LOCUS       sequence-349996-        7087 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses yeGFP under control of doxycycline-responsive TET4 
            promoter (contains four rtTA binding sites).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7087)
  AUTHORS   Barre B, Gladyshev V
  TITLE     Improved reverse tetracycline transactivator plasmids
  JOURNAL   (unpublished)
  PUBMED    27323850
REFERENCE   2  (bases 1 to 7087)
  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            "(unpublished)"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-58
FEATURES             Location/Qualifiers
     source          1..7087
                     /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    2108..2126
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2174..2192
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2258..2276
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2424..2447
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             join(2488..2490,2491..3201)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     terminator      3225..3412
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3280..3304)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3432..3448)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3432..3448)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3485..3505)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3485..3503)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3524..3540)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3524..3540)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3537..3559)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3548..3564
                     /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(3572..3578,3579..3596,3597..3602))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3617..3638
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3755..3772)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3926..4514)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4006..4025)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4685..5476,5477..5545))
                     /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     5308..5327
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5546..5650)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5677..7019
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(6640..6687)
                     /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     7060..7078
                     /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 agaccttctc tttggaactt tcagtaatac gcttaactgc tcattgctat
     2101 attgaagtcc ctatcagtga tagagatgcg tcctcgtctt caccggtcgc gttcctgaaa
     2161 cgcagatgtg ccttccctat cagtgataga gaaacaataa agattctaca atactagctt
     2221 ttatggttat gaagaggaaa aattggcagt aacctggtcc ctatcagtga tagagataat
     2281 taatcagcga agcgatgatt tttgatctat taacagatat ataaatggaa aagctgcata
     2341 accactttaa ctaatacttt caacattttc agtttgtatt acttcttatt caaatgtcat
     2401 aaaagtatca acaaaaaatt gttaatatac ctctatactt taacgtcaag gagaaaaaac
     2461 tataactaga gtgctggttt aattaacatg tcaaaaggcg aggaactatt cacaggtgtc
     2521 gtaccaatac tggtagaatt ggatggcgac gtaaacgggc ataagttttc tgtgagtggt
     2581 gagggtgaag gggacgcaac ttatggcaaa cttacactta agttcatctg caccacaggg
     2641 aagctgcctg tcccatggcc aactctagtt acaactctca cttacggagt acaatgtttt
     2701 tctcgatacc cagaccacat gaaacaacat gactttttca aatctgccat gcctgaggga
     2761 tatgtgcaag agagaactat tttctttaaa gatgatggta attacaaaac tcgtgctgaa
     2821 gtgaagtttg aaggagatac acttgttaat agaattgagt taaagggtat tgactttaag
     2881 gaagatggta atatcttagg tcacaaattg gaatacaact acaattcaca taacgtttac
     2941 atcatggcag ataaacagaa aaacggcata aaagttaatt tcaaaattag acataatatc
     3001 gaagatggtt ccgttcaact cgctgatcat tatcaacaaa atacaccaat cggagatgga
     3061 cctgttctat tgccagacaa ccattactta tctacacaga gcgccttgtc aaaagatcct
     3121 aatgaaaaga gagatcacat ggttttgtta gaatttgtca ccgctgcggg tataacgttg
     3181 ggcatggatg aattatacaa gtaaggcgcg ccacttctaa ataagcgaat ttcttatgat
     3241 ttatgatttt tattattaaa taagttataa aaaaaataag tgtatacaaa ttttaaagtg
     3301 actcttaggt tttaaaacga aaattcttat tcttgagtaa ctctttcctg taggtcaggt
     3361 tgctttctca ggtatagtat gaggtcgctc ttattgacca cacctctacc gggaattcct
     3421 gcagcccggg ggatccacta gttctagagc ggccgccacc gcggtggagc tccagctttt
     3481 gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg
     3541 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     3601 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     3661 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     3721 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     3781 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3841 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3901 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3961 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4021 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4081 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4141 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4201 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4261 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4321 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     4381 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4441 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4501 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4561 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4621 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4681 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     4741 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     4801 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     4861 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     4921 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     4981 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5041 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5101 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5161 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5221 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5281 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5341 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5401 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5461 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     5521 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     5581 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5641 gggttccgcg cacatttccc cgaaaagtgc cacctgaacg aagcatctgt gcttcatttt
     5701 gtagaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt
     5761 tttacagaac agaaatgcaa cgcgaaagcg ctattttacc aacgaagaat ctgtgcttca
     5821 tttttgtaaa acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct
     5881 gcatttttac agaacagaaa tgcaacgcga gagcgctatt ttaccaacaa agaatctata
     5941 cttctttttt gttctacaaa aatgcatccc gagagcgcta tttttctaac aaagcatctt
     6001 agattacttt ttttctcctt tgtgcgctct ataatgcagt ctcttgataa ctttttgcac
     6061 tgtaggtccg ttaaggttag aagaaggcta ctttggtgtc tattttctct tccataaaaa
     6121 aagcctgact ccacttcccg cgtttactga ttactagcga agctgcgggt gcattttttc
     6181 aagataaagg catccccgat tatattctat accgatgtgg attgcgcata ctttgtgaac
     6241 agaaagtgat agcgttgatg attcttcatt ggtcagaaaa ttatgaacgg tttcttctat
     6301 tttgtctcta tatactacgt ataggaaatg tttacatttt cgtattgttt tcgattcact
     6361 ctatgaatag ttcttactac aatttttttg tctaaagagt aatactagag ataaacataa
     6421 aaaatgtaga ggtcgagttt agatgcaagt tcaaggagcg aaaggtggat gggtaggtta
     6481 tatagggata tagcacagag atatatagca aagagatact tttgagcaat gtttgtggaa
     6541 gcggtattcg caatatttta gtagctcgtt acagtccggt gcgtttttgg ttttttgaaa
     6601 gtgcgtcttc agagcgcttt tggttttcaa aagcgctctg aagttcctat actttctaga
     6661 gaataggaac ttcggaatag gaacttcaaa gcgtttccga aaacgagcgc ttccgaaaat
     6721 gcaacgcgag ctgcgcacat acagctcact gttcacgtcg cacctatatc tgcgtgttgc
     6781 ctgtatatat atatacatga gaagaacggc atagtgcgtg tttatgctta aatgcgtact
     6841 tatatgcgtc tatttatgta ggatgaaagg tagtctagta cctcctgtga tattatccca
     6901 ttccatgcgg ggtatcgtat gcttccttca gcactaccct ttagctgttc tatatgctgc
     6961 cactcctcaa ttggattagt ctcatccttc aatgctatca tttcctttga tattggatca
     7021 tactaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     7081 tttcgtc
//
