LOCUS       sequence-457117-        7795 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive mutant rTetTA expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7795)
  AUTHORS   Tominaga M, Shima Y, Nozaki K, Ito Y, Someda M, Shoya Y, Hashii N, 
            Obata C, Matsumoto-Kitano M, Suematsu K, Matsukawa T, Hosoya K, 
            Hashiba N, Kondo A, Ishii J
  TITLE     Designing strong inducible synthetic promoters in yeasts.
  JOURNAL   Nat Commun. 2024 Dec 19;15(1):10653. doi: 
            10.1038/s41467-024-54865-z.
  PUBMED    39702268
REFERENCE   2  (bases 1 to 7795)
  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: "2024-12-19"; volume: "15(1):10653. doi"; pages: " 
            10.1038/s41467-024-54865-z"
COMMENT     Sequence Label: pMT1821
FEATURES             Location/Qualifiers
     source          1..7795
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    46..79
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            134..1490
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        134..477
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             478..1287
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      1293..1490
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    1553..1586
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1629..1648)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1630..1648)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1658..1674)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1667..1689)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(2336..2369)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(2839..2855)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2954..2971)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3125..3713)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3205..3224)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3835..4626,4627..4695))
                     /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     4458..4477
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4696..4787)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4854..4873
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5080..5113
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     terminator      complement(5830..6077)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     6043..6061
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             join(6748..7365,7366..7371,7372..7407,7408..7410,7411..7446,
                     7447..7449,7450..7485,7486..7494)
                     /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      7515..7785
                     /gene="S. cerevisae PGK1"
                     /label=PGK1 terminator
                     /note="transcription terminator for 3-phosphoglycerate 
                     kinase gene"
ORIGIN
        1 aattgcaaag ggaaaagctg aatgggcagt tcgaaaaccc ttaatataac ttcgtataat
       61 gtatgctata cgaagttatt aggtctagag atctgtttag cttgcctcgt ccccgccggg
      121 tcacccggcc agcgacatgg aggcccagaa taccctcctt gacagtcttg acgtgcgcag
      181 ctcaggggca tgatgtgact gtcgcccgta catttagccc atacatcccc atgtataatc
      241 atttgcatcc atacattttg atggccgcac ggcgcgaagc aaaaattacg gctcctcgct
      301 gcagacctgc gagcagggaa acgctcccct cacagacgcg ttgaattgtc cccacgccgc
      361 gcccctgtag agaaatataa aaggttagga tttgccactg aggttcttct ttcatatact
      421 tccttttaaa atcttgctag gatacagttc tcacatcaca tccgaacata aacaaccatg
      481 ggtaaggaaa agactcacgt ttcgaggccg cgattaaatt ccaacatgga tgctgattta
      541 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg
      601 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
      661 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
      721 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggc
      781 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
      841 ctggcagtgt tcctgcgccg gttacattcg attcctgttt gtaattgtcc ttttaacagc
      901 gaccgtgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
      961 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
