LOCUS       sequence-265286-        6683 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Cherry-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 6683)
  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 6683)
  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: Cherry-tTA-ER-C3
FEATURES             Location/Qualifiers
     source          1..6683
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      243..252
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             249..956
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(394..412)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     662..681
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             join(1005..1628,1629..2012)
                     /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(1081..1100)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             2031..2966
                     /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             2079..2969
                     /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    3107..3228
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3144..3163)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3198..3217
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3235..3690)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3372..3393)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3584..3603
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3717..3821
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3823..4180
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3823..4014
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3846..3866)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4031..4166
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4215..5009
                     /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             4215..5009
                     /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(4269..4288)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4879..4898
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5197..5216)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5241..5288
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5617..6205
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6106..6125
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        6380..6683
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg
      241 tcgccaccat ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc
      301 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
      361 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
      421 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg
      481 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
      541 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
      601 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
      661 gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg
      721 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
      781 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct
      841 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
      901 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtact
      961 cagatctcga gctcaagctt cgaattctgc agtcgacggt acccatggca tctagattag
     1021 ataaaagtaa agtgattaac agcgcattag agctgcttaa tgaggtcgga atcgaaggtt
     1081 taacaacccg taaactcgcc cagaagctag gtgtagagca gcctacattg tattggcatg
     1141 taaaaaataa gcgggctttg ctcgacgcct tagccattga gatgttagat aggcaccata
     1201 ctcacttttg ccctttagaa ggggaaagct ggcaagattt tttacgtaat aacgctaaaa
     1261 gttttagatg tgctttacta agtcatcgcg atggagcaaa agtacattta ggtacacggc
     1321 ctacagaaaa acagtatgaa actctcgaaa atcaattagc ctttttatgc caacaaggtt
     1381 tttcactaga gaatgcatta tatgcactca gcgctgtggg gcattttact ttaggttgcg
     1441 tattggaaga tcaagagcat caagtcgcta aagaagaaag ggaaacacct actactgata
     1501 gtatgccgcc attattacga caagctatcg aattatttga tcaccaaggt gcagagccag
     1561 ccttcttatt cggccttgaa ttgatcatat gcggattaga aaaacaactt aaatgtgaaa
     1621 gtgggtccgc gtacagccgc gcgcgtacga aaaacaatta cgggtctacc atcgagggcc
     1681 tgctcgatct cccggacgac gacgcccccg aagaggcggg gctggcggct ccgcgcctgt
     1741 cctttctccc cgcgggacac acgcgcagac tgtcgacggc ccccccgacc gatgtcagcc
     1801 tgggggacga gctccactta gacggcgagg acgtggcgat ggcgcatgcc gacgcgctag
     1861 acgatttcga tctggacatg ttgggggacg gggattcccc gggtccggga tttacccccc
     1921 acgactccgc cccctacggc gctctggata tggccgactt cgagtttgag cagatgttta
     1981 ccgatgccct tggaattgac gagtacggtg ggccgaattc cgatccatct gctggagaca
     2041 tgagagctgc caacctttgg ccaagcccgc tcatgatcaa acgctctaag aagaacagcc
     2101 tggccttgtc cctgacggcc gaccagatgg tcagtgcctt gttggatgct gagcccccca
     2161 tactctattc cgagtatgat cctaccagac ccttcagtga agcttcgatg atgggcttac
     2221 tgaccaacct ggcagacagg gagctggttc acatgatcaa ctgggcgaag agggtgccag
     2281 gctttgtgga tttgaccctc catgatcagg tccaccttct agaatgtgcc tggctagaga
     2341 tcctgatgat tggtctcgtc tggcgctcca tggagcaccc agtgaagcta ctgtttgctc
     2401 ctaacttgct cttggacagg aaccagggaa aatgtgtaga gggcatggtg gagatcttcg
     2461 acatgctgct ggctacatca tctcggttcc gcatgatgaa tctgcaggga gaggagtttg
     2521 tgtgcctcaa atctattatt ttgcttaatt ctggagtgta cacatttctg tccagcaccc
     2581 tgaagtctct ggaagagaag gaccatatcc accgagtcct ggacaagatc acagacactt
     2641 tgatccacct gatggccaag gcaggcctga ccctgcagca gcagcaccag cggctggccc
     2701 agctcctcct catcctctcc cacatcaggc acatgagtaa caaaggcatg gagcatctgt
     2761 acagcatgaa gtgcaagaac gtggtgcccc tctatgacct gctgctggag atgctggacg
     2821 cccaccgcct acatgcgccc actagccgtg gaggggcatc cgtggaggag acggaccaaa
     2881 gccacttggc cactgcgggc tctacttcat cgcattcctt gcaaaagtat tacatcacgg
     2941 gggaggcaga gggtttccct gccacagtcc gggcccggga tccaccggat ctagataact
     3001 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     3061 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     3121 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     3181 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaac gcgtaaattg
     3241 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     3301 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     3361 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     3421 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     3481 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     3541 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     3601 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     3661 ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg
     3721 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     3781 aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg gaaagaacca
     3841 gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
     3901 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
     3961 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
     4021 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     4081 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     4141 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa gagacaggat
     4201 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     4261 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     4321 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     4381 ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     4441 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     4501 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     4561 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     4621 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     4681 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     4741 cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     4801 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     4861 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     4921 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     4981 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     5041 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     5101 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     5161 gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg agacaatacc
     5221 ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg gtgttgggtc
     5281 gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag
     5341 accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc
     5401 gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcctcagg
     5461 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     5521 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     5581 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     5641 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     5701 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     5761 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     5821 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     5881 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     5941 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6001 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6061 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6121 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6181 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6241 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     6301 ccgtattacc gccatgcatt agttattaat agtaatcaat tacggggtca ttagttcata
     6361 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     6421 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     6481 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     6541 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     6601 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     6661 tattagtcat cgctattacc atg
//
