LOCUS       sequence-363924-        6064 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  cyclofen-inducible chicken EN2 (mut) activation via mammalian cell 
            transfection or mRNA synthesis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6064)
  AUTHORS   Rampon C, Gauron C, Lin T, Meda F, Dupont E, Cosson A, Ipendey E, 
            Frerot A, Aujard I, Le Saux T, Bensimon D, Jullien L, Volovitch M, 
            Vriz S, Joliot A
  TITLE     Control of brain patterning by Engrailed paracrine transfer: a new 
            function of the Pbx interaction domain.
  JOURNAL   Development. 2015 May 15;142(10):1840-9. doi: 10.1242/dev.114181. 
            Epub 2015 Apr 29.
  PUBMED    25926358
REFERENCE   2  (bases 1 to 6064)
  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"; doi: 
            "10.1242/dev.114181"; journalName: "Development"; date: "2015-05-15-
            15"; volume: "142"; issue: "10"; pages: "1840-9"
COMMENT     Sequence Label: pCSmEn25EERT(#232)
FEATURES             Location/Qualifiers
     source          1..6064
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     5'UTR           103..145
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             157..174
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             196..225
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1117..1137
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1150..2085
                     /codon_start=1
                     /product="mutated ligand-binding domain of the human 
                     estrogen receptor (Feil et al., 1997)"
                     /label=ERT2
                     /translation="AGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPIL
                     YSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEI
                     LMIGLVWRSMEHPVKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEF
                     VCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRL
                     AQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEET
                     DQSHLATAGSTSSHSLQKYYITGEAEGFPAT"
     CDS             1198..2091
                     /codon_start=1
                     /gene="ESR1"
                     /product="estrogen nuclear receptor alpha ligand-binding 
                     domain"
                     /label=ER-LBD
                     /translation="KRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMM
                     GLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPVKL
                     LFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTF
                     LSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNK
                     GMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSL
                     QKYYITGEAEGFPATA"
     polyA_signal    2116..2250
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2140..2159)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2194..2213
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2363..2383)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2363..2381)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2402..2418)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2402..2418)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2415..2437)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2426..2442
                     /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(2450..2456,2457..2474,2475..2480))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2495..2516
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2633..2650)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2804..3392)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2884..2903)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3563..4354,4355..4423))
                     /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     4186..4205
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4424..4528)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4554..5009)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4691..4712)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4903..4922
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5080..6064
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atcccatcga ttcgctagct tgcttgttct ttttgcagaa
      121 gctcagaata aacgctcaac tttggcagat accatgcatc accatcacca ccacgtggaa
      181 ttcatgactt cgaacgaaca gaaactgata tcagaggaag accttggatc catggaggag
      241 ggcggcaggt cgccccggga ggaggcggcc gaaccgcagg agtccggcgg ggacgcggaa
      301 cccggcggag ggcgtcgggc tctgctgctg ccccccggcg accccccgca cccccacccg
      361 caccctcacc gcatcaccaa cttcttcatc gacaacatcc tgaggcccga gttcggtcgg
      421 aggaaggaag cgggaggaac cgccggagaa ccccgcaggc ccggagcgga gagcaggagg
      481 agccccgcgg ccgccgcgcc cgctcccgga gctccggtgc ccggcggagg aggcggcggc
      541 gggggtggat cgccgggccg aggggagggc ggccccgccg ctctggccct gcacggagcg
      601 gccaagaagg ggggagaccc cgcggcgctg gaggcggccc tgaaagcgcg ggggctgagc
      661 ggcgcggagc tgtcggtgga agaggacgag gatgaagaac aggccggatc caacgccggg
      721 aaccaaccca tgctttggcc cgcttgggtg tactgcacgc ggtactcgga ccgaccgtct
      781 tcaggtcccc gctcccgcaa accaaagaag aagaacccca acaaggaaga caagcggccc
      841 cgcaccgcct tcacggccga gcagctgcag agactcaagg ccgagttcca gacgaaccgc
      901 tacctgacgg agcagcgccg gcagagcctg gcccaggagc tcggcctcaa cgagtcccag
      961 atcaagatct ggttccagaa caagagagcc aagatcaaga aggcgacggg cagcaagaac
     1021 tcgctggcgg tgcacctcat ggcccagggg ctctacaacc actccaccac ggcgaaagac
     1081 ggcaagtcgg acagcgaagc tttggcgccc gggccagaga acctgtactt ccaggggctc