     1021 aagcttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
     1081 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
     1141 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
     1201 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
     1261 catttgatgc tcgatgagtt tttctaatca gtactgacaa taaaaagatt cttgttttca
     1321 agaacttgtc atttgtatag tttttttata ttgtagttgt tctattttaa tcaaatgtta
     1381 gcgtgattta tatttttttt cgcctcgaca tcatctgccc agatgcgaag ttaagtgcgc
     1441 agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt cgctatactg ctgtcgattc
     1501 gatactaacg ccgccatcca gtgtcgaaaa cgagctctcg agaaccctta atataacttc
     1561 gtataatgta tgctatacga agttattagg tgatatcaga tccactagtg gcctatgcac
     1621 ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc
     1681 gtgactggga aaaccctggc gttacccctg caggactagt gctgaggcat taatctacta
     1741 tcggcgactc tctcgaaatt tttcttaacg cgtccttgta ctgcgtctaa cgcttttgcc
     1801 acttggattt ctattatagg aaatagtctc acttactggg cgacgaattt tcgcgttttg
     1861 atgaagcaca ggaagaattt cttttttttt tggcttcttc tggttccgtt ttttacgcgc
     1921 acaaatctaa aaaaagaaat aattataacc tagtctcgaa aattttcatc gatccattcg
     1981 ttcctttttt tcgatttttt cagatcaaaa ttcttgtttc tttctttgtc ttagtttata
     2041 ttaaaagata ttttgatttt actcctgaac tatttattct ttctaagaag gccagaacac
     2101 tacagctgtt ttaaccgact acgaagttct ccattctcga acactagcct tcatttacca
     2161 aacaggaact agcgtatatc attagtcctt attcgaaaag agattggtag atatttattg
     2221 tagtttgtga gaaggagaaa atactgtcat tggactgata gttagaggac attaacctct
     2281 cttacgttcg ctcattaaga cctcagcgcg gccgcaaatt taaataaaat gaagtgaagt
     2341 tcctatactt tctagagaat aggaacttct atagtgagtc gaataagggc gacacaaaat
     2401 ttattctaaa tgcataataa atactgataa catcttatag tttgtattat attttgtatt
     2461 atcgttgaca tgtataattt tgatatcaaa aactgatttt ccctttatta ttttcgagat
     2521 ttattttctt aattctcttt aacaaactag aaatattgta tatacaaaaa atcataaata
     2581 atagatgaat agtttaatta taggtgttca tcaatcgaaa aagcaacgta tcttatttaa
     2641 agtgcgttgc ttttttctca tttataaggt taaataattc tcatatatca agcaaagtga
     2701 caggcgccct taaatattct gacaaatgct ctttccctaa actcccccca taaaaaaacc
     2761 cgccgaagcg ggtttttacg ttatttgcgg attaacgatt actcgttatc agaaccgccc
     2821 agggggcccg agcttaagac tggccgtcgt tttacaacac agaaagagtt tgtagaaacg
     2881 caaaaaggcc atccgtcagg ggccttctgc ttagtttgat gcctggcagt tccctactct
     2941 cgccttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     3001 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     3061 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     3121 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     3181 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     3241 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     3301 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     3361 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     3421 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     3481 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     3541 gggctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     3601 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     3661 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     3721 ctttgatctt ttctacgggg tctgacgctc agtggaacga cgcgcgcgta actcacgtta
     3781 agggattttg gtcatgagct tgcgccgtcc cgtcaagtca gcgtaatgct ctgcttacca
     3841 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     3901 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagcgc
     3961 tgcgatgata ccgcgagaac cacgctcacc ggctccggat ttatcagcaa taaaccagcc
     4021 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     4081 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     4141 tgccatcgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     4201 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     4261 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     4321 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     4381 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     4441 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     4501 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     4561 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     4621 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     4681 atgttgaata ctcatattct tcctttttca atattattga agcatttatc agggttattg
