LOCUS       sequence-482146-        6373 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Middle entry vector for mini-Golden subcloning platform; creER; 
            acry-mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6373)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 6373)
  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"
COMMENT     Sequence Label: CreER-pA_FRT-acrymCherry
FEATURES             Location/Qualifiers
     source          1..6373
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(1..87)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     136..158
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     150..167
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     151..167
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             200..1228
                     /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="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     CDS             1241..2176
                     /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             1289..2182
                     /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    2212..2346
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2361..2394
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     polyA_signal    2395..2516
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(2639..3349)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEDDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGHHSTGGMDELYK"
     primer_bind     complement(2719..2742)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2917..2936)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3186..3204
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      3346..3355
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3991..4024
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             4453..5244
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      5337..5925
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5826..5845
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6079..6096
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(6255..6282)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc
       61 ttccgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc gcgttaacgc
      121 tagcatggat gttttcccag tcacgacgtt gtaaaacgac ggccagtctt aagctcgggc
      181 ccggtctctt cgcgccacca tgtccaattt actgaccgta caccaaaatt tgcctgcatt
      241 accggtcgat gcaacgagtg atgaggttcg caagaacctg atggacatgt tcagggatcg
      301 ccaggcgttt tctgagcata cctggaaaat gcttctgtcc gtttgccggt cgtgggcggc
      361 atggtgcaag ttgaataacc ggaaatggtt tcccgcagaa cctgaagatg ttcgcgatta
      421 tcttctatat cttcaggcgc gcggtctggc agtaaaaact atccagcaac atttgggcca
      481 gctaaacatg cttcatcgtc ggtccgggct gccacgacca agtgacagca atgctgtttc
      541 actggttatg cggcggatcc gaaaagaaaa cgttgatgcc ggtgaacgtg caaaacaggc
      601 tctagcgttc gaacgcactg atttcgacca ggttcgttca ctcatggaaa atagcgatcg
      661 ctgccaggat atacgtaatc tggcatttct ggggattgct tataacaccc tgttacgtat
      721 agccgaaatt gccaggatca gggttaaaga tatctcacgt actgacggtg ggagaatgtt
      781 aatccatatt ggcagaacga aaacgctggt tagcaccgca ggtgtagaga aggcacttag
      841 cctgggggta actaaactgg tcgagcgatg gatttccgtc tctggtgtag ctgatgatcc
      901 gaataactac ctgttttgcc gggtcagaaa aaatggtgtt gccgcgccat ctgccaccag
      961 ccagctatca actcgcgccc tggaagggat ttttgaagca actcatcgat tgatttacgg
     1021 cgctaaggat gactctggtc agagatacct ggcctggtct ggacacagtg cccgtgtcgg
     1081 agccgcgcga gatatggccc gcgctggagt ttcaataccg gagatcatgc aagctggtgg
     1141 ctggaccaat gtaaatattg tcatgaacta tatccgtaac ctggatagtg aaacaggggc
     1201 aatggtgcgc ctgctggaag atggcgatct cgagccatct gctggagaca tgagagctgc
     1261 caacctttgg ccaagcccgc tcatgatcaa acgctctaag aagaacagcc tggccttgtc
     1321 cctgacggcc gatcagatgg tcagtgcctt gttggatgct gagcccccca tactctattc
     1381 cgagtatgat cctaccagac ccttcagtga agcttcgatg atgggcttac tgaccaacct
     1441 ggcagacagg gagctggttc acatgatcaa ctgggcgaag agggtgccag gctttgtgga
     1501 tttgaccctc catgatcagg tccaccttct agaatgtgcc tggctagaga tcctgatgat
     1561 tggcctcgtc tggcgctcca tggagcaccc agtgaagcta ctgtttgctc ctaacttgct
     1621 cttggacagg aaccagggaa aatgtgtaga gggcatggtg gagatcttcg acatgctgct
     1681 ggctacatca tctcggttcc gcatgatgaa tctgcaggga gaggagtttg tgtgcctcaa
     1741 atctattatt ttgcttaatt ctggagtgta cacatttctg tccagcaccc tgaagtctct
     1801 ggaagagaag gaccatatcc accgagtcct ggacaagatc acagacactt tgatccacct
     1861 gatggccaag gcaggcctga ccctgcagca gcagcaccag cggctggccc agctcctcct
     1921 catcctctcc cacatcaggc acatgagtaa caaaggcatg gagcatctgt acagcatgaa
     1981 gtgcaagaac gtggtgcccc tctatgacct gctgctggag gcggcggacg cccaccgcct
     2041 acatgcgccc actagccgtg gaggggcatc cgtggaggag acggaccaaa gccacttggc
     2101 cactgcgggc tctacttcat cgcattcctt gcaaaagtat tacatcacgg gggaggcaga
     2161 gggtttccct gccacagctt aatctagaac tatagtgagt cgtattacgt agatccagac
