LOCUS       sequence-447557-        6409 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Middle entry vector containing tamoxifen-inducible estrogen receptor
            (ERT2) fused to both ends of CRE recombinase for tamoxifen-inducible
            Cre recombination.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6409)
  AUTHORS   Akerberg AA, Stewart S, Stankunas K
  TITLE     Spatial and temporal control of transgene expression in zebrafish.
  JOURNAL   PLoS One. 2014 Mar 18;9(3):e92217. doi: 
            10.1371/journal.pone.0092217. eCollection 2014.
  PUBMED    24643048
REFERENCE   2  (bases 1 to 6409)
  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: "2014-03-18"; volume: "9(3):e92217. doi"; pages: " 
            10.1371/journal.pone.0092217. eCollection 2014"
COMMENT     Sequence Label: pME-ERT2-Cre-ERT2
FEATURES             Location/Qualifiers
     source          1..6409
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     670..686
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        696..714
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     739..755
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     740..756
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             796..1731
                     /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             1741..2763
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="NLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLS
                     VCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPR
                     PSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLG
                     IAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERW
                     ISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRY
                     LAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDG
                     D"
     CDS             2776..3711
                     /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             2824..3717
                     /codon_start=1
                     /gene="ESR1"
                     /product="estrogen nuclear receptor alpha ligand-binding 
                     domain"
                     /label=ER-LBD
                     /translation="KRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMM
                     GLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPVKL
                     LFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTF
                     LSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNK
                     GMEHLYSMKCKNVVPLYDLLLEAADAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSL
                     QKYYITGEAEGFPATA"
     primer_bind     complement(3805..3824)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3870..3925
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3924..3943)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4286..4302)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4299..4321)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4310..4326
                     /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(4334..4340,4341..4358,4359..4364))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4379..4400
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4434..4450)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4434..4450)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4487..4507)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4487..4505)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    complement(4534..4633)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(4651..4669)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4674..4690)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4792..4811)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4803..5612
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4870..4889)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5759..6347
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6248..6267
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggcttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat
      721 tgggtaccgg gccccccctc gaggtcgacg gtatcgataa gcttgatatc gaattcctgc
      781 agcccggggg atctggctgg agacatgaga gctgccaacc tttggccaag cccgctcatg
      841 atcaaacgct ctaagaagaa cagcctggcc ttgtccctga cggccgacca gatggtcagt
      901 gccttgttgg atgctgagcc ccccatactc tattccgagt atgatcctac cagacccttc
      961 agtgaagctt cgatgatggg cttactgacc aacctggcag acagggagct ggttcacatg
     1021 atcaactggg cgaagagggt gccaggcttt gtggatttga ccctccatga tcaggtccac
     1081 cttctagaat gtgcctggct agagatcctg atgattggtc tcgtctggcg ctccatggag
     1141 cacccagtga agctactgtt tgctcctaac ttgctcttgg acaggaacca gggaaaatgt
     1201 gtagagggca tggtggagat cttcgacatg ctgctggcta catcatctcg gttccgcatg
     1261 atgaatctgc agggagagga gtttgtgtgc ctcaaatcta ttattttgct taattctgga
     1321 gtgtacacat ttctgtccag caccctgaag tctctggaag agaaggacca tatccaccga
     1381 gtcctggaca agatcacaga cactttgatc cacctgatgg ccaaggcagg cctgaccctg
     1441 cagcagcagc accagcggct ggcccagctc ctcctcatcc tctcccacat caggcacatg
     1501 agtaacaaag gcatggagca tctgtacagc atgaagtgca agaacgtggt gcccctctat
     1561 gacctgctgc tggaggcggc ggacgcccac cgcctacatg cgcccactag ccgtggaggg
     1621 gcatccgtgg aggagacgga ccaaagccac ttggccactg cgggctctac ttcatcgcat
     1681 tccttgcaaa agtattacat cacgggggag gcagagggtt tccctgccac agctgtcgac
     1741 aatttactga ccgtacacca aaatttgcct gcattaccgg tcgatgcaac gagtgatgag
