LOCUS       sequence-394650-        6421 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini circle middle entry plasmid for P2A-CreER-pA (ORF 1) 
            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 6421)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6421)
  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            "Unpublished"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 ORF1 FRT-acry:mCherry-pA-FRT
FEATURES             Location/Qualifiers
     source          1..6421
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             40..96
                     /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             97..1122
                     /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             1135..2070
                     /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             1183..2076
                     /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    2106..2240
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2255..2288
                     /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    2289..2410
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(2533..3243)
                     /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(2613..2636)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2811..2830)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3080..3098
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      3240..3249
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3885..3918
                     /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             4347..5138
                     /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      5231..5819
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5720..5739
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5973..5990
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(6149..6176)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(6268..6354)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(6403..6421,1..4)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(6417..6421,1..13)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     join(6418..6421,1..13)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 aacgacggcc agtcttaagc tcgggcccgg tctctagtcg ctacaaactt ctctctgctg
       61 aagcaggctg gagacgtgga ggagaaccct ggaccttcca atttactgac cgtacaccaa
      121 aatttgcctg cattaccggt cgatgcaacg agtgatgagg ttcgcaagaa cctgatggac
      181 atgttcaggg atcgccaggc gttttctgag catacctgga aaatgcttct gtccgtttgc
      241 cggtcgtggg cggcatggtg caagttgaat aaccggaaat ggtttcccgc agaacctgaa
      301 gatgttcgcg attatcttct atatcttcag gcgcgcggtc tggcagtaaa aactatccag
      361 caacatttgg gccagctaaa catgcttcat cgtcggtccg ggctgccacg accaagtgac
      421 agcaatgctg tttcactggt tatgcggcgg atccgaaaag aaaacgttga tgccggtgaa
      481 cgtgcaaaac aggctctagc gttcgaacgc actgatttcg accaggttcg ttcactcatg
      541 gaaaatagcg atcgctgcca ggatatacgt aatctggcat ttctggggat tgcttataac
      601 accctgttac gtatagccga aattgccagg atcagggtta aagatatctc acgtactgac
      661 ggtgggagaa tgttaatcca tattggcaga acgaaaacgc tggttagcac cgcaggtgta
      721 gagaaggcac ttagcctggg ggtaactaaa ctggtcgagc gatggatttc cgtctctggt
      781 gtagctgatg atccgaataa ctacctgttt tgccgggtca gaaaaaatgg tgttgccgcg
      841 ccatctgcca ccagccagct atcaactcgc gccctggaag ggatttttga agcaactcat
      901 cgattgattt acggcgctaa ggatgactct ggtcagagat acctggcctg gtctggacac
      961 agtgcccgtg tcggagccgc gcgagatatg gcccgcgctg gagtttcaat accggagatc
     1021 atgcaagctg gtggctggac caatgtaaat attgtcatga actatatccg taacctggat
     1081 agtgaaacag gggcaatggt gcgcctgctg gaagatggcg atctcgagcc atctgctgga
     1141 gacatgagag ctgccaacct ttggccaagc ccgctcatga tcaaacgctc taagaagaac
     1201 agcctggcct tgtccctgac ggccgaccag atggtcagtg ccttgttgga tgctgagccc
     1261 cccatactct attccgagta tgatcctacc agacccttca gtgaagcttc gatgatgggc
     1321 ttactgacca acctggcaga cagggagctg gttcacatga tcaactgggc gaagagggtg
     1381 ccaggctttg tggatttgac cctccatgat caggtccacc ttctagaatg tgcctggcta
     1441 gagatcctga tgattggcct cgtctggcgc tccatggagc acccagtgaa gctactgttt
     1501 gctcctaact tgctcttgga caggaaccag ggaaaatgtg tagagggcat ggtggagatc
     1561 ttcgacatgc tgctggctac atcatctcgg ttccgcatga tgaatctgca gggagaggag
     1621 tttgtgtgcc tcaaatctat tattttgctt aattctggag tgtacacatt tctgtccagc
     1681 accctgaagt ctctggaaga gaaggaccat atccaccgag tcctggacaa gatcacagac
     1741 actttgatcc acctgatggc caaggcaggc ctgaccctgc agcagcagca ccagcggctg
     1801 gcccagctcc tcctcatcct ctcccacatc aggcacatga gtaacaaagg catggagcat
     1861 ctgtacagca tgaagtgcaa gaacgtggtg cccctctatg acctgctgct ggaggcggcg
     1921 gacgcccacc gcctacatgc gcccactagc cgtggagggg catccgtgga ggagacggac
     1981 caaagccact tggccactgc gggctctact tcatcgcatt ccttgcaaaa gtattacatc
     2041 acgggggagg cagagggttt ccctgccaca gcttaatcta gaactatagt gagtcgtatt
     2101 acgtagatcc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     2161 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     2221 taagctgcaa taaacaagtt aacaacaaca attcgaagtt cctattctct agaaagtata
