LOCUS       sequence-394651-        6422 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini circle middle entry plasmid for P2A-CreER-pA (ORF 2) 
            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 6422)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6422)
  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            "Unpublished"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 ORF2 FRT-acry:mCherry-pA-FRT
FEATURES             Location/Qualifiers
     source          1..6422
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     18..40
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     32..49
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     33..49
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             77..133
                     /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             134..1159
                     /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             1172..2107
                     /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             1220..2113
                     /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    2143..2277
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2292..2325
                     /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    2326..2447
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(2570..3280)
                     /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(2650..2673)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2848..2867)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3117..3135
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      3277..3286
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3922..3955
                     /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             4384..5175
                     /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      5268..5856
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5757..5776
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6010..6027
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(6186..6213)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(6305..6391)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gctagcatgg atgttttccc agtcacgacg ttgtaaaacg acggccagtc ttaagctcgg
       61 gcccggtctc tagtccgcta caaacttctc tctgctgaag caggctggag acgtggagga
      121 gaaccctgga ccttccaatt tactgaccgt acaccaaaat ttgcctgcat taccggtcga
      181 tgcaacgagt gatgaggttc gcaagaacct gatggacatg ttcagggatc gccaggcgtt
      241 ttctgagcat acctggaaaa tgcttctgtc cgtttgccgg tcgtgggcgg catggtgcaa
      301 gttgaataac cggaaatggt ttcccgcaga acctgaagat gttcgcgatt atcttctata
      361 tcttcaggcg cgcggtctgg cagtaaaaac tatccagcaa catttgggcc agctaaacat
      421 gcttcatcgt cggtccgggc tgccacgacc aagtgacagc aatgctgttt cactggttat
      481 gcggcggatc cgaaaagaaa acgttgatgc cggtgaacgt gcaaaacagg ctctagcgtt
      541 cgaacgcact gatttcgacc aggttcgttc actcatggaa aatagcgatc gctgccagga
      601 tatacgtaat ctggcatttc tggggattgc ttataacacc ctgttacgta tagccgaaat
      661 tgccaggatc agggttaaag atatctcacg tactgacggt gggagaatgt taatccatat
      721 tggcagaacg aaaacgctgg ttagcaccgc aggtgtagag aaggcactta gcctgggggt
      781 aactaaactg gtcgagcgat ggatttccgt ctctggtgta gctgatgatc cgaataacta
      841 cctgttttgc cgggtcagaa aaaatggtgt tgccgcgcca tctgccacca gccagctatc
      901 aactcgcgcc ctggaaggga tttttgaagc aactcatcga ttgatttacg gcgctaagga
      961 tgactctggt cagagatacc tggcctggtc tggacacagt gcccgtgtcg gagccgcgcg
     1021 agatatggcc cgcgctggag tttcaatacc ggagatcatg caagctggtg gctggaccaa
     1081 tgtaaatatt gtcatgaact atatccgtaa cctggatagt gaaacagggg caatggtgcg
     1141 cctgctggaa gatggcgatc tcgagccatc tgctggagac atgagagctg ccaacctttg
     1201 gccaagcccg ctcatgatca aacgctctaa gaagaacagc ctggccttgt ccctgacggc
     1261 cgaccagatg gtcagtgcct tgttggatgc tgagcccccc atactctatt ccgagtatga
     1321 tcctaccaga cccttcagtg aagcttcgat gatgggctta ctgaccaacc tggcagacag
     1381 ggagctggtt cacatgatca actgggcgaa gagggtgcca ggctttgtgg atttgaccct
     1441 ccatgatcag gtccaccttc tagaatgtgc ctggctagag atcctgatga ttggcctcgt
     1501 ctggcgctcc atggagcacc cagtgaagct actgtttgct cctaacttgc tcttggacag
     1561 gaaccaggga aaatgtgtag agggcatggt ggagatcttc gacatgctgc tggctacatc
     1621 atctcggttc cgcatgatga atctgcaggg agaggagttt gtgtgcctca aatctattat
     1681 tttgcttaat tctggagtgt acacatttct gtccagcacc ctgaagtctc tggaagagaa
     1741 ggaccatatc caccgagtcc tggacaagat cacagacact ttgatccacc tgatggccaa
     1801 ggcaggcctg accctgcagc agcagcacca gcggctggcc cagctcctcc tcatcctctc
     1861 ccacatcagg cacatgagta acaaaggcat ggagcatctg tacagcatga agtgcaagaa
     1921 cgtggtgccc ctctatgacc tgctgctgga ggcggcggac gcccaccgcc tacatgcgcc
     1981 cactagccgt ggaggggcat ccgtggagga gacggaccaa agccacttgg ccactgcggg
     2041 ctctacttca tcgcattcct tgcaaaagta ttacatcacg ggggaggcag agggtttccc
     2101 tgccacagct taatctagaa ctatagtgag tcgtattacg tagatccaga catgataaga
