LOCUS       sequence-370762-        9425 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plugging the mechanism of tetracycline-mediated repression of GAL 
            promoters in Saccharomyces cerevisiae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9425)
  AUTHORS   Peng B, Bandari NC, Lu Z, Howard CB, Scott C, Trau M, Dumsday G, 
            Vickers CE
  TITLE     Engineering eukaryote-like regulatory circuits to expand artificial 
            control mechanisms for metabolic engineering in Saccharomyces 
            cerevisiae.
  JOURNAL   Commun Biol. 2022 Feb 16;5(1):135. doi: 10.1038/s42003-022-03070-z.
  PUBMED    35173283
REFERENCE   2  (bases 1 to 9425)
  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: "Commun 
            Biol."; date: "2022-02-16"; volume: "5(1):135. doi"; pages: " 
            10.1038/s42003-022-03070-z"
COMMENT     Sequence Label: pIRTetR-GAL80
FEATURES             Location/Qualifiers
     source          1..9425
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    497..515
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    524..542
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    558..576
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    584..602
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     primer_bind     complement(1549..1565)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1549..1565)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1562..1584)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1573..1589
                     /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(1597..1603,1604..1621,1622..1627))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1642..1663
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1780..1797)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1951..2539)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2031..2050)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2710..3501,3502..3570))
                     /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     3333..3352
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3571..3675)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3743..3761
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3799..3821)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3921..3940
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4134..4156
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4148..4165
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4149..4165
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     gene            4507..5863
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        4507..4850
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             4851..5660
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4918..4937)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      5666..5863
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
ORIGIN
        1 tttaccttct gtattaatca catgtatatc gcggccgcca gaaagcgacc acccaacttt
       61 ggctgataat agcgtataaa caatgcatac tttgtacgtt caaaatacaa tgcagtagat
      121 atatttatgc atattacata taatacatat cacataggaa gcaacaggcg cgttggactt
      181 ttaattttcg aggaccgcga atccttacat cacacccaat cccccacaag tgatccccca
      241 cacaccatag cttcaaaatg tttctactcc ttttttactc ttccagattt tctcggactc
      301 cgcgcatcgc cgtaccactt caaaacaccc aagcacagca tactaaattt cccctctttc
      361 ttcctctagg gtgtcgttaa ttacccgtac taaaggtttg gaaaagaaaa aagagaccgc
      421 ctcgtttctt tttcttcgtc gaaaaaggca ataaaaattt ttatcacgtt ctacctgaat
      481 ctaagattgg gccctaactc tatcattgat agagtaattg gtatccctat cagtgataga
      541 gataagcggg ggcggtatcc ctatcagtga tagagagggc ttaactctat cattgataga
      601 gtgccggcaa tcctcgagca gattcttttt cttgaaaatt tttttttttg atttttttct
      661 ctttcgatga cctcccattg atatttaagt taataaacgg tcttcaattt ctcaagtttc
      721 agtttcattt ttcttgttct attacaactt tttttacttc ttgctcatta gaaagaaagc
      781 atagcaatct aatctaagtt ttaattacaa aactagtaaa atggactaca acaagagatc
      841 ttcggtctca accgtgccta atgcagctcc cataagagtc ggattcgtcg gtctcaacgc
      901 agccaaagga tgggcaatca agacacatta ccccgccata ctgcaactat cgtcacaatt
      961 tcaaatcact gccttataca gtccaaaaat tgagacttct attgccacca tccagcgtct
     1021 aaaattgagt aatgccactg cttttcccac tttagagtca tttgcatcat cttccactat
     1081 agatatgata gtgatagcta tccaagtggc cagtcattat gacgttgtta tgcctctctt
     1141 ggaattctcc aaaaataatc cgaacctcaa gtatcttttc gtagaatggg cccttgcatg
     1201 ttcactagat caagccgaat ccatttataa ggctgctgct gaacgtgggg ttcaaaccat
     1261 catctcttta caaggtcgta aatcaccata tattttgaga gcaaaagaat taatatctca
     1321 aggctatatc ggcgacatta attctatcga gattgctgga aatggcggtt ggtacggcta
     1381 cgaaaggcct gttaaatcac caaaatacat ctatgaaatc gggaacggtg tagatctggt
