LOCUS       sequence-394489-        6423 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini circle middle entry plasmid for P2A-CreER-pA (ORF 3) 
            FRT-acry:mCherry-pA-FRT with flanking BsaI cut sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6423)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6423)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pMC-ME_P2A-CreER-pA ORF3 FRT-acry:mCherry-pA-FRT
FEATURES             Location/Qualifiers
     source          1..6423
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(17..103)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     152..174
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     166..183
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     167..183
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             212..268
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             269..1294
                     /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="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     CDS             1307..2242
                     /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             1355..2248
                     /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    2278..2412
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2427..2460
                     /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    2461..2582
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(2705..3415)
                     /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(2785..2808)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2983..3002)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3252..3270
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      3412..3421
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4057..4090
                     /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             4519..5310
                     /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      5403..5991
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5892..5911
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6145..6162
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(6321..6348)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa
       61 aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg gcagttccct
      121 actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa aacgacggcc
      181 agtcttaagc tcgggcccgg tctctagtcc cgctacaaac ttctctctgc tgaagcaggc
      241 tggagacgtg gaggagaacc ctggaccttc caatttactg accgtacacc aaaatttgcc
      301 tgcattaccg gtcgatgcaa cgagtgatga ggttcgcaag aacctgatgg acatgttcag
      361 ggatcgccag gcgttttctg agcatacctg gaaaatgctt ctgtccgttt gccggtcgtg
      421 ggcggcatgg tgcaagttga ataaccggaa atggtttccc gcagaacctg aagatgttcg
      481 cgattatctt ctatatcttc aggcgcgcgg tctggcagta aaaactatcc agcaacattt
      541 gggccagcta aacatgcttc atcgtcggtc cgggctgcca cgaccaagtg acagcaatgc
      601 tgtttcactg gttatgcggc ggatccgaaa agaaaacgtt gatgccggtg aacgtgcaaa
      661 acaggctcta gcgttcgaac gcactgattt cgaccaggtt cgttcactca tggaaaatag
      721 cgatcgctgc caggatatac gtaatctggc atttctgggg attgcttata acaccctgtt
      781 acgtatagcc gaaattgcca ggatcagggt taaagatatc tcacgtactg acggtgggag
      841 aatgttaatc catattggca gaacgaaaac gctggttagc accgcaggtg tagagaaggc
      901 acttagcctg ggggtaacta aactggtcga gcgatggatt tccgtctctg gtgtagctga
      961 tgatccgaat aactacctgt tttgccgggt cagaaaaaat ggtgttgccg cgccatctgc
     1021 caccagccag ctatcaactc gcgccctgga agggattttt gaagcaactc atcgattgat
     1081 ttacggcgct aaggatgact ctggtcagag atacctggcc tggtctggac acagtgcccg
     1141 tgtcggagcc gcgcgagata tggcccgcgc tggagtttca ataccggaga tcatgcaagc
     1201 tggtggctgg accaatgtaa atattgtcat gaactatatc cgtaacctgg atagtgaaac
     1261 aggggcaatg gtgcgcctgc tggaagatgg cgatctcgag ccatctgctg gagacatgag
     1321 agctgccaac ctttggccaa gcccgctcat gatcaaacgc tctaagaaga acagcctggc
     1381 cttgtccctg acggccgacc agatggtcag tgccttgttg gatgctgagc cccccatact
     1441 ctattccgag tatgatccta ccagaccctt cagtgaagct tcgatgatgg gcttactgac
     1501 caacctggca gacagggagc tggttcacat gatcaactgg gcgaagaggg tgccaggctt
     1561 tgtggatttg accctccatg atcaggtcca ccttctagaa tgtgcctggc tagagatcct
     1621 gatgattggc ctcgtctggc gctccatgga gcacccagtg aagctactgt ttgctcctaa
     1681 cttgctcttg gacaggaacc agggaaaatg tgtagagggc atggtggaga tcttcgacat
     1741 gctgctggct acatcatctc ggttccgcat gatgaatctg cagggagagg agtttgtgtg
     1801 cctcaaatct attattttgc ttaattctgg agtgtacaca tttctgtcca gcaccctgaa
     1861 gtctctggaa gagaaggacc atatccaccg agtcctggac aagatcacag acactttgat
     1921 ccacctgatg gccaaggcag gcctgaccct gcagcagcag caccagcggc tggcccagct
     1981 cctcctcatc ctctcccaca tcaggcacat gagtaacaaa ggcatggagc atctgtacag
     2041 catgaagtgc aagaacgtgg tgcccctcta tgacctgctg ctggaggcgg cggacgccca
     2101 ccgcctacat gcgcccacta gccgtggagg ggcatccgtg gaggagacgg accaaagcca
     2161 cttggccact gcgggctcta cttcatcgca ttccttgcaa aagtattaca tcacggggga
     2221 ggcagagggt ttccctgcca cagcttaatc tagaactata gtgagtcgta ttacgtagat
