LOCUS       sequence-354577-        7690 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 7690)
  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 7690)
  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: pSSa31
FEATURES             Location/Qualifiers
     source          1..7690
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      620..807
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(675..699)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        828..883
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    834..852
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    864..882
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(1083..1102)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1084..1102)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1112..1129)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1112..1128)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1121..1143)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1269..1724
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1356..1375)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1566..1587
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1855..2658)
                     /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(2659..2874)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(2898..2917)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3017..3039
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3077..3095)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    3136..3639
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        3676..3780
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3781..3849,3850..4641)
                     /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(3999..4018)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4812..5400
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5301..5320
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5554..5571
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5688..5709
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5724..5729,5730..5747,5748..5754)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5762..5778
                     /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     5767..5789
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5786..5802
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5786..5802
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5821..5841
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5823..5841
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5866..6509
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     6403..6426
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     6515..6531
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6515..6531
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
ORIGIN
        1 tgtcatacta acccattttt atagaagaag ttgctgcttt agttatcgat aacggttctc
       61 atatgaaaat ttcggtgatc cctgagcagg tggcggaaac attggatgct gagaaccatt
      121 tcattgttcg tgaagtgttc gatgtgcacc tatccgacca aggctttgaa ctatctacca
      181 gaagtgtgag cccctaccgg aaggattaca tctcggatga tgactctgat gaagactctg
      241 cttgctatgg cgcattcatc gaccaagagc ttgtcgggaa gattgaactc aactcaacat
      301 ggaacgatct agcctctatc gaacacattg ttgtgtcgca cacgcaccga ggcaaaggag
      361 tcgcgcacag tctcatcgaa tttgcgaaaa agtgggcact aagcagacag ctccttggca
      421 tacgattaga gacacaaacg aacaatgtac ctgcctgcaa tttgtacgca aaatgtggct
      481 ttactctcgg cggcattgac ctcttcacgt ataaaactag acctcaagtc tcgaacgaaa
      541 cagcgatgta ctggtactgg ttctcgggag cacaggatga cgcctaacat atagatacgt
      601 tgttgacact tctaaataag cgaatttctt atgatttatg atttttatta ttaaataagt
      661 tataaaaaaa ataagtgtat acaaatttta aagtgactct taggttttaa aacgaaaatt
      721 cttattcttg agtaactctt tcctgtaggt caggttgctt tctcaggtat agcatgaggt
      781 cgctcttatt gaccacacct ctaccggcat gcgagctcct aatttttgtt gacactctat
      841 cattgataga gttattttac cactccctat cagtgataga gaaaagtgga tcctatgttt
      901 ttaacctggg cggctaaatt gtactttata tatagttgaa acaacaggga aagggcagga
      961 taaagaaaga aattgactgc agtttcctat gatcatattt catttttgta attaacaata
     1021 taatataata cacttataaa cggtttaaaa acagctttat ctcagaaaag gtacccaatt
     1081 cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact
     1141 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     1201 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     1261 gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     1321 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     1381 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     1441 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     1501 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     1561 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     1621 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     1681 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttcctgat gcggtatttt
     1741 ctccttacgc atctgtgcgg tatttcacac cgcatagggt aataactgat ataattaaat
     1801 tgaagctcta atttgtgagt ttagtataca tgcatttact tataatacag ttttttagtt
     1861 ttgctggccg catcttctca aatatgcttc ccagcctgct tttctgtaac gttcaccctc
     1921 taccttagca tcccttccct ttgcaaatag tcctcttcca acaataataa tgtcagatcc
     1981 tgtagagacc acatcatcca cggttctata ctgttgaccc aatgcgtctc ccttgtcatc
     2041 taaacccaca ccgggtgtca taatcaacca atcgtaacct tcatctcttc cacccatgtc
     2101 tctttgagca ataaagccga taacaaaatc tttgtcgctc ttcgcaatgt caacagtacc
     2161 cttagtatat tctccagtag atagggagcc cttgcatgac aattctgcta acatcaaaag
     2221 gcctctaggt tcctttgtta cttcttctgc cgcctgcttc aaaccgctaa caatacctgg
     2281 gcccaccaca ccgtgtgcat tcgtaatgtc tgcccattct gctattctgt atacacccgc
     2341 agagtactgc aatttgactg tattaccaat gtcagcaaat tttctgtctt cgaagagtaa
     2401 aaaattgtac ttggcggata atgcctttag cggcttaact gtgccctcca tggaaaaatc
     2461 agtcaagata tccacatgtg tttttagtaa acaaattttg ggacctaatg cttcaactaa