     1141 gagccatctg ctggagacat gagagctgcc aacctttggc caagcccgct catgatcaaa
     1201 cgctctaaga agaacagcct ggccttgtcc ctgacggccg accagatggt cagtgccttg
     1261 ttggatgctg agccccccat actctattcc gagtatgatc ctaccagacc cttcagtgaa
     1321 gcttcgatga tgggcttact gaccaacctg gcagacaggg agctggttca catgatcaac
     1381 tgggcgaaga gggtgccagg ctttgtggat ttgaccctcc atgatcaggt ccaccttcta
     1441 gaatgtgcct ggctagagat cctgatgatt ggtctcgtct ggcgctccat ggagcaccca
     1501 gtgaagctac tgtttgctcc taacttgctc ttggacagga accagggaaa atgtgtagag
     1561 ggcatggtgg agatcttcga catgctgctg gctacatcat ctcggttccg catgatgaat
     1621 ctgcagggag aggagtttgt gtgcctcaaa tctattattt tgcttaattc tggagtgtac
     1681 acatttctgt ccagcaccct gaagtctctg gaagagaagg accatatcca ccgagtcctg
     1741 gacaagatca cagacacttt gatccacctg atggccaagg caggcctgac cctgcagcag
     1801 cagcaccagc ggctggccca gctcctcctc atcctctccc acatcaggca catgagtaac
     1861 aaaggcatgg agcatctgta cagcatgaag tgcaagaacg tggtgcccct ctatgacctg
     1921 ctgctggagg cggcggacgc ccaccgccta catgcgccca ctagccgtgg aggggcatcc
     1981 gtggaggaga cggaccaaag ccacttggcc actgcgggct ctacttcatc gcattccttg
     2041 caaaagtatt acatcacggg ggaggcagag ggtttccctg ccacagcttg atgaagatct
     2101 gagggcccgt ttgtagatcc agacatgata agatacattg atgagtttgg acaaaccaca
     2161 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     2221 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     2281 caggttcagg gggaggtgtg ggaggttttt taattcgcgg ccgcggcgcc aatgcattgg
     2341 gcccggtacc cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat
     2401 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     2461 ccgggagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     2521 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     2581 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     2641 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     2701 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     2761 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     2821 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     2881 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     2941 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3001 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3061 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3121 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3181 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3241 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3301 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3361 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3421 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     3481 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     3541 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     3601 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     3661 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     3721 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     3781 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     3841 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     3901 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     3961 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4021 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4081 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     4141 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     4201 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     4261 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     4321 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     4381 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     4441 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     4501 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt
     4561 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     4621 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     4681 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     4741 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     4801 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     4861 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     4921 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     4981 cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg
     5041 ggaagggcga tcggtgcggg cctcttcgct attacgccag tcgaccgcca attcaatatg
     5101 gcgtatatgg actcatgcca attcaatatg gtggatctgg acctgtgcca attcaatatg
     5161 gcgtatatgg actcgtgcca attcaatatg gtggatctgg accccagcca attcaatatg
     5221 gcggacttgg caccatgcca attcaatatg gcggacctgg cactgtgcca actggggagg
     5281 ggtctacttg gcacggtgcc aagtttgagg aggggtcttg gccctgtgcc aagtccgcca
     5341 tattgaattg gcatggtgcc aataatggcg gccatattgg ctatatgcca ggatcaatat
     5401 ataggcaata tccaatatgg ccctatgcca atatggctat tggccaggtt caatactatg
     5461 tattggccct atgccatata gtattccata tatgggtttt cctattgacg tagatagccc
     5521 ctcccaatgg gcggtcccat ataccatata tggggcttcc taataccgcc catagccact
     5581 cccccattga cgtcaatggt ctctatatat ggtctttcct attgacgtca tatgggcggt
     5641 cctattgacg tatatggcgc ctcccccatt gacgtcaatt acggtaaatg gcccgcctgg
     5701 ctcaatgccc attgacgtca ataggaccac ccaccattga cgtcaatggg atggctcatt
     5761 gcccattcat atccgttctc acgcccccta ttgacgtcaa tgacggtaaa tggcccactt
     5821 ggcagtacat caatatctat taatagtaac ttggcaagta cattactatt ggaagtacgc
     5881 cagggtacat tggcagtact cccattgacg tcaatggcgg taaatggccc gcgatggctg
     5941 ccaagtacat ccccattgac gtcaatgggg aggggcaatg acgcaaatgg gcgttccatt
     6001 gacgtaaatg ggcggtaggc gtgcctaatg ggaggtctat ataagcaatg ctcgtttagg
     6061 gaac
//
