LOCUS       sequence-394480-        6511 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mini circle middle entry plasmid for CreER-pA-FRT-acry:venus-pA-FRT 
            with flanking BsaI cut sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6511)
  AUTHORS   Kang Lab
  TITLE     mini-Golden version1
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6511)
  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            "Unpublished"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_CreER-pA-FRT-acry:venus-pA-FRT
FEATURES             Location/Qualifiers
     source          1..6511
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(9..95)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     144..166
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     158..175
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     159..175
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             208..1236
                     /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             1249..2184
                     /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             1297..2190
                     /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    2220..2354
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2244..2263)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2298..2317
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    2369..2402
                     /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    2409..2616
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2605..2622
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(2640..3353,3354..3356,3357..3359))
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2685..2706)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3034..3053
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     protein_bind    4137..4170
                     /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             4599..5390
                     /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      5483..6071
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5972..5991
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6225..6242
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(6401..6428)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cccggcggat ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag
       61 gcccagtctt ccgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc
      121 gttaacgcta gcatggatgt tttcccagtc acgacgttgt aaaacgacgg ccagtcttaa
      181 gctcgggccc ggtctctagt cgccaccatg tccaatttac tgaccgtaca ccaaaatttg
      241 cctgcattac cggtcgatgc aacgagtgat gaggttcgca agaacctgat ggacatgttc
      301 agggatcgcc aggcgttttc tgagcatacc tggaaaatgc ttctgtccgt ttgccggtcg
      361 tgggcggcat ggtgcaagtt gaataaccgg aaatggtttc ccgcagaacc tgaagatgtt
      421 cgcgattatc ttctatatct tcaggcgcgc ggtctggcag taaaaactat ccagcaacat
      481 ttgggccagc taaacatgct tcatcgtcgg tccgggctgc cacgaccaag tgacagcaat
      541 gctgtttcac tggttatgcg gcggatccga aaagaaaacg ttgatgccgg tgaacgtgca
      601 aaacaggctc tagcgttcga acgcactgat ttcgaccagg ttcgttcact catggaaaat
      661 agcgatcgct gccaggatat acgtaatctg gcatttctgg ggattgctta taacaccctg
      721 ttacgtatag ccgaaattgc caggatcagg gttaaagata tctcacgtac tgacggtggg
      781 agaatgttaa tccatattgg cagaacgaaa acgctggtta gcaccgcagg tgtagagaag
      841 gcacttagcc tgggggtaac taaactggtc gagcgatgga tttccgtctc tggtgtagct
      901 gatgatccga ataactacct gttttgccgg gtcagaaaaa atggtgttgc cgcgccatct
      961 gccaccagcc agctatcaac tcgcgccctg gaagggattt ttgaagcaac tcatcgattg
     1021 atttacggcg ctaaggatga ctctggtcag agatacctgg cctggtctgg acacagtgcc
     1081 cgtgtcggag ccgcgcgaga tatggcccgc gctggagttt caataccgga gatcatgcaa
     1141 gctggtggct ggaccaatgt aaatattgtc atgaactata tccgtaacct ggatagtgaa
     1201 acaggggcaa tggtgcgcct gctggaagat ggcgatctcg agccatctgc tggagacatg
     1261 agagctgcca acctttggcc aagcccgctc atgatcaaac gctctaagaa gaacagcctg
     1321 gccttgtccc tgacggccga ccagatggtc agtgccttgt tggatgctga gccccccata
     1381 ctctattccg agtatgatcc taccagaccc ttcagtgaag cttcgatgat gggcttactg
     1441 accaacctgg cagacaggga gctggttcac atgatcaact gggcgaagag ggtgccaggc
     1501 tttgtggatt tgaccctcca tgatcaggtc caccttctag aatgtgcctg gctagagatc
     1561 ctgatgattg gcctcgtctg gcgctccatg gagcacccag tgaagctact gtttgctcct
     1621 aacttgctct tggacaggaa ccagggaaaa tgtgtagagg gcatggtgga gatcttcgac
     1681 atgctgctgg ctacatcatc tcggttccgc atgatgaatc tgcagggaga ggagtttgtg
     1741 tgcctcaaat ctattatttt gcttaattct ggagtgtaca catttctgtc cagcaccctg
     1801 aagtctctgg aagagaagga ccatatccac cgagtcctgg acaagatcac agacactttg
     1861 atccacctga tggccaaggc aggcctgacc ctgcagcagc agcaccagcg gctggcccag
     1921 ctcctcctca tcctctccca catcaggcac atgagtaaca aaggcatgga gcatctgtac
     1981 agcatgaagt gcaagaacgt ggtgcccctc tatgacctgc tgctggaggc ggcggacgcc
     2041 caccgcctac atgcgcccac tagccgtgga ggggcatccg tggaggagac ggaccaaagc
     2101 cacttggcca ctgcgggctc tacttcatcg cattccttgc aaaagtatta catcacgggg
     2161 gaggcagagg gtttccctgc cacagcttaa tctagaacta tagtgagtcg tattacgtag
