LOCUS       sequence-263893-        6698 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a CFP-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 6698)
  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 6698)
  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: CFP-tTA-ER-C3
FEATURES             Location/Qualifiers
     source          1..6698
                     /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"
     CDS             join(288..911,912..1295)
                     /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(364..383)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             1314..2249
                     /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             1362..2252
                     /codon_start=1
                     /gene="ESR1"
                     /product="estrogen nuclear receptor alpha ligand-binding 
                     domain"
                     /label=ER-LBD
                     /translation="KRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMM
                     GLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPVKL
                     LFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTF
                     LSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNK
                     GMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSL
                     QKYYITGEAEGFPATV"
     regulatory      2274..2283
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2280..2282,2283..2285,2286..2999)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2325..2346)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2586..2605)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2933..2954
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3122..3243
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3159..3178)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3213..3232
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3250..3705)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3387..3408)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3599..3618
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3732..3836
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3838..4195
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3838..4029
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3861..3881)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4046..4181
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4230..5024
                     /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             4230..5024
                     /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(4284..4303)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4894..4913
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5212..5231)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5256..5303
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5632..6220
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6121..6140
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        6395..6698
                     /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 actcagatct cgagctcaag cttcgaattc tgcagtcgac ggtacccatg gcatctagat
      301 tagataaaag taaagtgatt aacagcgcat tagagctgct taatgaggtc ggaatcgaag
      361 gtttaacaac ccgtaaactc gcccagaagc taggtgtaga gcagcctaca ttgtattggc
      421 atgtaaaaaa taagcgggct ttgctcgacg ccttagccat tgagatgtta gataggcacc
      481 atactcactt ttgcccttta gaaggggaaa gctggcaaga ttttttacgt aataacgcta
      541 aaagttttag atgtgcttta ctaagtcatc gcgatggagc aaaagtacat ttaggtacac
      601 ggcctacaga aaaacagtat gaaactctcg aaaatcaatt agccttttta tgccaacaag
      661 gtttttcact agagaatgca ttatatgcac tcagcgctgt ggggcatttt actttaggtt
      721 gcgtattgga agatcaagag catcaagtcg ctaaagaaga aagggaaaca cctactactg
      781 atagtatgcc gccattatta cgacaagcta tcgaattatt tgatcaccaa ggtgcagagc
      841 cagccttctt attcggcctt gaattgatca tatgcggatt agaaaaacaa cttaaatgtg
      901 aaagtgggtc cgcgtacagc cgcgcgcgta cgaaaaacaa ttacgggtct accatcgagg
      961 gcctgctcga tctcccggac gacgacgccc ccgaagaggc ggggctggcg gctccgcgcc
     1021 tgtcctttct ccccgcggga cacacgcgca gactgtcgac ggcccccccg accgatgtca
     1081 gcctggggga cgagctccac ttagacggcg aggacgtggc gatggcgcat gccgacgcgc
     1141 tagacgattt cgatctggac atgttggggg acggggattc cccgggtccg ggatttaccc
     1201 cccacgactc cgccccctac ggcgctctgg atatggccga cttcgagttt gagcagatgt
     1261 ttaccgatgc ccttggaatt gacgagtacg gtgggccgaa ttccgatcca tctgctggag
     1321 acatgagagc tgccaacctt tggccaagcc cgctcatgat caaacgctct aagaagaaca
     1381 gcctggcctt gtccctgacg gccgaccaga tggtcagtgc cttgttggat gctgagcccc
     1441 ccatactcta ttccgagtat gatcctacca gacccttcag tgaagcttcg atgatgggct
     1501 tactgaccaa cctggcagac agggagctgg ttcacatgat caactgggcg aagagggtgc
     1561 caggctttgt ggatttgacc ctccatgatc aggtccacct tctagaatgt gcctggctag
     1621 agatcctgat gattggtctc gtctggcgct ccatggagca cccagtgaag ctactgtttg
     1681 ctcctaactt gctcttggac aggaaccagg gaaaatgtgt agagggcatg gtggagatct
     1741 tcgacatgct gctggctaca tcatctcggt tccgcatgat gaatctgcag ggagaggagt
     1801 ttgtgtgcct caaatctatt attttgctta attctggagt gtacacattt ctgtccagca
     1861 ccctgaagtc tctggaagag aaggaccata tccaccgagt cctggacaag atcacagaca
     1921 ctttgatcca cctgatggcc aaggcaggcc tgaccctgca gcagcagcac cagcgactgg
     1981 cccagctcct cctcatcctc tcccacatca ggcacatgag taacaaaggc atggagcatc
     2041 tgtacagcat gaagtgcaag aacgtggtgc ccctctatga cctgctgctg gagatgctgg
     2101 acgcccaccg cctacatgcg cccactagcc gtggaggggc atccgtggag gagacggacc
     2161 aaagccactt ggccactgcg ggctctactt catcgcattc cttgcaaaag tattacatca
     2221 cgggggaggc agagggtttc cctgccacag tccgggcccg ggatccaccg gtcgccacca
     2281 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     2341 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     2401 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     2461 tcgtgaccac cctgacctgg ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     2521 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     2581 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     2641 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     2701 agctggagta caactacatc agccacaacg tctatatcac cgccgacaag cagaagaacg
     2761 gcatcaaggc caacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     2821 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     2881 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     2941 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa
     3001 gcggccgcga ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt
     3061 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     3121 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     3181 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     3241 ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     3301 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     3361 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     3421 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     3481 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     3541 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     3601 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     3661 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
     3721 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     3781 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg
     3841 aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     3901 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     3961 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     4021 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     4081 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     4141 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg
     4201 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     4261 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     4321 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     4381 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
     4441 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     4501 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     4561 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     4621 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     4681 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     4741 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     4801 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     4861 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     4921 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     4981 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     5041 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     5101 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     5161 gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg
     5221 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     5281 gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt
     5341 cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc
     5401 ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc
     5461 tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt
     5521 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     5581 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5641 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     5701 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     5761 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     5821 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     5881 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     5941 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6001 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6061 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     6121 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     6181 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     6241 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     6301 tgattctgtg gataaccgta ttaccgccat gcattagtta ttaatagtaa tcaattacgg
     6361 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     6421 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     6481 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     6541 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     6601 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     6661 ggcagtacat ctacgtatta gtcatcgcta ttaccatg
//