     1801 gttcgcaaga acctgatgga catgttcagg gatcgccagg cgttttctga gcatacctgg
     1861 aaaatgcttc tgtccgtttg ccggtcgtgg gcggcatggt gcaagttgaa taaccggaaa
     1921 tggtttcccg cagaacctga agatgttcgc gattatcttc tatatcttca ggcgcgcggt
     1981 ctggcagtaa aaactatcca gcaacatttg ggccagctaa acatgcttca tcgtcggtcc
     2041 gggctgccac gaccaagtga cagcaatgct gtttcactgg ttatgcggcg gatccgaaaa
     2101 gaaaacgttg atgccggtga acgtgcaaaa caggctctag cgttcgaacg cactgatttc
     2161 gaccaggttc gttcactcat ggaaaatagc gatcgctgcc aggatatacg taatctggca
     2221 tttctgggga ttgcttataa caccctgtta cgtatagccg aaattgccag gatcagggtt
     2281 aaagatatct cacgtactga cggtgggaga atgttaatcc atattggcag aacgaaaacg
     2341 ctggttagca ccgcaggtgt agagaaggca cttagcctgg gggtaactaa actggtcgag
     2401 cgatggattt ccgtctctgg tgtagctgat gatccgaata actacctgtt ttgccgggtc
     2461 agaaaaaatg gtgttgccgc gccatctgcc accagccagc tatcaactcg cgccctggaa
     2521 gggatttttg aagcaactca tcgattgatt tacggcgcta aggatgactc tggtcagaga
     2581 tacctggcct ggtctggaca cagtgcccgt gtcggagccg cgcgagatat ggcccgcgct
     2641 ggagtttcaa taccggagat catgcaagct ggtggctgga ccaatgtaaa tattgtcatg
     2701 aactatatcc gtaacctgga tagtgaaaca ggggcaatgg tgcgcctgct ggaagatggc
     2761 gatctcgagc catctgctgg agacatgaga gctgccaacc tttggccaag cccgctcatg
     2821 atcaaacgct ctaagaagaa cagcctggcc ttgtccctga cggccgacca gatggtcagt
     2881 gccttgttgg atgctgagcc ccccatactc tattccgagt atgatcctac cagacccttc
     2941 agtgaagctt cgatgatggg cttactgacc aacctggcag acagggagct ggttcacatg
     3001 atcaactggg cgaagagggt gccaggcttt gtggatttga ccctccatga tcaggtccac
     3061 cttctagaat gtgcctggct agagatcctg atgattggtc tcgtctggcg ctccatggag
     3121 cacccagtga agctactgtt tgctcctaac ttgctcttgg acaggaacca gggaaaatgt
     3181 gtagagggca tggtggagat cttcgacatg ctgctggcta catcatctcg gttccgcatg
     3241 atgaatctgc agggagagga gtttgtgtgc ctcaaatcta ttattttgct taattctgga
     3301 gtgtacacat ttctgtccag caccctgaag tctctggaag agaaggacca tatccaccga
     3361 gtcctggaca agatcacaga cactttgatc cacctgatgg ccaaggcagg cctgaccctg
     3421 cagcagcagc accagcggct ggcccagctc ctcctcatcc tctcccacat caggcacatg
     3481 agtaacaaag gcatggagca tctgtacagc atgaagtgca agaacgtggt gcccctctat
     3541 gacctgctgc tggaggcggc ggacgcccac cgcctacatg cgcccactag ccgtggaggg
     3601 gcatccgtgg aggagacgga ccaaagccac ttggccactg cgggctctac ttcatcgcat
     3661 tccttgcaaa agtattacat cacgggggag gcagagggtt tccctgccac agcttgatag
     3721 cggccgcact cctcaggtgc aggctgccta tcagaaggtg gtggctggtg tggccaatgc
     3781 cctggctcac aaataccact gagatctttt tccctctgcc aaaaattatg gggacatcat
     3841 gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt
     3901 gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa
     3961 acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc tggctgccat
     4021 gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc
     4081 cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt
     4141 atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct
     4201 cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat ccctcgacct
     4261 gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     4321 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     4381 gagtgagcta actcacatta ggatcgggac cctctagttt aaacgcctac gcggatccac
     4441 tagttctaga gcggccgcca ccgcggtgga gctccagctt ttgttccctt tagtgagggt
     4501 taattgcgcg cttggcgtaa tcatggtcat agcacccagc tttcttgtac aaagttggca
     4561 ttataagaaa gcattgctta tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata
     4621 aaatcattat ttgccatcca gctgatatcc cctatagtga gtcgtattac atggtcatag
     4681 ctgtttcctg gcagctctgg cccgtgtctc aaaatctctg atgttacatt gcacaagata
     4741 aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg
     4801 ttatgagcca tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg
     4861 atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc
     4921 gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg
     4981 ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc
     5041 cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc
     5101 ccggaaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg
     5161 atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta
     5221 acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg
     5281 atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa
     5341 tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg
     5401 ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa
     5461 tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt
     5521 cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc
     5581 agtttcattt gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaacact
     5641 ggcagagcat tacgctgact tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg
     5701 tgagttacgc gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     5761 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     5821 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     5881 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     5941 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6001 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6061 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6121 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6181 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     6241 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6301 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6361 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtt
//