     2221 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     2281 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     2341 caagttaaca acaacaattc gaagttccta ttctctagaa agtataggaa cttctaagat
     2401 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     2461 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     2521 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
     2581 gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tctagagtcg cggccgcttt
     2641 acttgtacag ctcgtccatg ccgccggtgg agtggtggcc ctcggcgcgc tcgtactgtt
     2701 ccacgatggt gtagtcctcg ttgtgggagg tgatgtccag cttgatgttg acgttgtagg
     2761 cgccgggcag ctgcacgggc ttcttggcct tgtaggtggt cttgacctcg gcgtcgtagt
     2821 ggccgccgtc cttcagcttc agcctctgct tgatctcgcc cttcagggcg ccgtcctcgg
     2881 ggtacatccg ctcggaggag gcctcccagc ccatggtctt cttctgcatt acggggccgt
     2941 cggaggggaa gttggtgccg cgcagcttca ccttgtagat gaactcgccg tcctgcaggg
     3001 aggagtcctg ggtcacggtc accacgccgc cgtcctcgaa gttcatcacg cgctcccact
     3061 tgaagccctc ggggaaggac agcttcaagt agtcggggat gtcggcgggg tgcttcacgt
     3121 aggccttgga gccgtacatg aactgagggg acaggatgtc ccaggcgaag ggcagggggc
     3181 cgcccttggt caccttcagc ttggcggtct gggtgccctc gtaggggcgg ccctcgccct
     3241 cgccctcgat ctcgaactcg tggccgttca cggagccctc catgtgcacc ttgaagcgca
     3301 tgaactcctt gatgatggcc atgttgtcat cctcgccctt gctcaccatg gtggcgaccg
     3361 gtaatgtcag acctggtaac tccttactgt aacttattgc acctgatgaa caaaagatgt
     3421 gaactgaacg caagtaggag gctaatagag gctagtacag tacaaagatc cccaaatgat
     3481 gtgtgccggc cttagtggag gcagacccct ggttatatac ctgtgctgca gaaagagggg
     3541 cttacaaatg gatctctctg gaatggcatt atctcatggt gggaacagtg ttgtcagcag
     3601 aatcagcggg gtggactctg acctgagaat gacagtctag cactgaatgg ctcagacaat
     3661 actcttgtgg ccaactatca ccggaaaata agtgcctgcc aaaacaaacc agaccagatg
     3721 aagctacatg gctgtagaac gtcagtattt aatgtgttgt ggccacttgg ttcaccagat
     3781 ttaagagcta atttttttaa atatcacaga taatcaatcg cttttttttt tcttgactga
     3841 aaagcttaga ctggaaaatt ctttgaaaaa gtttgcacta cagaaaataa atacataaca
     3901 tcctgcactg aatgcacact attaatagaa aagagagcat tattgtagga aatgtattgt
     3961 atatgtacat tctgtgcatt tcacaatttt gaagttccta ttctctagaa agtataggaa
     4021 cttcgaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaat
     4081 tcctcaagag acctctagac cagccaggac agaaatgcct cgacttcgct gctacccaag
     4141 gttgccgggt gacgcacacc gtggaaacgg atgaaggcac gaacccagtg gacataagcc
     4201 tgttcggttc gtaagctgta atgcaagtag cgtatgcgct cacgcaactg gtccagaacc
     4261 ttgaccgaac gcagcggtgg taacggcgca gtggcggttt tcatggcttg ttatgactgt
     4321 ttttttgggg tacagtctat gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt
     4381 cgatgtttga tgttatggag cagcaacgat gttacgcagc agggcagtcg ccctaaaaca
     4441 aagttaaaca ttatgaggga agcggtgatc gccgaagtat cgactcaact atcagaggta
     4501 gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg ccgtacattt gtacggctcc
     4561 gcagtggatg gcggcctgaa gccacacagt gatattgatt tgctggttac ggtgaccgta
     4621 aggcttgatg aaacaacgcg gcgagctttg atcaacgacc ttttggaaac ttcggcttcc
     4681 cctggagaga gcgagattct ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc
     4741 attccgtggc gttatccagc taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac
     4801 attcttgcag gtatcttcga gccagccacg atcgacattg atctggctat cttgctgaca
     4861 aaagcaagag aacatagcgt tgccttggta ggtccagcgg cggaggaact ctttgatccg
     4921 gttcctgaac aggatctatt tgaggcgcta aatgaaacct taacgctatg gaactcgccg
     4981 cccgactggg ctggcgatga gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc
     5041 gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg actgggcaat ggagcgcctg
     5101 ccggcccagt atcagcccgt catacttgaa gctagacagg cttatcttgg acaagaagaa
     5161 gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg tccactacgt gaaaggcgag
     5221 atcaccaagg tagtcggcaa ataaccctcg agccacccat gaccaaaatc ccttaacgtg
     5281 agttacgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5341 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5401 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5461 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     5521 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5581 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5641 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5701 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5761 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5821 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5881 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5941 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     6001 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     6061 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     6121 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     6181 gactggaaag cgggcagtga gcgcaacgca attaatacgc gtaccgctag ccaggaagag
     6241 tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt ctgcttagtt tgatgcctgg
     6301 cagtttatgg cgggcgtcct gcccgccacc ctccgggccg ttgcttcaca acgttcaaat
     6361 ccgctcccgg cgg
//
