LOCUS       sequence-263908-        6452 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a YFP-tTA (tetracycline transcriptional activator)-ER 
            (estrogen receptor hormone binding domain) fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6452)
  AUTHORS   Rafalska-Metcalf IU, Powers SL, Joo LM, LeRoy G, Janicki SM
  TITLE     Single cell analysis of transcriptional activation dynamics.
  JOURNAL   PLoS One. 2010 Apr 21;5(4):e10272. doi: 
            10.1371/journal.pone.0010272.
  PUBMED    20422051
REFERENCE   2  (bases 1 to 6452)
  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: "PLoS One.";
            date: "2010-04-21"; volume: "5(4):e10272. doi"; pages: " 
            10.1371/journal.pone.0010272"
COMMENT     Sequence Label: YFP-tTA-ER-C3
FEATURES             Location/Qualifiers
     source          1..6452
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        77..406
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      257..392
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      425..434
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(431..433,434..436,437..1147)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(476..497)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(737..756)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1084..1105
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             join(1196..1819,1820..2203)
                     /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="MASRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGST
                     IEGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAH
                     ADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     primer_bind     complement(1272..1291)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             2222..3157
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             2270..3160
                     /codon_start=1
                     /gene="ESR1"
                     /product="estrogen nuclear receptor alpha ligand-binding 
                     domain"
                     /label=ER-LBD
                     /translation="KRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMM
                     GLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPVKL
                     LFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTF
                     LSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNK
                     GMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSL
                     QKYYITGEAEGFPATV"
     polyA_signal    3298..3419
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3335..3354)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3389..3408
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3426..3881)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3563..3584)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3775..3794
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3908..4012
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4014..4371
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4014..4205
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     rep_origin      4222..4357
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4406..5200
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4406..5200
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4460..4479)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5070..5089
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5388..5407)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5432..5479
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5808..6396
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6297..6316
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcatta
       61 gttattactg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
      121 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
      181 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
      241 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
      301 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
      361 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tagcgctacc
      421 ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
      481 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
      541 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
      601 ctggcccacc ctcgtgacca ccttcggcta cggcctgcag tgcttcgccc gctaccccga
      661 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
      721 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
      781 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
      841 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
      901 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
      961 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     1021 cgacaaccac tacctgagct accagtccgc cctgagcaaa gaccccaacg agaagcgcga
     1081 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     1141 gtacaagtac tcagatctcg agctcaagct tcgaattctg cagtcgacgg tacccatggc
     1201 atctagatta gataaaagta aagtgattaa cagcgcatta gagctgctta atgaggtcgg
     1261 aatcgaaggt ttaacaaccc gtaaactcgc ccagaagcta ggtgtagagc agcctacatt
     1321 gtattggcat gtaaaaaata agcgggcttt gctcgacgcc ttagccattg agatgttaga
     1381 taggcaccat actcactttt gccctttaga aggggaaagc tggcaagatt ttttacgtaa
     1441 taacgctaaa agttttagat gtgctttact aagtcatcgc gatggagcaa aagtacattt
     1501 aggtacacgg cctacagaaa aacagtatga aactctcgaa aatcaattag cctttttatg
     1561 ccaacaaggt ttttcactag agaatgcatt atatgcactc agcgctgtgg ggcattttac
     1621 tttaggttgc gtattggaag atcaagagca tcaagtcgct aaagaagaaa gggaaacacc
     1681 tactactgat agtatgccgc cattattacg acaagctatc gaattatttg atcaccaagg
     1741 tgcagagcca gccttcttat tcggccttga attgatcata tgcggattag aaaaacaact
     1801 taaatgtgaa agtgggtccg cgtacagccg cgcgcgtacg aaaaacaatt acgggtctac
     1861 catcgagggc ctgctcgatc tcccggacga cgacgccccc gaagaggcgg ggctggcggc
     1921 tccgcgcctg tcctttctcc ccgcgggaca cacgcgcaga ctgtcgacgg cccccccgac
     1981 cgatgtcagc ctgggggacg agctccactt agacggcgag gacgtggcga tggcgcatgc
     2041 cgacgcgcta gacgatttcg atctggacat gttgggggac ggggattccc cgggtccggg
     2101 atttaccccc cacgactccg ccccctacgg cgctctggat atggccgact tcgagtttga
     2161 gcagatgttt accgatgccc ttggaattga cgagtacggt gggccgaatt ccgatccatc
     2221 tgctggagac atgagagctg ccaacctttg gccaagcccg ctcatgatca aacgctctaa
     2281 gaagaacagc ctggccttgt ccctgacggc cgaccagatg gtcagtgcct tgttggatgc
     2341 tgagcccccc atactctatt ccgagtatga tcctaccaga cccttcagtg aagcttcgat
     2401 gatgggctta ctgaccaacc tggcagacag ggagctggtt cacatgatca actgggcgaa
     2461 gagggtgcca ggctttgtgg atttgaccct ccatgatcag gtccaccttc tagaatgtgc
     2521 ctggctagag atcctgatga ttggtctcgt ctggcgctcc atggagcacc cagtgaagct
     2581 actgtttgct cctaacttgc tcttggacag gaaccaggga aaatgtgtag agggcatggt
     2641 ggagatcttc gacatgctgc tggctacatc atctcggttc cgcatgatga atctgcaggg
     2701 agaggagttt gtgtgcctca aatctattat tttgcttaat tctggagtgt acacatttct
     2761 gtccagcacc ctgaagtctc tggaagagaa ggaccatatc caccgagtcc tggacaagat
     2821 cacagacact ttgatccacc tgatggccaa ggcaggcctg accctgcagc agcagcacca
     2881 gcggctggcc cagctcctcc tcatcctctc ccacatcagg cacatgagta acaaaggcat
     2941 ggagcatctg tacagcatga agtgcaagaa cgtggtgccc ctctatgacc tgctgctgga
     3001 gatgctggac gcccaccgcc tacatgcgcc cactagccgt ggaggggcat ccgtggagga
     3061 gacggaccaa agccacttgg ccactgcggg ctctacttca tcgcattcct tgcaaaagta
     3121 ttacatcacg ggggaggcag agggtttccc tgccacagtc cgggcccggg atccaccgga
     3181 tctagataac tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     3241 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     3301 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     3361 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa
     3421 ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     3481 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     3541 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     3601 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     3661 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     3721 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     3781 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     3841 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg
     3901 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3961 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc
     4021 ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     4081 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     4141 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     4201 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     4261 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     4321 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca
     4381 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     4441 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     4501 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     4561 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     4621 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4681 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4741 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4801 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4861 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     4921 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4981 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     5041 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     5101 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     5161 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     5221 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     5281 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     5341 ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag
     5401 gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac
     5461 ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat
     5521 accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac
     5581 cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc
     5641 atagcctcag gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5701 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5761 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5821 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5881 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5941 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6001 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6061 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6121 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6181 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6241 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6301 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6361 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6421 cggttcctgg ccttttgctg gccttttgct ca
//