     4741 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggtcagtgt
     4801 tacaaccaat taaccaattc tgaacattat cgcgagccca tttatacctg aatatggctc
     4861 ataacacccc ttgtttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga
     4921 actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gactccccat gcgagagtag
     4981 ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgcccg
     5041 ggctaattat ggggtgtcgc ccttattcga ctctatagtg aagttcctat tctctagaaa
     5101 gtataggaac ttctgaagtg gggatttaaa tgcggccgcg ctgagggttt aatcggcatg
     5161 caaacatcta cacaattagc aagggcaatc catattttgt cttttcgcgc cctggaaagg
     5221 cctaagtaat gtcgtaaacg cattctatct gtacttcaac tctcctctgt gcattggttt
     5281 gtgcaaatca cattttacga tactgccaga tatatgcaaa aagagaaaac caagggacca
     5341 gaacaaagca aaattacgat attcttcgaa ttccttcgtg cttgactaag acaaagggat
     5401 ggacgtagcg atttttagcg ggccaagaac tggttccgaa aaagcacagg tacaccgaac
     5461 cctcagctaa ggagggacag caccgatgcg gaaggacaaa ctttcttttt gcctatcaca
     5521 gtatcttatc gagctaacta ttttcgacac acatgaaaaa gcagaaatat taacgaaaaa
     5581 gaaaagaaag accatgtcat gtacgggcaa tcagaatctg taacaagcgc catttttttt
     5641 tctgtatcgg gccctcctta ctgctctcct tccgtgtaac gcgttatgat taagtcctca
     5701 gcgagctcgc atggaatgcg tgcgatgagc gacctcatgc tatacctgag aaagcaacct
     5761 gacctacagg aaagagttac tcaagaataa gaattttcgt tttaaaacct aagagtcact
     5821 ttaaaatttg taaattaaag ccttcgagcg tcccaaaacc ttctcaagca aggttttcag
     5881 tataatgtta catgcgtaca cgcgtctgta cagaaaaaaa agaaaaattt gaaatataaa
     5941 taacgttctt aatactaaca taactataaa aaaataaata gggacctaga cttcaggttg
     6001 tctaactcct tccttttcgg ttagagcgga tgtgggggga gggcgtgaat gtaagcgtga
     6061 cataactaat tacatgacaa agatgccgat ttgggcgcga atcctttatt ttggcttcac
     6121 cctcatacta ttatcagggc cagaaaaagg aagtgtttcc ctccttcttg aattgatgtt
     6181 accctcataa agcacgtggc ctcttatcga gaaagaaatt accgtcgctc gtgatttgtt
     6241 tgcaaaaaga acaaaactga aaaaacccag acacgctcga cttcctgtct tcctattgat
     6301 tgcagcttcc aatttcgtca cacaacaagg tcctagcgac ggctcacagg ttttgtaaca
     6361 agcaatcgaa ggttctggaa tggcgggaaa gggtttagta ccacatgcta tgatgcccac
     6421 tgtgatctcc agagcaaagt tcgttcgatc gtactgttac tctctctctt tcaaacagaa
     6481 ttgtccgaat cgtgtgacaa caacagcctg ttctcacaca ctcttttctt ctaaccaagg
     6541 gggtggttta gtttagtaga acctcgtgaa acttacattt acatatatat aaacttgcat
     6601 aaattggtca atgcaagaaa tacatatttg gtcttttcta attcgtagtt tttcaagttc
     6661 ttagatgctt tctttttctc ttttttacag atcatcaagg aagtaattat ctacttttta
     6721 caacaaatat aaaacgctag cgtcgacatg tctagactgg acaagagcaa tgtcataaac
     6781 ggcgctctgg aattactcaa tggagtcggt atcgaaggcc tgacgacaag gaaactcgct
     6841 caaaagctgg gagttgagca gcctaccctg tactggcacg tgaagaacaa gcgggccctg
     6901 ctcgatgccc tgccaatcga gatgctggac aggcatcata cccacttctg ccccctggaa
     6961 ggcgagtcat ggcaagactt tctgcggaac aacgccaagt cattccgctg tgctctcctc
     7021 tcacatcgcg acggggctaa agtgcatctc ggcacccgcc caacagagaa acagtacgaa
     7081 accctggaaa atcagctcgc gttcctgtgt cagcaaggct tctccctgga gaacgcacta
     7141 tacgctctgt ccgccgtggg ccactttaca ctgggctgcg tattggagga acaggagcat
     7201 caagtagcaa aagaggaaag agagacacct accaccgatt ctatgccccc acttctgaga
     7261 caagcaattg agctgttcga ccggcaggga gccgaacctg ccttcctttt cggcctggaa
     7321 ctaatcatat gtggcctgga gaaacagcta aagtgcgaaa gcggcgggcc ggccgacgcc
     7381 cttgacgatt ttgacttaga catgctccca gccgatgccc ttgacgactt tgaccttgat
     7441 atgctgcctg ctgacgctct tgacgatttt gaccttgaca tgctccccgg gtaactaagt
     7501 aagatctgaa ataaattgaa ttgaattgaa atcgatagat caattttttt cttttctctt
     7561 tccccatcct ttacgctaaa ataatagttt attttatttt ttgaatattt tttatttata
     7621 tacgtatata tagactatta tttatctttt aatgattatt aagattttta ttaaaaaaaa
     7681 attcgctcct cttttaatgc ctttatgcag tttttttttc ccattcgata tttctatgtt
     7741 cgggttcagc gtattttaag tttaataact cgaaaattct gcgttcgtta aagct
//
