LOCUS       sequence-349994-        7582 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses optimized unleaky reverse tetracycline transactivator 
            (rtTA-M2-SE-G72P) under control of yeast TDH3 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7582)
  AUTHORS   Barre B, Gladyshev V
  TITLE     Improved reverse tetracycline transactivator plasmids
  JOURNAL   (unpublished)
  PUBMED    27323850
REFERENCE   2  (bases 1 to 7582)
  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: 
            "(unpublished)"
COMMENT     Sequence Label: p2B-55
FEATURES             Location/Qualifiers
     source          1..7582
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        194..409
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             410..1213
                     /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"
     rep_origin      complement(1344..1799)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1481..1502)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1693..1712
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1925..1947
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1939..1956
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1940..1956
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1966..1985
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1966..1984
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2009..2025
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2010..2026
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2035..2702
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     2572..2595
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     CDS             join(2703..3323,3324..3707)
                     /codon_start=1
                     /product="tetracycline-controlled transactivator, 
                     comprising a fusion of the tetracycline repressor TetR with
                     the C-terminal activation domain of herpes simplex virus 
                     VP16"
                     /label=tTA
                     /note="In the Tet-Off(R) system, tTA binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="MSRLDKSKIINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEPESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTI
                     EGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHA
                     DALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     primer_bind     complement(2776..2795)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3927..3943)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3927..3943)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3980..4000)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3980..3998)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4019..4035)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4019..4035)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4032..4054)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4043..4059
                     /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(4067..4073,4074..4091,4092..4097))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4112..4133
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4250..4267)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4421..5009)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4501..4520)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5180..5971,5972..6040))
                     /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     5803..5822
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6041..6145)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6172..7514
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(7135..7182)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     7555..7573
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataccac cttttcaatt catcattttt tttttattct tttttttgat ttcggtttcc
      241 ttgaaatttt tttgattcgg taatctccga acagaaggaa gaacgaagga aggagcacag
      301 acttagattg gtatatatac gcatatgtag tgttgaagaa acatgaaatt gcccagtatt
      361 cttaacccaa ctgcacagaa caaaaacctg caggaaacga agataaatca tgtcgaaagc
      421 tacatataag gaacgtgctg ctactcatcc tagtcctgtt gctgccaagc tatttaatat
      481 catgcacgaa aagcaaacaa acttgtgtgc ttcattggat gttcgtacca ccaaggaatt
      541 actggagtta gttgaagcat taggtcccaa aatttgttta ctaaaaacac atgtggatat
      601 cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat ccgccaagta
      661 caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag tcaaattgca
      721 gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg cacacggtgt
      781 ggtgggccca ggtattgtta gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc
      841 tagaggcctt ttgatgttag cagaattgtc atgcaagggc tccctatcta ctggagaata
      901 tactaagggt actgttgaca ttgcgaagag cgacaaagat tttgttatcg gctttattgc
      961 tcaaagagac atgggtggaa gagatgaagg ttacgattgg ttgattatga cacccggtgt
     1021 gggtttagat gacaagggag acgcattggg tcaacagtat agaaccgtgg atgatgtggt
     1081 ctctacagga tctgacatta ttattgttgg aagaggacta tttgcaaagg gaagggatgc
     1141 taaggtagag ggtgaacgtt acagaaaagc aggctgggaa gcatatttga gaagatgcgg
     1201 ccagcaaaac taaaaaactg tattataagt aaatgcatgt atactaaact cacaaattag
     1261 agcttcaatt taattatatc agttattacc ctatgcggtg tgaaataccg cacagatgcg
     1321 taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt
     1381 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     1441 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     1501 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     1561 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     1621 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     1681 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     1741 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     1801 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     1861 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     1921 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta
     1981 tagggcgaat tgggtaccgg gccccccctc gaggtcgacg gtatcgataa gctttcatta
     2041 tcaatactgc catttcaaag aatacgtaaa taattaatag tagtgatttt cctaacttta
     2101 tttagtcaaa aaattagcct tttaattctg ctgtaacccg tacatgccca aaataggggg