     2281 ggaacttcta agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     2341 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     2401 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     2461 ggaggttttt taaagcaagt aaaacctcta caaatgtggt atggctgatt atgatctaga
     2521 gtcgcggccg ctttacttgt acagctcgtc catgccgccg gtggagtggt ggccctcggc
     2581 gcgctcgtac tgttccacga tggtgtagtc ctcgttgtgg gaggtgatgt ccagcttgat
     2641 gttgacgttg taggcgccgg gcagctgcac gggcttcttg gccttgtagg tggtcttgac
     2701 ctcggcgtcg tagtggccgc cgtccttcag cttcagcctc tgcttgatct cgcccttcag
     2761 ggcgccgtcc tcggggtaca tccgctcgga ggaggcctcc cagcccatgg tcttcttctg
     2821 cattacgggg ccgtcggagg ggaagttggt gccgcgcagc ttcaccttgt agatgaactc
     2881 gccgtcctgc agggaggagt cctgggtcac ggtcaccacg ccgccgtcct cgaagttcat
     2941 cacgcgctcc cacttgaagc cctcggggaa ggacagcttc aagtagtcgg ggatgtcggc
     3001 ggggtgcttc acgtaggcct tggagccgta catgaactga ggggacagga tgtcccaggc
     3061 gaagggcagg gggccgccct tggtcacctt cagcttggcg gtctgggtgc cctcgtaggg
     3121 gcggccctcg ccctcgccct cgatctcgaa ctcgtggccg ttcacggagc cctccatgtg
     3181 caccttgaag cgcatgaact ccttgatgat ggccatgttg tcatcctcgc ccttgctcac
     3241 catggtggcg accggtaatg tcagacctgg taactcctta ctgtaactta ttgcacctga
     3301 tgaacaaaag atgtgaactg aacgcaagta ggaggctaat agaggctagt acagtacaaa
     3361 gatccccaaa tgatgtgtgc cggccttagt ggaggcagac ccctggttat atacctgtgc
     3421 tgcagaaaga ggggcttaca aatggatctc tctggaatgg cattatctca tggtgggaac
     3481 agtgttgtca gcagaatcag cggggtggac tctgacctga gaatgacagt ctagcactga
     3541 atggctcaga caatactctt gtggccaact atcaccggaa aataagtgcc tgccaaaaca
     3601 aaccagacca gatgaagcta catggctgta gaacgtcagt atttaatgtg ttgtggccac
     3661 ttggttcacc agatttaaga gctaattttt ttaaatatca cagataatca atcgcttttt
     3721 tttttcttga ctgaaaagct tagactggaa aattctttga aaaagtttgc actacagaaa
     3781 ataaatacat aacatcctgc actgaatgca cactattaat agaaaagaga gcattattgt
     3841 aggaaatgta ttgtatatgt acattctgtg catttcacaa ttttgaagtt cctattctct
     3901 agaaagtata ggaacttcga attgcattca ttttatgttt caggttcagg gggaggtgtg
     3961 ggaggttttt taattcctca agagacctct agaccagcca ggacagaaat gcctcgactt
     4021 cgctgctacc caaggttgcc gggtgacgca caccgtggaa acggatgaag gcacgaaccc
     4081 agtggacata agcctgttcg gttcgtaagc tgtaatgcaa gtagcgtatg cgctcacgca
     4141 actggtccag aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg
     4201 cttgttatga ctgttttttt ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc
     4261 gttacgccgt gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg cagcagggca
     4321 gtcgccctaa aacaaagtta aacattatga gggaagcggt gatcgccgaa gtatcgactc
     4381 aactatcaga ggtagttggc gtcatcgagc gccatctcga accgacgttg ctggccgtac
     4441 atttgtacgg ctccgcagtg gatggcggcc tgaagccaca cagtgatatt gatttgctgg
     4501 ttacggtgac cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg
     4561 aaacttcggc ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc accattgttg
     4621 tgcacgacga catcattccg tggcgttatc cagctaagcg cgaactgcaa tttggagaat
     4681 ggcagcgcaa tgacattctt gcaggtatct tcgagccagc cacgatcgac attgatctgg
     4741 ctatcttgct gacaaaagca agagaacata gcgttgcctt ggtaggtcca gcggcggagg
     4801 aactctttga tccggttcct gaacaggatc tatttgaggc gctaaatgaa accttaacgc
     4861 tatggaactc gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc
     4921 gcatttggta cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg
     4981 caatggagcg cctgccggcc cagtatcagc ccgtcatact tgaagctaga caggcttatc
     5041 ttggacaaga agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgtccact
     5101 acgtgaaagg cgagatcacc aaggtagtcg gcaaataacc ctcgagccac ccatgaccaa
     5161 aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta gaaaagatca
     5221 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     5281 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     5341 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     5401 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     5461 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5521 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     5581 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     5641 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     5701 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5761 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     5821 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     5881 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     5941 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     6001 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     6061 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat acgcgtaccg
     6121 ctagccagga agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg ccttctgctt
     6181 agtttgatgc ctggcagttt atggcgggcg tcctgcccgc caccctccgg gccgttgctt
     6241 cacaacgttc aaatccgctc ccggcggatt tgtcctactc aggagagcgt tcaccgacaa
     6301 acaacagata aaacgaaagg cccagtcttc cgactgagcc tttcgtttta tttgatgcct
     6361 ggcagttccc tactctcgcg ttaacgctag catggatgtt ttcccagtca cgacgttgta
     6421 a
//