     1441 aaccacaaca tttggtcaca caatcgatat tttacaatac atgacaagtt cgtacttttc
     1501 caggataaat gcaatggttt aaacgcatgc aagcttggcg taatcatggt catagctgtt
     1561 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     1621 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     1681 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     1741 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     1801 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     1861 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     1921 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     1981 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     2041 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     2101 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     2161 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     2221 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     2281 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     2341 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     2401 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     2461 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     2521 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     2581 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     2641 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     2701 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     2761 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     2821 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     2881 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     2941 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     3001 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     3061 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     3121 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     3181 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     3241 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     3301 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     3361 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     3421 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     3481 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     3541 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     3601 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     3661 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     3721 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt
     3781 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     3841 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     3901 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg
     3961 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt
     4021 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     4081 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
     4141 acgacgttgt aaaacgacgg ccagtgaatt cgtttaaacc aacctaaagg tattaacttc
     4201 ttcactataa gaaaatcaca cgagcgcccg gacgatgtct ctgtttaaat ggcgcaagtt
     4261 ttccgctttg taatatatat ttatacccct ttcttctctc ccctgcaata taatagttta
     4321 attctaatat taataatatc ctatattttc ttcatttacc ggcgcactct cgcccgaacg
     4381 acctcaaaat gtctgctaca ttcataataa ccaaaagctc ataacagggt aatgaacctg
     4441 aatatatata catcattaat tccatgggtt tagcttgcct cgtccccgcc gggtcacccg
     4501 gccagcgaca tggaggccca gaataccctc cttgacagtc ttgacgtgcg cagctcaggg
     4561 gcatgatgtg actgtcgccc gtacatttag cccatacatc cccatgtata atcatttgca
     4621 tccatacatt ttgatggccg cacggcgcga agcaaaaatt acggctcctc gctgcagacc
     4681 tgcgagcagg gaaacgctcc cctcacagac gcgttgaatt gtccccacgc cgcgcccctg
     4741 tagagaaata taaaaggtta ggatttgcca ctgaggttct tctttcatat acttcctttt
     4801 aaaatcttgc taggatacag ttctcacatc acatccgaac ataaacaacc atgggtaagg
     4861 aaaagactca cgtttcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt
     4921 ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga
     4981 agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta
     5041 cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc
     5101 attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc ggcaaaacag
     5161 cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag
     5221 tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg
     5281 tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt
     5341 ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataagcttt
     5401 tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt
     5461 ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat
     5521 accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac
     5581 ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga
     5641 tgctcgatga gtttttctaa tcagtactga caataaaaag attcttgttt tcaagaactt