     2161 cgggttacac agaatatata acatcgtagg tgtctgggtg aacagtttat tcctggcatc
     2221 cactaaatat aatggagccc gctttttaag ctggcatcca gaaaaaaaaa agaatcccag
     2281 caccaaaata ttgttttctt caccaaccat cagttcatag gtccattctc ttagcgcaac
     2341 tacagagaac aggggcacaa acaggcaaaa aacgggcaca acctcaatgg agtgatgcaa
     2401 cctgcctgga gtaaatgatg acacaaggca attgacccac gcatgtatct atctcatttt
     2461 cttacacctt ctattacctt ctgctctctc tgatttggaa aaagctgaaa aaaaaggttg
     2521 aaaccagttc cctgaaatta ttcccctact tgactaataa gtatataaag acggtaggta
     2581 ttgattgtaa ttctgtaaat ctatttctta aacttcttaa attctacttt tatagttagt
     2641 ctttttttta gttttaaaac accaagaact tagtttcgaa taaacacaca taaacaaaca
     2701 aaatgtctag attagataaa agtaaaatta ttaacggcgc attagagctg cttaatgggg
     2761 tcggaatcga aggtttaaca acccgtaaac tcgcccagaa gctaggtgta gagcagccta
     2821 cattgtattg gcatgtaaaa aataagcggg ctttgctcga cgccttaccc attgagatgt
     2881 tagataggca ccatactcac tcttgccctt tagaacctga aagctggcaa gattttttac
     2941 gtaataacgc taaaagttat agatgtgctt tactaagtca tcgcgatgga gcaaaagtac
     3001 atttaggtac acggcctaca gaaaaacagt atgaaactct cgaaaatcaa ttagcctttt
     3061 tatgccaaca aggtttttca ctagagaatg cattatatgc actcagcgct gtggggcact
     3121 ttactttagg ttgcgtattg gaagaacaag agcatcaagt cgctaaagaa gaaagggaaa
     3181 cacctactac tgatagtatg ccgccattat taaaacaagc tatcgaatta tttgatcgcc
     3241 aaggtgcaga gccagccttc ttattcggcc ttgaattgat catatgcgga ttagaaaaac
     3301 aacttaaatg tgaaagtggg tccgcgtaca gccgcgcgcg tacgaaaaac aattacgggt
     3361 ctaccatcga gggcctgctc gatctcccgg acgacgacgc ccccgaagag gcggggctgg
     3421 cggctccgcg cctgtccttt ctccccgcgg gacacacgcg cagactgtcg acggcccccc
     3481 cgaccgatgt cagcctgggg gacgagctcc acttagacgg cgaggacgtg gcgatggcgc
     3541 atgccgacgc gctagacgat ttcgatctgg acatgttggg ggacggggat tccccgggtc
     3601 cgggatttac cccccacgac tccgccccct acggcgctct ggatatggcc gacttcgagt
     3661 ttgagcagat gtttaccgat gcccttggaa ttgacgagta cggtgggact agataaattg
     3721 aattgaattg aaatcgatag atcaattttt ttcttttctc tttccccatc ctttacgcta
     3781 aaataatagt ttattttatt ttttgaatat tttttattta tatacgtata tatagactat
     3841 tatttatctt ttaatgatta ttaagatttt tattaaaaaa aaattcgctc ctcttttaat
     3901 gcctttagaa ttcctgcagc ccgggggatc cactagttct agagcggccg ccaccgcggt
     3961 ggagctccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt
     4021 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4081 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4141 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4201 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4261 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4321 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4381 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4441 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4501 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4561 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4621 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4681 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4741 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4801 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4861 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4921 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4981 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5041 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5101 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5161 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5221 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5281 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5341 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5401 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5461 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5521 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5581 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5641 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5701 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5761 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5821 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5881 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5941 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6001 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6061 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6121 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gaacgaagca
     6181 tctgtgcttc attttgtaga acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag
     6241 aatctgagct gcatttttac agaacagaaa tgcaacgcga aagcgctatt ttaccaacga
     6301 agaatctgtg cttcattttt gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa
     6361 caaagaatct gagctgcatt tttacagaac agaaatgcaa cgcgagagcg ctattttacc
     6421 aacaaagaat ctatacttct tttttgttct acaaaaatgc atcccgagag cgctattttt
     6481 ctaacaaagc atcttagatt actttttttc tcctttgtgc gctctataat gcagtctctt
     6541 gataactttt tgcactgtag gtccgttaag gttagaagaa ggctactttg gtgtctattt
     6601 tctcttccat aaaaaaagcc tgactccact tcccgcgttt actgattact agcgaagctg
     6661 cgggtgcatt ttttcaagat aaaggcatcc ccgattatat tctataccga tgtggattgc
     6721 gcatactttg tgaacagaaa gtgatagcgt tgatgattct tcattggtca gaaaattatg
     6781 aacggtttct tctattttgt ctctatatac tacgtatagg aaatgtttac attttcgtat
     6841 tgttttcgat tcactctatg aatagttctt actacaattt ttttgtctaa agagtaatac
     6901 tagagataaa cataaaaaat gtagaggtcg agtttagatg caagttcaag gagcgaaagg
     6961 tggatgggta ggttatatag ggatatagca cagagatata tagcaaagag atacttttga
     7021 gcaatgtttg tggaagcggt attcgcaata ttttagtagc tcgttacagt ccggtgcgtt
     7081 tttggttttt tgaaagtgcg tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt
     7141 cctatacttt ctagagaata ggaacttcgg aataggaact tcaaagcgtt tccgaaaacg
     7201 agcgcttccg aaaatgcaac gcgagctgcg cacatacagc tcactgttca cgtcgcacct
     7261 atatctgcgt gttgcctgta tatatatata catgagaaga acggcatagt gcgtgtttat
     7321 gcttaaatgc gtacttatat gcgtctattt atgtaggatg aaaggtagtc tagtacctcc
     7381 tgtgatatta tcccattcca tgcggggtat cgtatgcttc cttcagcact accctttagc
     7441 tgttctatat gctgccactc ctcaattgga ttagtctcat ccttcaatgc tatcatttcc
     7501 tttgatattg gatcatacta agaaaccatt attatcatga cattaaccta taaaaatagg
     7561 cgtatcacga ggccctttcg tc
//
