LOCUS       sequence-354576-        7667 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tet-on genome insertion system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7667)
  AUTHORS   Schrevens S, Sanglard D
  TITLE     A novel Candida glabrata doxycycline-inducible system for in 
            vitro/in vivo use.
  JOURNAL   FEMS Yeast Res. 2022 Sep 24;22(1):foac046. doi: 
            10.1093/femsyr/foac046.
  PUBMED    36047937
REFERENCE   2  (bases 1 to 7667)
  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: "FEMS Yeast 
            Res."; date: "2022-09-24"; volume: "22(1):foac046. doi"; pages: " 
            10.1093/femsyr/foac046"
COMMENT     Sequence Label: pSSa32
FEATURES             Location/Qualifiers
     source          1..7667
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      74..261
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(129..153)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        282..337
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    288..306
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    318..336
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(514..533)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(515..533)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(543..560)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(543..559)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(552..574)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      700..1155
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(787..806)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     997..1018
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1286..2089)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(2090..2305)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(2329..2348)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2448..2470
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2508..2526)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    2567..3070
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        3107..3211
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3212..3280,3281..4072)
                     /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     complement(3430..3449)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4243..4831
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4732..4751
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4985..5002
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5119..5140
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5155..5160,5161..5178,5179..5185)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5193..5209
                     /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)."
     primer_bind     5198..5220
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5217..5233
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5217..5233
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5252..5272
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5254..5272
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5297..5940
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     5834..5857
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     5946..5962
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5946..5962
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
ORIGIN
        1 tgtactggta ctggttctcg ggagcacagg atgacgccta acatatagat acgttgttga
       61 cacttctaaa taagcgaatt tcttatgatt tatgattttt attattaaat aagttataaa
      121 aaaaataagt gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt
      181 cttgagtaac tctttcctgt aggtcaggtt gctttctcag gtatagcatg aggtcgctct
      241 tattgaccac acctctaccg gcatgcgagc tcctaatttt tgttgacact ctatcattga
      301 tagagttatt ttaccactcc ctatcagtga tagagaaaag tggatccaat tgtactttat
      361 atatagttga aacaacaggg aaagggcagg ataaagaaag aaattgactg cagtttccta
      421 tgatcatatt tcatttttgt aattaacaat ataatataat acacttataa acggtttaaa
      481 aacagcttta tctcagaaaa ggtacccaat tcgccctata gtgagtcgta ttacgcgcgc
      541 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
      601 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
      661 cgcccttccc aacagttgcg cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat
      721 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
      781 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
      841 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
      901 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
      961 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1021 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1081 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1141 taacgcttac aatttcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     1201 ccgcataggg taataactga tataattaaa ttgaagctct aatttgtgag tttagtatac
     1261 atgcatttac ttataataca gttttttagt tttgctggcc gcatcttctc aaatatgctt
     1321 cccagcctgc ttttctgtaa cgttcaccct ctaccttagc atcccttccc tttgcaaata
     1381 gtcctcttcc aacaataata atgtcagatc ctgtagagac cacatcatcc acggttctat
     1441 actgttgacc caatgcgtct cccttgtcat ctaaacccac accgggtgtc ataatcaacc
     1501 aatcgtaacc ttcatctctt ccacccatgt ctctttgagc aataaagccg ataacaaaat
     1561 ctttgtcgct cttcgcaatg tcaacagtac ccttagtata ttctccagta gatagggagc
     1621 ccttgcatga caattctgct aacatcaaaa ggcctctagg ttcctttgtt acttcttctg
     1681 ccgcctgctt caaaccgcta acaatacctg ggcccaccac accgtgtgca ttcgtaatgt
     1741 ctgcccattc tgctattctg tatacacccg cagagtactg caatttgact gtattaccaa
     1801 tgtcagcaaa ttttctgtct tcgaagagta aaaaattgta cttggcggat aatgccttta
     1861 gcggcttaac tgtgccctcc atggaaaaat cagtcaagat atccacatgt gtttttagta
     1921 aacaaatttt gggacctaat gcttcaacta actccagtaa ttccttggtg gtacgaacat
     1981 ccaatgaagc acacaagttt gtttgctttt cgtgcatgat attaaatagc ttggcagcaa
     2041 caggactagg atgagtagca gcacgttcct tatatgtagc tttcgacatg atttatcttc
     2101 gtttcctgca ggtttttgtt ctgtgcagtt gggttaagaa tactgggcaa tttcatgttt
     2161 cttcaacact acatatgcgt atatatacca atctaagtct gtgctccttc cttcgttctt
     2221 ccttctgttc ggagattacc gaatcaaaaa aatttcaagg aaaccgaaat caaaaaaaag
     2281 aataaaaaaa aaatgatgaa ttgaaaaggt ggtatggtgc actctcagta caatctgctc