     2281 ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     2341 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     2401 aataaacaag ttaacaacaa caattcgaag ttcctattct ctagaaagta taggaacttc
     2461 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
     2521 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
     2581 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt
     2641 tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga ttatgatcta gagtcgcggc
     2701 cgctttactt gtacagctcg tccatgccgc cggtggagtg gtggccctcg gcgcgctcgt
     2761 actgttccac gatggtgtag tcctcgttgt gggaggtgat gtccagcttg atgttgacgt
     2821 tgtaggcgcc gggcagctgc acgggcttct tggccttgta ggtggtcttg acctcggcgt
     2881 cgtagtggcc gccgtccttc agcttcagcc tctgcttgat ctcgcccttc agggcgccgt
     2941 cctcggggta catccgctcg gaggaggcct cccagcccat ggtcttcttc tgcattacgg
     3001 ggccgtcgga ggggaagttg gtgccgcgca gcttcacctt gtagatgaac tcgccgtcct
     3061 gcagggagga gtcctgggtc acggtcacca cgccgccgtc ctcgaagttc atcacgcgct
     3121 cccacttgaa gccctcgggg aaggacagct tcaagtagtc ggggatgtcg gcggggtgct
     3181 tcacgtaggc cttggagccg tacatgaact gaggggacag gatgtcccag gcgaagggca
     3241 gggggccgcc cttggtcacc ttcagcttgg cggtctgggt gccctcgtag gggcggccct
     3301 cgccctcgcc ctcgatctcg aactcgtggc cgttcacgga gccctccatg tgcaccttga
     3361 agcgcatgaa ctccttgatg atggccatgt tgtcatcctc gcccttgctc accatggtgg
     3421 cgaccggtaa tgtcagacct ggtaactcct tactgtaact tattgcacct gatgaacaaa
     3481 agatgtgaac tgaacgcaag taggaggcta atagaggcta gtacagtaca aagatcccca
     3541 aatgatgtgt gccggcctta gtggaggcag acccctggtt atatacctgt gctgcagaaa
     3601 gaggggctta caaatggatc tctctggaat ggcattatct catggtggga acagtgttgt
     3661 cagcagaatc agcggggtgg actctgacct gagaatgaca gtctagcact gaatggctca
     3721 gacaatactc ttgtggccaa ctatcaccgg aaaataagtg cctgccaaaa caaaccagac
     3781 cagatgaagc tacatggctg tagaacgtca gtatttaatg tgttgtggcc acttggttca
     3841 ccagatttaa gagctaattt ttttaaatat cacagataat caatcgcttt tttttttctt
     3901 gactgaaaag cttagactgg aaaattcttt gaaaaagttt gcactacaga aaataaatac
     3961 ataacatcct gcactgaatg cacactatta atagaaaaga gagcattatt gtaggaaatg
     4021 tattgtatat gtacattctg tgcatttcac aattttgaag ttcctattct ctagaaagta
     4081 taggaacttc gaattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt
     4141 tttaattcct caagagacct ctagaccagc caggacagaa atgcctcgac ttcgctgcta
     4201 cccaaggttg ccgggtgacg cacaccgtgg aaacggatga aggcacgaac ccagtggaca
     4261 taagcctgtt cggttcgtaa gctgtaatgc aagtagcgta tgcgctcacg caactggtcc
     4321 agaaccttga ccgaacgcag cggtggtaac ggcgcagtgg cggttttcat ggcttgttat
     4381 gactgttttt ttggggtaca gtctatgcct cgggcatcca agcagcaagc gcgttacgcc
     4441 gtgggtcgat gtttgatgtt atggagcagc aacgatgtta cgcagcaggg cagtcgccct
     4501 aaaacaaagt taaacattat gagggaagcg gtgatcgccg aagtatcgac tcaactatca
     4561 gaggtagttg gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt acatttgtac
     4621 ggctccgcag tggatggcgg cctgaagcca cacagtgata ttgatttgct ggttacggtg
     4681 accgtaaggc ttgatgaaac aacgcggcga gctttgatca acgacctttt ggaaacttcg
     4741 gcttcccctg gagagagcga gattctccgc gctgtagaag tcaccattgt tgtgcacgac
     4801 gacatcattc cgtggcgtta tccagctaag cgcgaactgc aatttggaga atggcagcgc
     4861 aatgacattc ttgcaggtat cttcgagcca gccacgatcg acattgatct ggctatcttg
     4921 ctgacaaaag caagagaaca tagcgttgcc ttggtaggtc cagcggcgga ggaactcttt
     4981 gatccggttc ctgaacagga tctatttgag gcgctaaatg aaaccttaac gctatggaac
     5041 tcgccgcccg actgggctgg cgatgagcga aatgtagtgc ttacgttgtc ccgcatttgg
     5101 tacagcgcag taaccggcaa aatcgcgccg aaggatgtcg ctgccgactg ggcaatggag
     5161 cgcctgccgg cccagtatca gcccgtcata cttgaagcta gacaggctta tcttggacaa
     5221 gaagaagatc gcttggcctc gcgcgcagat cagttggaag aatttgtcca ctacgtgaaa
     5281 ggcgagatca ccaaggtagt cggcaaataa ccctcgagcc acccatgacc aaaatccctt
     5341 aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5401 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5461 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     5521 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     5581 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5641 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     5701 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     5761 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     5821 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     5881 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     5941 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     6001 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     6061 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     6121 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     6181 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     6241 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atacgcgtac cgctagccag
     6301 gaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc ttagtttgat
     6361 gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt
     6421 tca
//