     5701 gtcatttgta tagttttttt atattgtagt tgttctattt taatcaaatg ttagcgtgat
     5761 ttatattttt tttcgcctcg acatcatctg cccagatgcg aagttaagtg cgcagaaagt
     5821 aatatcatgc gtcaatcgta tgtgaatgct ggtcgctata ctgctgtcga ttcgatacta
     5881 acgccgccat ccagtgtcga aaactaggga taacagggta atgaacctga atatatatac
     5941 atcattaatt aatacaatga aaaacaacac caagctgcca aaagctgtat attaaagtta
     6001 aagctccctc atttttagta aggacgcttt cttttcccac aaaagggacg aggaaaaaaa
     6061 gaaaaacctg gaaatatagt cacgtgacat gaataaacaa gacctagggc aatatggaaa
     6121 aagaagctag gataaaaata catttatgag ggttgtaagg caaagtggct cagcattaag
     6181 tagggctaaa aagagtcgca aatatacatc gtaccgagtg atgaacgtgg ttttgaacac
     6241 cttgttctct tttgttctcg ctccctacat tcactatata ttagaagaaa tttcacctca
     6301 atggacaact cgagaaatga atacagaaat atgtttttta gcgaaatttt cctttcttct
     6361 tgtcttcttg ttttatttaa acttccaagg ctttaactca gtgtcaaaca taacaacctc
     6421 ctcctccccc acctacgaca acaaccgcca ctccgcggat catgtccaga ttggataagt
     6481 ccaaggttat taactctgcc ttggaattgt tgaacgaagt tggtattgaa ggtttgacca
     6541 ctagaaagtt ggctcaaaaa ttgggtgttg aacaacctac cttgtactgg catgttaaga
     6601 acaaaagagc tttgttggac gctttggcca ttgaaatgtt ggatagacat catacccatt
     6661 tctgtccatt ggaaggtgaa tcatggcaag atttcttgag aaacaacgcc aagtctttca
     6721 gatgtgcttt gttgtctcat agagatggtg ctaaagttca cttgggtact agaccaactg
     6781 aaaagcaata cgaaactttg gaaaaccagt tggctttctt gtgtcaacaa ggtttctctt
     6841 tagaaaacgc cttgtatgct ttgtctgctg ttggtcattt taccttgggt tgtgttttgg
     6901 aagatcaaga acatcaagtc gccaaagaag aaagggaaac tccaactact gattctatgc
     6961 caccattatt gagacaagcc attgagttgt ttgatcatca aggtgctgaa ccagcttttt
     7021 tgtttggttt ggaattgatc atctgcggtt tggagaaaca gttgaaatgt gaatctggct
     7081 cctctacctc tacaactgct aaaagaaaga agagaaagtt gactgccagc gtttcgaata
     7141 cgcagaataa gctgaatgag cttctcgatg ccatcagaca ggagtttctc caagtctcac
     7201 aagaggcaaa tacctaccgt cttcaaaacc aaaaggatta cgatttcaaa atgaaccagc
     7261 agctggctga gatgcagcag ataagaaaca ccgtctacga actggagcta actcacagga
     7321 aaatgaagga cgcgtacgaa gaagagatca agcacttgaa actagggctg gagcaaagag
     7381 accatcaaat tgcatctttg accgtccagc aacagcagca acagcaacag cagcaacagc
     7441 aacagcagca acaggtccag cagcatttac aacagcaaca acagcagcta gccgctgcat
     7501 ctgcatctgt tccagttgcg caacaaccac cggctactac ttcggccacc gccactccag
     7561 cagcaaacac aactactggt tcgccatcgg ccttcccagt acaagctagc cgtcctaatc
     7621 tggttggctc acagttgcct accaccactt tgcctgtggt gtcctcaaac gcccaacaac
     7681 aactaccaca acagcaactg caacagcagc aacttcaaca acagcaacca cctccccagg
     7741 tttccgtggc accattgagt aacacagcca tcaacggatc tcctacttct aaagagacca
     7801 ctactttacc ctctgtcaag gcacctgaat ctacgttgaa agaaactgaa ccggaaaata
     7861 ataatacctc gaagataaat gacaccggat ccgccaccac ggccaccact accaccgcaa
     7921 ctgaaactga aatcaaacct aaggaggaag acgccacccc ggctagtttg caccaggatc
     7981 actacttagt cccttataat caaagagcaa accactctaa acctatccca cctttccttt
     8041 tggatctaga ttcccagtct gttcccgatg ctctgaagaa gcaaacaaat gattattata
     8101 ttttatacaa cccggcacta ccaagagaaa ttgacgttga gttacacaaa tctttggatc
     8161 atacttcagt tgtttgttgc gtgaagttca gtaacgatgg tgaatactta gccacaggct
     8221 gcaacaaaac tactcaagtg tatcgcgttt cagacggttc tctggtggcc cgtttatctg
     8281 acgattctgc tgccaataac catcgaaatt cgatcactga aaataacacc accacgtcca
     8341 cggataacaa tacaatgaca accactacta ccaccacaat tactaccaca gcgatgactt
     8401 cggcagcaga attggcaaaa gatgtggaaa acctgaacac ttcgtcttcc ccatcatccg
     8461 acttgtatat ccgttcagtg tgtttttctc cagatgggaa atttttggca acaggtgctg
     8521 aagacagact gattagaatt tgggatattg aaaatagaaa gattgttatg attcttcaag
     8581 gccacgaaca agatatttat tcattggact actttccctc aggtgacaaa ttagtctccg
     8641 gttctggtga ccgtaccgtt cgtatttggg acttacgtac aggccagtgt tcattgactt
     8701 tatccattga agatggtgtt accaccgtcg ctgtatcacc aggtgatggt aaatacatcg
     8761 ctgctggttc tctagatcgt gctgtgagag tttgggattc cgagaccgga ttcttggtgg
     8821 aaagactaga ttcggaaaac gaatccggta caggccacaa ggactctgtt tatagcgttg
     8881 tcttcacgag agatggacaa agcgttgtat ccggctcatt agatagatct gttaagctct
     8941 ggaatttgca gaatgcaaac aacaagagcg attcgaaaac tccaaattcc ggcacttgtg
     9001 aagttacgta tatcgggcat aaagactttg tattgtccgt ggccaccaca caaaatgatg
     9061 agtacatctt gtccggttcc aaagatcgtg gtgtcctgtt ttgggataag aaatccggca
     9121 atccgttatt gatgttgcaa ggtcatagga attcagttat atctgtggct gtggcaaacg
     9181 ggtctccgct gggtccagaa tataacgttt ttgctactgg tagcggtgat tgtaaagcaa
     9241 ggatttggaa gtataaaaaa atagcgccaa attaacctgc aggataccca gttgagaaga
     9301 cgagctattt ttctcatgtt tcttttgttt attcactttt ctatactttt tcttttctat
     9361 tgaaatatat attatatctt atgtatgttt ttttgtatta taaacaagca aatttcgtct
     9421 tgcca
//
