
     1501 ccggtcaagc cgctcaaacc cttgccatcc aaggtttggc acacactggt gacaacatcg
     1561 tttccacttc ttacttatac ggtggtactt ataaccagtt caaaatctcg ttcaaaagat
     1621 ttggtatcga ggctagattt gttgaaggtg acaatccaga agaattcgaa aaggtctttg
     1681 atgaaagaac caaggctgtt tatttggaaa ccattggtaa tccaaagtac aatgttccgg
     1741 attttgaaaa aattgttgca attgctcaca aacacggtat tccagttgtc gttgacaaca
     1801 catttggtgc cggtggttac ttctgtcagc caattaaata cggtgctgat attgtaacac
     1861 attctgctac caaatggatt ggtggtcatg gtactactat cggtggtatt attgttgact
     1921 ctggtaagtt cccatggaag gactacccag aaaagttccc tcaattctct caacctgccg
     1981 aaggatatca cggtactatc tacaatgaag cctacggtaa cttggcatac atcgttcatg
     2041 ttagaactga actattaaga gatttgggtc cattgatgaa cccatttgcc tctttcttgc
     2101 tactacaagg tgttgaaaca ttatctttga gagctgaaag acacggtgaa aatgcattga
     2161 agttagccaa atggttagaa caatccccat acgtatcttg ggtttcatac cctggtttag
     2221 catctcattc tcatcatgaa aatgctaaga agtatctatc taacggtttc ggtggtgtct
     2281 tatctttcgg tgtaaaagac ttaccaaatg ccgacaagga aactgaccca ttcaaacttt
     2341 ctggtgctca agttgttgac aatttaaagc ttgcctctaa cttggccaat gttggtgatg
     2401 ccaagacctt agtcattgct ccatacttca ctacccacaa acaattaaat gacaaagaaa
     2461 agttggcatc tggtgttacc aaggacttaa ttcgtgtctc tgttggtatc gaatttattg
     2521 atgacattat tgcagacttc cagcaatctt ttgaaactgt tttcgctggc caaaaaccat
     2581 gagtgtgcgt aatgagttgt aaaattatgt ataaacctac tttctctcac aagtactata
     2641 cttttataaa acgaacttta ttgaaatgaa tatccttttt ttcccttgtt acatgtcgtg
     2701 actcgtactt tgaacctaaa ttgttctaac atcaaagaac agtgttaatt cgcagtcgag
     2761 aagtcgagaa agatgccgat ttgggcgcga atcctttatt ttggcttcac cctcatacta
     2821 ttatcagggc cagaaaaagg aagtgtttcc ctccttcttg aattgatgtt accctcataa
     2881 agcacgtggc ctcttatcga gaaagaaatt accgtcgctc gtgatttgtt tgcaaaaaga
     2941 acaaaactga aaaaacccag acacgctcga cttcctgtct tcctattgat tgcagcttcc
     3001 aatttcgtca cacaacaagg tcctagcgac ggctcacagg ttttgtaaca agcaatcgaa
     3061 ggttctggaa tggcgggaaa gggtttagta ccacatgcta tgatgcccac tgtgatctcc
     3121 agagcaaagt tcgttcgatc gtactgttac tctctctctt tcaaacagaa ttgtccgaat
     3181 cgtgtgacaa caacagcctg ttctcacaca ctcttttctt ctaaccaagg gggtggttta
     3241 gtttagtaga acctcgtgaa acttacattt acatatatat aaacttgcat aaattggtca
     3301 atgcaagaaa tacatatttg gtcttttcta attcgtagtt tttcaagttc ttagatgctt
     3361 tctttttctc ttttttacag atcatcaagg aagtaattat ctacttttta caacaaatat
     3421 aaaacgctag cgtcgacatg tctagactgg acaagagcaa tgtcataaac ggcgctctgg
     3481 aattactcaa tggagtcggt atcgaaggcc tgacgacaag gaaactcgct caaaagctgg
     3541 gagttgagca gcctaccctg tactggcacg tgaagaacaa gcgggccctg ctcgatgccc
     3601 tgccaatcga gatgctggac aggcatcata cccacttctg ccccctggaa ggcgagtcat
     3661 ggcaagactt tctgcggaac aacgccaagt cattccgctg tgctctcctc tcacatcgcg
     3721 acggggctaa agtgcatctc ggcacccgcc caacagagaa acagtacgaa accctggaaa
     3781 atcagctcgc gttcctgtgt cagcaaggct tctccctgga gaacgcacta tacgctctgt
     3841 ccgccgtggg ccactttaca ctgggctgcg tattggagga acaggagcat caagtagcaa
     3901 aagaggaaag agagacacct accaccgatt ctatgccccc acttctgaga caagcaattg
     3961 agctgttcga ccggcaggga gccgaacctg ccttcctttt cggcctggaa ctaatcatat
     4021 gtggcctgga gaaacagcta aagtgcgaaa gcggcgggcc ggccgacgcc cttgacgatt
     4081 ttgacttaga catgctccca gccgatgccc ttgacgactt tgaccttgat atgctgcctg
     4141 ctgacgctct tgacgatttt gaccttgaca tgctccccgg gtaactaagt aagatctgaa
     4201 ataaattgaa ttgaattgaa atcgatagat caattttttt cttttctctt tccccatcct
     4261 ttacgctaaa ataatagttt attttatttt ttgaatattt tttatttata tacgtatata
     4321 tagactatta tttatctttt aatgattatt aagattttta ttaaaaaaaa attcgctcct
     4381 cttttaatgc ctttatgcag tttttttttc ccattcgata tttctatgtt cgggttcagc
     4441 gtattttaag tttaataact cgaaaattct gcgttcgtta aagctgcggc cgccaccgcg
     4501 gtggagctcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg
     4561 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4621 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4681 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4741 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4801 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4861 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4921 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4981 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5041 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5101 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5161 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5221 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5281 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5341 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5401 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5461 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5521 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5581 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5641 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5701 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5761 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5821 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5881 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5941 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6001 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6061 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6121 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6181 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6241 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6301 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6361 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6421 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6481 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6541 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6601 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6661 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctg
//
