LOCUS       sequence-349993-        7014 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses yeGFP under control of doxycycline-responsive TET3 
            promoter (contains three rtTA binding sites).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7014)
  AUTHORS   Barre B, Gladyshev V
  TITLE     Improved reverse tetracycline transactivator plasmids
  JOURNAL   (unpublished)
  PUBMED    27323850
REFERENCE   2  (bases 1 to 7014)
  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            "(unpublished)"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-57
FEATURES             Location/Qualifiers
     source          1..7014
                     /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"
     protein_bind    2035..2053
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2101..2119
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2185..2203
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2351..2374
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             join(2415..2417,2418..3128)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     terminator      3152..3339
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3207..3231)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3359..3375)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3359..3375)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3412..3432)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3412..3430)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3451..3467)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3451..3467)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3464..3486)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3475..3491
                     /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(3499..3505,3506..3523,3524..3529))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3544..3565
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3682..3699)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3853..4441)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3933..3952)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4612..5403,5404..5472))
                     /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     5235..5254
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5473..5577)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5604..6946
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(6567..6614)
                     /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     6987..7005
                     /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 gctttcccta
     2041 tcagtgatag agatgcgtcc tcgtcttcac cggtcgcgtt cctgaaacgc agatgtgcct
     2101 tccctatcag tgatagagaa acaataaaga ttctacaata ctagctttta tggttatgaa
     2161 gaggaaaaat tggcagtaac ctggtcccta tcagtgatag agataattaa tcagcgaagc
     2221 gatgattttt gatctattaa cagatatata aatggaaaag ctgcataacc actttaacta
     2281 atactttcaa cattttcagt ttgtattact tcttattcaa atgtcataaa agtatcaaca
     2341 aaaaattgtt aatatacctc tatactttaa cgtcaaggag aaaaaactat aactagagtg
     2401 ctggtttaat taacatgtca aaaggcgagg aactattcac aggtgtcgta ccaatactgg
     2461 tagaattgga tggcgacgta aacgggcata agttttctgt gagtggtgag ggtgaagggg
     2521 acgcaactta tggcaaactt acacttaagt tcatctgcac cacagggaag ctgcctgtcc
     2581 catggccaac tctagttaca actctcactt acggagtaca atgtttttct cgatacccag
     2641 accacatgaa acaacatgac tttttcaaat ctgccatgcc tgagggatat gtgcaagaga
     2701 gaactatttt ctttaaagat gatggtaatt acaaaactcg tgctgaagtg aagtttgaag
     2761 gagatacact tgttaataga attgagttaa agggtattga ctttaaggaa gatggtaata
     2821 tcttaggtca caaattggaa tacaactaca attcacataa cgtttacatc atggcagata
     2881 aacagaaaaa cggcataaaa gttaatttca aaattagaca taatatcgaa gatggttccg
     2941 ttcaactcgc tgatcattat caacaaaata caccaatcgg agatggacct gttctattgc
     3001 cagacaacca ttacttatct acacagagcg ccttgtcaaa agatcctaat gaaaagagag
     3061 atcacatggt tttgttagaa tttgtcaccg ctgcgggtat aacgttgggc atggatgaat
     3121 tatacaagta aggcgcgcca cttctaaata agcgaatttc ttatgattta tgatttttat
     3181 tattaaataa gttataaaaa aaataagtgt atacaaattt taaagtgact cttaggtttt
     3241 aaaacgaaaa ttcttattct tgagtaactc tttcctgtag gtcaggttgc tttctcaggt
     3301 atagtatgag gtcgctctta ttgaccacac ctctaccggg aattcctgca gcccggggga
     3361 tccactagtt ctagagcggc cgccaccgcg gtggagctcc agcttttgtt ccctttagtg
     3421 agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     3481 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     3541 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3601 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     3661 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     3721 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     3781 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     3841 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     3901 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     3961 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4021 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4081 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4141 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4201 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4261 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4321 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4381 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4441 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4501 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4561 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     4621 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     4681 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     4741 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     4801 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     4861 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     4921 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     4981 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5041 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5101 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5161 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5221 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5281 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5341 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5401 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5461 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5521 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5581 atttccccga aaagtgccac ctgaacgaag catctgtgct tcattttgta gaacaaaaat
     5641 gcaacgcgag agcgctaatt tttcaaacaa agaatctgag ctgcattttt acagaacaga
     5701 aatgcaacgc gaaagcgcta ttttaccaac gaagaatctg tgcttcattt ttgtaaaaca
     5761 aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca tttttacaga
     5821 acagaaatgc aacgcgagag cgctatttta ccaacaaaga atctatactt cttttttgtt
     5881 ctacaaaaat gcatcccgag agcgctattt ttctaacaaa gcatcttaga ttactttttt
     5941 tctcctttgt gcgctctata atgcagtctc ttgataactt tttgcactgt aggtccgtta
     6001 aggttagaag aaggctactt tggtgtctat tttctcttcc ataaaaaaag cctgactcca
     6061 cttcccgcgt ttactgatta ctagcgaagc tgcgggtgca ttttttcaag ataaaggcat
     6121 ccccgattat attctatacc gatgtggatt gcgcatactt tgtgaacaga aagtgatagc
     6181 gttgatgatt cttcattggt cagaaaatta tgaacggttt cttctatttt gtctctatat
     6241 actacgtata ggaaatgttt acattttcgt attgttttcg attcactcta tgaatagttc
     6301 ttactacaat ttttttgtct aaagagtaat actagagata aacataaaaa atgtagaggt
     6361 cgagtttaga tgcaagttca aggagcgaaa ggtggatggg taggttatat agggatatag
     6421 cacagagata tatagcaaag agatactttt gagcaatgtt tgtggaagcg gtattcgcaa
     6481 tattttagta gctcgttaca gtccggtgcg tttttggttt tttgaaagtg cgtcttcaga
     6541 gcgcttttgg ttttcaaaag cgctctgaag ttcctatact ttctagagaa taggaacttc
     6601 ggaataggaa cttcaaagcg tttccgaaaa cgagcgcttc cgaaaatgca acgcgagctg
     6661 cgcacataca gctcactgtt cacgtcgcac ctatatctgc gtgttgcctg tatatatata
     6721 tacatgagaa gaacggcata gtgcgtgttt atgcttaaat gcgtacttat atgcgtctat
     6781 ttatgtagga tgaaaggtag tctagtacct cctgtgatat tatcccattc catgcggggt
     6841 atcgtatgct tccttcagca ctacccttta gctgttctat atgctgccac tcctcaattg
     6901 gattagtctc atccttcaat gctatcattt cctttgatat tggatcatac taagaaacca
     6961 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtc
//