     2161 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     2221 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     2281 aacaacaatt cgaagttcct attctctaga aagtatagga acttctaaga tacattgatg
     2341 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     2401 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     2461 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa
     2521 acctctacaa atgtggtatg gctgattatg atctagagtc gcggccgctt tacttgtaca
     2581 gctcgtccat gccgccggtg gagtggtggc cctcggcgcg ctcgtactgt tccacgatgg
     2641 tgtagtcctc gttgtgggag gtgatgtcca gcttgatgtt gacgttgtag gcgccgggca
     2701 gctgcacggg cttcttggcc ttgtaggtgg tcttgacctc ggcgtcgtag tggccgccgt
     2761 ccttcagctt cagcctctgc ttgatctcgc ccttcagggc gccgtcctcg gggtacatcc
     2821 gctcggagga ggcctcccag cccatggtct tcttctgcat tacggggccg tcggagggga
     2881 agttggtgcc gcgcagcttc accttgtaga tgaactcgcc gtcctgcagg gaggagtcct
     2941 gggtcacggt caccacgccg ccgtcctcga agttcatcac gcgctcccac ttgaagccct
     3001 cggggaagga cagcttcaag tagtcgggga tgtcggcggg gtgcttcacg taggccttgg
     3061 agccgtacat gaactgaggg gacaggatgt cccaggcgaa gggcaggggg ccgcccttgg
     3121 tcaccttcag cttggcggtc tgggtgccct cgtaggggcg gccctcgccc tcgccctcga
     3181 tctcgaactc gtggccgttc acggagccct ccatgtgcac cttgaagcgc atgaactcct
     3241 tgatgatggc catgttgtca tcctcgccct tgctcaccat ggtggcgacc ggtaatgtca
     3301 gacctggtaa ctccttactg taacttattg cacctgatga acaaaagatg tgaactgaac
     3361 gcaagtagga ggctaataga ggctagtaca gtacaaagat ccccaaatga tgtgtgccgg
     3421 ccttagtgga ggcagacccc tggttatata cctgtgctgc agaaagaggg gcttacaaat
     3481 ggatctctct ggaatggcat tatctcatgg tgggaacagt gttgtcagca gaatcagcgg
     3541 ggtggactct gacctgagaa tgacagtcta gcactgaatg gctcagacaa tactcttgtg
     3601 gccaactatc accggaaaat aagtgcctgc caaaacaaac cagaccagat gaagctacat
     3661 ggctgtagaa cgtcagtatt taatgtgttg tggccacttg gttcaccaga tttaagagct
     3721 aattttttta aatatcacag ataatcaatc gctttttttt ttcttgactg aaaagcttag
     3781 actggaaaat tctttgaaaa agtttgcact acagaaaata aatacataac atcctgcact
     3841 gaatgcacac tattaataga aaagagagca ttattgtagg aaatgtattg tatatgtaca
     3901 ttctgtgcat ttcacaattt tgaagttcct attctctaga aagtatagga acttcgaatt
     3961 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa ttcctcaaga
     4021 gacctctaga ccagccagga cagaaatgcc tcgacttcgc tgctacccaa ggttgccggg
     4081 tgacgcacac cgtggaaacg gatgaaggca cgaacccagt ggacataagc ctgttcggtt
     4141 cgtaagctgt aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa
     4201 cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttggg
     4261 gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg
     4321 atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaaac
     4381 attatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc
     4441 atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat
     4501 ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat
     4561 gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag
     4621 agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg
     4681 cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca
     4741 ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga
     4801 gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa
     4861 caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg
     4921 gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc
     4981 ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag
     5041 tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg
     5101 gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag
     5161 gtagtcggca aataaccctc gagccaccca tgaccaaaat cccttaacgt gagttacgcg
     5221 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5281 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5341 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5401 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     5461 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     5521 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5581 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     5641 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5701 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5761 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5821 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5881 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5941 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     6001 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     6061 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     6121 gcgggcagtg agcgcaacgc aattaatacg cgtaccgcta gccaggaaga gtttgtagaa
     6181 acgcaaaaag gccatccgtc aggatggcct tctgcttagt ttgatgcctg gcagtttatg
     6241 gcgggcgtcc tgcccgccac cctccgggcc gttgcttcac aacgttcaaa tccgctcccg
     6301 gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc
     6361 agtcttccga ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcgtta
     6421 ac
//