     2221 atccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     2281 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     2341 gcaataaaca agttaacaac aacaattgga agttcctatt ctctagaaag tataggaact
     2401 tcgatctctc cccagcatgc ctgctattgt cttcccaatc ctcccccttg ctgtcctgcc
     2461 ccaccccacc ccccagaata gaatgacacc tactcagaca atgcgatgca atttcctcat
     2521 tttattagga aaggacagtg ggagtggcac cttccagggt caaggaaggc acgggggagg
     2581 ggcaaacaac agatggctgg caactagaag gcacagtcga ggctgatcag cgggccgctt
     2641 tacttgtaca gctcgtccat gccgagagtg atcccggcgg cggtcacgaa ctccagcagg
     2701 accatgtgat cgcgcttctc gttggggtct ttgctcaggg cggactggta gctcaggtag
     2761 tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg
     2821 gcgagctgca cgccgccgtc ctcgatgttg tggcggatct tgaagttggc cttgatgccg
     2881 ttcttctgct tgtcggcggt gatatagacg ttgtggctgt tgtagttgta ctccagcttg
     2941 tgccccagga tgttgccgtc ctccttgaag tcgatgccct tcagctcgat gcggttcacc
     3001 agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt agttgccgtc gtccttgaag
     3061 aagatggtgc gctcctggac gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc
     3121 ttcatgtggt cggggtagcg ggcgaagcac tgcaggccgt agcccagggt ggtcacgagg
     3181 gtgggccagg gcacgggcag cttgccggtg gtgcagatca gcttcagggt cagcttgccg
     3241 taggtggcat cgccctcgcc ctcgccggac acgctgaact tgtggccgtt tacgtcgccg
     3301 tccagctcga ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcaccatg
     3361 ttgtcagacc tggtaactcc ttactgtaac ttattgcacc tgatgaacaa aagatgtgaa
     3421 ctgaacgcaa gtaggaggct aatagaggct agtacagtac aaagatcccc aaatgatgtg
     3481 tgccggcctt agtggaggca gacccctggt tatatacctg tgctgcagaa agaggggctt
     3541 acaaatggat ctctctggaa tggcattatc tcatggtggg aacagtgttg tcagcagaat
     3601 cagcggggtg gactctgacc tgagaatgac agtctagcac tgaatggctc agacaatact
     3661 cttgtggcca actatcaccg gaaaataagt gcctgccaaa acaaaccaga ccagatgaag
     3721 ctacatggct gtagaacgtc agtatttaat gtgttgtggc cacttggttc accagattta
     3781 agagctaatt tttttaaata tcacagataa tcaatcgctt ttttttttct tgactgaaaa
     3841 gcttagactg gaaaattctt tgaaaaagtt tgcactacag aaaataaata cataacatcc
     3901 tgcactgaat gcacactatt aatagaaaag agagcattat tgtaggaaat gtattgtata
     3961 tgtacattct gtgcatttca caattttatg ttgaaaagca tataaatctg gatttattgg
     4021 atcaaactat tataaacata taatactgta agatagtcat tacagtttga ataactacat
     4081 atgcataggc tgaaactatt taccaatgca agtttctctg aagatcgttt attgccgaag
     4141 ttcctattct ctagaaagta taggaacttc caattgcatt cattttatgt ttcaggttca
     4201 gggggaggtg tgggaggttt tttaattcct caagagacct ctagaccagc caggacagaa
     4261 atgcctcgac ttcgctgcta cccaaggttg ccgggtgacg cacaccgtgg aaacggatga
     4321 aggcacgaac ccagtggaca taagcctgtt cggttcgtaa gctgtaatgc aagtagcgta
     4381 tgcgctcacg caactggtcc agaaccttga ccgaacgcag cggtggtaac ggcgcagtgg
     4441 cggttttcat ggcttgttat gactgttttt ttggggtaca gtctatgcct cgggcatcca
     4501 agcagcaagc gcgttacgcc gtgggtcgat gtttgatgtt atggagcagc aacgatgtta
     4561 cgcagcaggg cagtcgccct aaaacaaagt taaacattat gagggaagcg gtgatcgccg
     4621 aagtatcgac tcaactatca gaggtagttg gcgtcatcga gcgccatctc gaaccgacgt
     4681 tgctggccgt acatttgtac ggctccgcag tggatggcgg cctgaagcca cacagtgata
     4741 ttgatttgct ggttacggtg accgtaaggc ttgatgaaac aacgcggcga gctttgatca
     4801 acgacctttt ggaaacttcg gcttcccctg gagagagcga gattctccgc gctgtagaag
     4861 tcaccattgt tgtgcacgac gacatcattc cgtggcgtta tccagctaag cgcgaactgc
     4921 aatttggaga atggcagcgc aatgacattc ttgcaggtat cttcgagcca gccacgatcg
     4981 acattgatct ggctatcttg ctgacaaaag caagagaaca tagcgttgcc ttggtaggtc
     5041 cagcggcgga ggaactcttt gatccggttc ctgaacagga tctatttgag gcgctaaatg
     5101 aaaccttaac gctatggaac tcgccgcccg actgggctgg cgatgagcga aatgtagtgc
     5161 ttacgttgtc ccgcatttgg tacagcgcag taaccggcaa aatcgcgccg aaggatgtcg
     5221 ctgccgactg ggcaatggag cgcctgccgg cccagtatca gcccgtcata cttgaagcta
     5281 gacaggctta tcttggacaa gaagaagatc gcttggcctc gcgcgcagat cagttggaag
     5341 aatttgtcca ctacgtgaaa ggcgagatca ccaaggtagt cggcaaataa ccctcgagcc
     5401 acccatgacc aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg
     5461 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5521 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5581 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     5641 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5701 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5761 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5821 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5881 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5941 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     6001 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6061 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     6121 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     6181 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     6241 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     6301 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     6361 atacgcgtac cgctagccag gaagagtttg tagaaacgca aaaaggccat ccgtcaggat
     6421 ggccttctgc ttagtttgat gcctggcagt ttatggcggg cgtcctgccc gccaccctcc
     6481 gggccgttgc ttcacaacgt tcaaatccgc t
//