     2341 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     2401 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     2461 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     2521 cctattttta taggttaatg tcatgataat aatggtttct tagacggatc gcttgcctgt
     2581 aacttacacg cgcctcgtat cttttaatga tggaataatt tgggaattta ctctgtgttt
     2641 atttattttt atgttttgta tttggatttt agaaagtaaa taaagaaggt agaagagtta
     2701 cggaatgaag aaaaaaaaat aaacaaaggt ttaaaaaatt tcaacaaaaa gcgtacttta
     2761 catatatatt tattagacaa gaaaagcaga ttaaatagat atacattcga ttaacgataa
     2821 gtaaaatgta aaatcacagg attttcgtgt gtggtcttct acacagacaa gatgaaacaa
     2881 ttcggcatta atacctgaga gcaggaagag caagataaaa ggtagtattt gttggcgatc
     2941 cccctagagt cttttacatc ttcggaaaac aaaaactatt ttttctttaa tttctttttt
     3001 tactttctat ttttaattta tatatttata ttaaaaaatt taaattataa ttatttttat
     3061 agcacgtgat gaaaaggacc caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3121 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3181 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     3241 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     3301 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     3361 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     3421 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     3481 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     3541 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     3601 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     3661 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     3721 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     3781 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     3841 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     3901 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     3961 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     4021 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     4081 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     4141 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     4201 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4261 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4321 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4381 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4441 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4501 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     4561 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     4621 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4681 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     4741 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     4801 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     4861 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     4921 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     4981 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     5041 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     5101 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     5161 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     5221 aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa
     5281 gctggagctc agtttatcat tatcaatact cgccatttca aagaatacgt aaataattaa
     5341 tagtagtgat tttcctaact ttatttagtc aaaaaattag ccttttaatt ctgctgtaac
     5401 ccgtacatgc ccaaaatagg gggcgggtta cacagaatat ataacatcgt aggtgtctgg
     5461 gtgaacagtt tattcctggc atccactaaa tataatggag cccgcttttt aagctggcat
     5521 ccagaaaaaa aaagaatccc agcaccaaaa tattgttttc ttcaccaacc atcagttcat
     5581 aggtccattc tcttagcgca actacagaga acaggggcac aaacaggcaa aaaacgggca
     5641 caacctcaat ggagtgatgc aacctgcctg gagtaaatga tgacacaagg caattgaccc
     5701 acgcatgtat ctatctcatt ttcttacacc ttctattacc ttctgctctc tctgatttgg
     5761 aaaaagctga aaaaaaaggt tgaaaccagt tccctgaaat tattccccta cttgactaat
     5821 aagtatataa agacggtagg tattgattgt aattctgtaa atctatttct taaacttctt
     5881 aaattctact tttatagtta gtcttttttt tagttttaaa acaccagaac ttagtttcga
     5941 cggattctag aactagtgga tcccccgggc tgcaggaatt ccctgtcaaa actagagaat
     6001 aataaagaaa acgatctttt caaaaagaaa aaacctttta gttttccttt gttgttgttg
     6061 tgggtgtgtg ctatttatat tatatagttt actcataata ccataaaata ttcggtttga
     6121 ttaggttatt ttaataagct aatttgtttc taatcgtgta atttatgctg tgtatattaa
     6181 gtagtgtgtg cactgcccaa aaatgtttgt tgtttatagt cggttaaaga gaaaaaagaa
     6241 aaaaagatcc atacacacac gttaattagt tgttcaacgt aatacactca tattttgttc
     6301 ttatttgctt tcggtcgctg ttctcaccaa gatttattgc caacgaaaca attttttttt
     6361 atatattttc agatttttct ttttttcctt tcctttcctt ttctaatttt cactcctggt
     6421 tttctttctt tcttagaaac attatctcga tattaatatt aaaaaaatat aatcattcaa
     6481 aatggacggt ggtatgtttt agtttagctt caattctaat tgattgatta atcagttgat
     6541 tggtttcaat atgacaaatg ggtagggtgg gaaaacttca ttttcaattc agatcaaact
     6601 tttttgttgt cgacataata tttctcgttt gggatgttac tgtcacatta ataatacaca
     6661 cacatcagct tataattttg aaagtaattt atcagatatg ttgtgacgat cagtggaaat
     6721 ggctaacttc aatgtatctg ttcttcccct ttttcaaagt tcacgttttt tgattgattg
     6781 attgatctgt cggcagtggt ttcaaaacca ttcggtgagt aatcctatca atcaatgtta
     6841 cgacaaaagg ctcaatattc aaaattgcaa tgttttatgt tttcctacgt gtacttgtgc
     6901 aaggcaattg attcaacatt gcttttggtg tttgacgagt ttctagtttg gacttgtgtt
     6961 gttatctggg ctatacagat ttcccggctc actatgaatt ttttttttcg acgctcagtg
     7021 cacacaacta taaacaacac aaacacaaac acagcaagaa aaaaaaaaaa cgaacattga
     7081 attgaaacca agccaactga aaaattcctt atttaaatga ctgtcatact aacccatttt
     7141 tatagaagaa gttgctgctt tagttatcga taacggttct catatgaaaa tttcggtgat
     7201 ccctgagcag gtggcggaaa cattggatgc tgagaaccat ttcattgttc gtgaagtgtt
     7261 cgatgtgcac ctatccgacc aaggctttga actatctacc agaagtgtga gcccctaccg
     7321 gaaggattac atctcggatg atgactctga tgaagactct gcttgctatg gcgcattcat
     7381 cgaccaagag cttgtcggga agattgaact caactcaaca tggaacgatc tagcctctat
     7441 cgaacacatt gttgtgtcgc acacgcaccg aggcaaagga gtcgcgcaca gtctcatcga
     7501 atttgcgaaa aagtgggcac taagcagaca gctccttggc atacgattag agacacaaac
     7561 gaacaatgta cctgcctgca atttgtacgc aaaatgtggc tttactctcg gcggcattga
     7621 cctcttcacg tataaaacta gacctcaagt ctcgaacgaa acagcga
//
