LOCUS       sequence-349995-        7414 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses optimized unleaky reverse tetracycline transactivator 
            (rtTA-M2-SE-G72P) under control of yeast MYO2 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7414)
  AUTHORS   Barre B, Gladyshev V
  TITLE     Improved reverse tetracycline transactivator plasmids
  JOURNAL   (unpublished)
  PUBMED    27323850
REFERENCE   2  (bases 1 to 7414)
  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            "(unpublished)"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-56
FEATURES             Location/Qualifiers
     source          1..7414
                     /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"
     CDS             join(2535..3155,3156..3539)
                     /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(2608..2627)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3759..3775)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3759..3775)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3812..3832)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3812..3830)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3851..3867)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3851..3867)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3864..3886)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3875..3891
                     /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(3899..3905,3906..3923,3924..3929))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3944..3965
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4082..4099)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4253..4841)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4333..4352)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5012..5803,5804..5872))
                     /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     5635..5654
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5873..5977)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6004..7346
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(6967..7014)
                     /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     7387..7405
                     /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 gcttccgttt
     2041 ctcgatgctt atctggttta gtttacgctg ttaaaaccaa aaccccaaca gattttcgac
     2101 cctaacgtat gtagggctaa aatagatatt gagtaggtta caattaatta ttggcaattg
     2161 cacctagtga cacatttacg aaaacgtagg gcaaaaacta ttacccgacc cagggctatt
     2221 ttgtgatttt ttcctttttt ttgtttatga tcgcgcttct cgaaaagcca aatatcagaa
     2281 atcccaaaca cgccttcatt tgatacgatt cgtagcctgc gtttcagaga tctatcaact
     2341 ttgcaaggcc aatcagagaa caaaaaagtc tcgcaaagtc atttcacttt tctcgcttga
     2401 aattattcgt tcgatttctg gctgcttgct tgttttttgt tttctaaggt actattcgac
     2461 accattccat tggacagcga tacttatacc attgtacata taggacataa aaacagcaga
     2521 tattacagcg tataatgtct agattagata aaagtaaaat tattaacggc gcattagagc
     2581 tgcttaatgg ggtcggaatc gaaggtttaa caacccgtaa actcgcccag aagctaggtg
     2641 tagagcagcc tacattgtat tggcatgtaa aaaataagcg ggctttgctc gacgccttac
     2701 ccattgagat gttagatagg caccatactc actcttgccc tttagaacct gaaagctggc
     2761 aagatttttt acgtaataac gctaaaagtt atagatgtgc tttactaagt catcgcgatg
     2821 gagcaaaagt acatttaggt acacggccta cagaaaaaca gtatgaaact ctcgaaaatc
     2881 aattagcctt tttatgccaa caaggttttt cactagagaa tgcattatat gcactcagcg
     2941 ctgtggggca ctttacttta ggttgcgtat tggaagaaca agagcatcaa gtcgctaaag
     3001 aagaaaggga aacacctact actgatagta tgccgccatt attaaaacaa gctatcgaat
     3061 tatttgatcg ccaaggtgca gagccagcct tcttattcgg ccttgaattg atcatatgcg
     3121 gattagaaaa acaacttaaa tgtgaaagtg ggtccgcgta cagccgcgcg cgtacgaaaa
     3181 acaattacgg gtctaccatc gagggcctgc tcgatctccc ggacgacgac gcccccgaag
     3241 aggcggggct ggcggctccg cgcctgtcct ttctccccgc gggacacacg cgcagactgt
     3301 cgacggcccc cccgaccgat gtcagcctgg gggacgagct ccacttagac ggcgaggacg
     3361 tggcgatggc gcatgccgac gcgctagacg atttcgatct ggacatgttg ggggacgggg
     3421 attccccggg tccgggattt accccccacg actccgcccc ctacggcgct ctggatatgg
     3481 ccgacttcga gtttgagcag atgtttaccg atgcccttgg aattgacgag tacggtggga
     3541 ctagataaat tgaattgaat tgaaatcgat agatcaattt ttttcttttc tctttcccca
     3601 tcctttacgc taaaataata gtttatttta ttttttgaat attttttatt tatatacgta
     3661 tatatagact attatttatc ttttaatgat tattaagatt tttattaaaa aaaaattcgc
     3721 tcctctttta atgcctttag aattcctgca gcccggggga tccactagtt ctagagcggc
     3781 cgccaccgcg gtggagctcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg
     3841 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     3901 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     3961 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4021 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4081 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4141 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4201 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4261 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4321 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4381 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4441 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4501 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4561 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4621 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4681 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4741 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4801 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4861 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4921 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     4981 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5041 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5101 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5161 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5221 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5281 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5341 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5401 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5461 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5521 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5581 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5641 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5701 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5761 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5821 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5881 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5941 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6001 ctgaacgaag catctgtgct tcattttgta gaacaaaaat gcaacgcgag agcgctaatt
     6061 tttcaaacaa agaatctgag ctgcattttt acagaacaga aatgcaacgc gaaagcgcta
     6121 ttttaccaac gaagaatctg tgcttcattt ttgtaaaaca aaaatgcaac gcgagagcgc
     6181 taatttttca aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgagag
     6241 cgctatttta ccaacaaaga atctatactt cttttttgtt ctacaaaaat gcatcccgag
     6301 agcgctattt ttctaacaaa gcatcttaga ttactttttt tctcctttgt gcgctctata
     6361 atgcagtctc ttgataactt tttgcactgt aggtccgtta aggttagaag aaggctactt
     6421 tggtgtctat tttctcttcc ataaaaaaag cctgactcca cttcccgcgt ttactgatta
     6481 ctagcgaagc tgcgggtgca ttttttcaag ataaaggcat ccccgattat attctatacc
     6541 gatgtggatt gcgcatactt tgtgaacaga aagtgatagc gttgatgatt cttcattggt
     6601 cagaaaatta tgaacggttt cttctatttt gtctctatat actacgtata ggaaatgttt
     6661 acattttcgt attgttttcg attcactcta tgaatagttc ttactacaat ttttttgtct
     6721 aaagagtaat actagagata aacataaaaa atgtagaggt cgagtttaga tgcaagttca
     6781 aggagcgaaa ggtggatggg taggttatat agggatatag cacagagata tatagcaaag
     6841 agatactttt gagcaatgtt tgtggaagcg gtattcgcaa tattttagta gctcgttaca
     6901 gtccggtgcg tttttggttt tttgaaagtg cgtcttcaga gcgcttttgg ttttcaaaag
     6961 cgctctgaag ttcctatact ttctagagaa taggaacttc ggaataggaa cttcaaagcg
     7021 tttccgaaaa cgagcgcttc cgaaaatgca acgcgagctg cgcacataca gctcactgtt
     7081 cacgtcgcac ctatatctgc gtgttgcctg tatatatata tacatgagaa gaacggcata
     7141 gtgcgtgttt atgcttaaat gcgtacttat atgcgtctat ttatgtagga tgaaaggtag
     7201 tctagtacct cctgtgatat tatcccattc catgcggggt atcgtatgct tccttcagca
     7261 ctacccttta gctgttctat atgctgccac tcctcaattg gattagtctc atccttcaat
     7321 gctatcattt cctttgatat tggatcatac taagaaacca ttattatcat gacattaacc
     7381 tataaaaata ggcgtatcac gaggcccttt cgtc
//