     2521 ctccagtaat tccttggtgg tacgaacatc caatgaagca cacaagtttg tttgcttttc
     2581 gtgcatgata ttaaatagct tggcagcaac aggactagga tgagtagcag cacgttcctt
     2641 atatgtagct ttcgacatga tttatcttcg tttcctgcag gtttttgttc tgtgcagttg
     2701 ggttaagaat actgggcaat ttcatgtttc ttcaacacta catatgcgta tatataccaa
     2761 tctaagtctg tgctccttcc ttcgttcttc cttctgttcg gagattaccg aatcaaaaaa
     2821 atttcaagga aaccgaaatc aaaaaaaaga ataaaaaaaa aatgatgaat tgaaaaggtg
     2881 gtatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     2941 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3001 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3061 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     3121 atggtttctt agacggatcg cttgcctgta acttacacgc gcctcgtatc ttttaatgat
     3181 ggaataattt gggaatttac tctgtgttta tttattttta tgttttgtat ttggatttta
     3241 gaaagtaaat aaagaaggta gaagagttac ggaatgaaga aaaaaaaata aacaaaggtt
     3301 taaaaaattt caacaaaaag cgtactttac atatatattt attagacaag aaaagcagat
     3361 taaatagata tacattcgat taacgataag taaaatgtaa aatcacagga ttttcgtgtg
     3421 tggtcttcta cacagacaag atgaaacaat tcggcattaa tacctgagag caggaagagc
     3481 aagataaaag gtagtatttg ttggcgatcc ccctagagtc ttttacatct tcggaaaaca
     3541 aaaactattt tttctttaat ttcttttttt actttctatt tttaatttat atatttatat
     3601 taaaaaattt aaattataat tatttttata gcacgtgatg aaaaggaccc aggtggcact
     3661 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3721 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3781 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3841 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     3901 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     3961 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     4021 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     4081 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     4141 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     4201 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     4261 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     4321 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     4381 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     4441 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     4501 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     4561 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     4621 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     4681 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4741 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4801 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4861 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4921 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     4981 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5041 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     5101 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     5161 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     5221 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     5281 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     5341 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     5401 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     5461 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     5521 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     5581 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     5641 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     5701 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     5761 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc
     5821 gcaattaacc ctcactaaag ggaacaaaag ctggagctca gtttatcatt atcaatactc
     5881 gccatttcaa agaatacgta aataattaat agtagtgatt ttcctaactt tatttagtca
     5941 aaaaattagc cttttaattc tgctgtaacc cgtacatgcc caaaataggg ggcgggttac
     6001 acagaatata taacatcgta ggtgtctggg tgaacagttt attcctggca tccactaaat
     6061 ataatggagc ccgcttttta agctggcatc cagaaaaaaa aagaatccca gcaccaaaat
     6121 attgttttct tcaccaacca tcagttcata ggtccattct cttagcgcaa ctacagagaa
     6181 caggggcaca aacaggcaaa aaacgggcac aacctcaatg gagtgatgca acctgcctgg
     6241 agtaaatgat gacacaaggc aattgaccca cgcatgtatc tatctcattt tcttacacct
     6301 tctattacct tctgctctct ctgatttgga aaaagctgaa aaaaaaggtt gaaaccagtt
     6361 ccctgaaatt attcccctac ttgactaata agtatataaa gacggtaggt attgattgta
     6421 attctgtaaa tctatttctt aaacttctta aattctactt ttatagttag tctttttttt
     6481 agttttaaaa caccagaact tagtttcgac ggattctaga actagtggat cccccgggct
     6541 gcaggaattc cctgtcaaaa ctagagaata ataaagaaaa cgatcttttc aaaaagaaaa
     6601 aaccttttag ttttcctttg ttgttgttgt gggtgtgtgc tatttatatt atatagttta
     6661 ctcataatac cataaaatat tcggtttgat taggttattt taataagcta atttgtttct
     6721 aatcgtgtaa tttatgctgt gtatattaag tagtgtgtgc actgcccaaa aatgtttgtt
     6781 gtttatagtc ggttaaagag aaaaaagaaa aaaagatcca tacacacacg ttaattagtt
     6841 gttcaacgta atacactcat attttgttct tatttgcttt cggtcgctgt tctcaccaag
     6901 atttattgcc aacgaaacaa ttttttttta tatattttca gatttttctt tttttccttt
     6961 cctttccttt tctaattttc actcctggtt ttctttcttt cttagaaaca ttatctcgat
     7021 attaatatta aaaaaatata atcattcaaa atggacggtg gtatgtttta gtttagcttc
     7081 aattctaatt gattgattaa tcagttgatt ggtttcaata tgacaaatgg gtagggtggg
     7141 aaaacttcat tttcaattca gatcaaactt ttttgttgtc gacataatat ttctcgtttg
     7201 ggatgttact gtcacattaa taatacacac acatcagctt ataattttga aagtaattta
     7261 tcagatatgt tgtgacgatc agtggaaatg gctaacttca atgtatctgt tcttcccctt
     7321 tttcaaagtt cacgtttttt gattgattga ttgatctgtc ggcagtggtt tcaaaaccat
     7381 tcggtgagta atcctatcaa tcaatgttac gacaaaaggc tcaatattca aaattgcaat
     7441 gttttatgtt ttcctacgtg tacttgtgca aggcaattga ttcaacattg cttttggtgt
     7501 ttgacgagtt tctagtttgg acttgtgttg ttatctgggc tatacagatt tcccggctca
     7561 ctatgaattt tttttttcga cgctcagtgc acacaactat aaacaacaca aacacaaaca
     7621 cagcaagaaa aaaaaaaaac gaacattgaa ttgaaaccaa gccaactgaa aaattcctta
     7681 tttaaatgac
//
