LOCUS       sequence-482041-        6423 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Middle entry vector for mini-Golden subcloning platform; creER.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6423)
  TITLE     Mini-Golden Subcloning Platform
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"
COMMENT     Sequence Label: P2A-CreER-pA-ORF3-FRT-acry mCherry
FEATURES             Location/Qualifiers
     source          1..6423
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             46..102
                     /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             103..1128
                     /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             1141..2076
                     /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             1189..2082
                     /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    2112..2246
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2261..2294
                     /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    2295..2416
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(2539..3249)
                     /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(2619..2642)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2817..2836)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3086..3104
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      3246..3255
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3891..3924
                     /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             4353..5144
                     /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      5237..5825
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5726..5745
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5979..5996
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(6155..6182)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(6274..6360)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(6409..6423,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(6423,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc ccggtctctt cgcccgctac aaacttctct
       61 ctgctgaaac aagctggcga cgtggaggag aaccctggac cttccaattt actgaccgta
      121 caccaaaatt tgcctgcatt accggtcgat gcaacgagtg atgaggttcg caagaacctg
      181 atggacatgt tcagggatcg ccaggcgttt tctgagcata cctggaaaat gcttctgtcc
      241 gtttgccggt cgtgggcggc atggtgcaag ttgaataacc ggaaatggtt tcccgcagaa
      301 cctgaagatg ttcgcgatta tcttctatat cttcaggcgc gcggtctggc agtaaaaact
      361 atccagcaac atttgggcca gctaaacatg cttcatcgtc ggtccgggct gccacgacca
      421 agtgacagca atgctgtttc actggttatg cggcggatcc gaaaagaaaa cgttgatgcc
      481 ggtgaacgtg caaaacaggc tctagcgttc gaacgcactg atttcgacca ggttcgttca
      541 ctcatggaaa atagcgatcg ctgccaggat atacgtaatc tggcatttct ggggattgct
      601 tataacaccc tgttacgtat agccgaaatt gccaggatca gggttaaaga tatctcacgt
      661 actgacggtg ggagaatgtt aatccatatt ggcagaacga aaacgctggt tagcaccgca
      721 ggtgtagaga aggcacttag cctgggggta actaaactgg tcgagcgatg gatttccgtc
      781 tctggtgtag ctgatgatcc gaataactac ctgttttgcc gggtcagaaa aaatggtgtt
      841 gccgcgccat ctgccaccag ccagctatca actcgcgccc tggaagggat ttttgaagca
      901 actcatcgat tgatttacgg cgctaaggat gactctggtc agagatacct ggcctggtct
      961 ggacacagtg cccgtgtcgg agccgcgcga gatatggccc gcgctggagt ttcaataccg
     1021 gagatcatgc aagctggtgg ctggaccaat gtaaatattg tcatgaacta tatccgtaac
     1081 ctggatagtg aaacaggggc aatggtgcgc ctgctggaag atggcgatct cgagccatct
     1141 gctggagaca tgagagctgc caacctttgg ccaagcccgc tcatgatcaa acgctctaag
     1201 aagaacagcc tggccttgtc cctgacggcc gaccagatgg tcagtgcctt gttggatgct
     1261 gagcccccca tactctattc cgagtatgat cctaccagac ccttcagtga agcttcgatg
     1321 atgggcttac tgaccaacct ggcagacagg gagctggttc acatgatcaa ctgggcgaag
     1381 agggtgccag gctttgtgga tttgaccctc catgatcagg tccaccttct agaatgtgcc
     1441 tggctagaga tcctgatgat tggcctcgtc tggcgctcca tggagcaccc agtgaagcta
     1501 ctgtttgctc ctaacttgct cttggacagg aaccagggaa aatgtgtaga gggcatggtg
     1561 gagatcttcg acatgctgct ggctacatca tctcggttcc gcatgatgaa tctgcaggga
     1621 gaggagtttg tgtgcctcaa atctattatt ttgcttaatt ctggagtgta cacatttctg
     1681 tccagcaccc tgaagtctct ggaagagaag gaccatatcc accgagtcct ggacaagatc
     1741 acagacactt tgatccacct gatggccaag gcaggcctga ccctgcagca gcagcaccag
     1801 cggctggccc agctcctcct catcctctcc cacatcaggc acatgagtaa caaaggcatg
     1861 gagcatctgt acagcatgaa gtgcaagaac gtggtgcccc tctatgacct gctgctggag
     1921 gcggcggacg cccaccgcct acatgcgccc actagccgtg gaggggcatc cgtggaggag
     1981 acggaccaaa gccacttggc cactgcgggc tctacttcat cgcattcctt gcaaaagtat
     2041 tacatcacgg gggaggcaga gggtttccct gccacagctt aatctagaac tatagtgagt
     2101 cgtattacgt agatccagac atgataagat acattgatga gtttggacaa accacaacta
     2161 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     2221 ccattataag ctgcaataaa caagttaaca acaacaattc gaagttccta ttctctagaa
     2281 agtataggaa cttctaagat acattgatga gtttggacaa accacaacta gaatgcagtg
     2341 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     2401 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     2461 ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga
     2521 tctagagtcg cggccgcttt acttgtacag ctcgtccatg ccgccggtgg agtggtggcc
     2581 ctcggcgcgc tcgtactgtt ccacgatggt gtagtcctcg ttgtgggagg tgatgtccag
     2641 cttgatgttg acgttgtagg cgccgggcag ctgcacgggc ttcttggcct tgtaggtggt
     2701 cttgacctcg gcgtcgtagt ggccgccgtc cttcagcttc agcctctgct tgatctcgcc
     2761 cttcagggcg ccgtcctcgg ggtacatccg ctcggaggag gcctcccagc ccatggtctt
     2821 cttctgcatt acggggccgt cggaggggaa gttggtgccg cgcagcttca ccttgtagat
     2881 gaactcgccg tcctgcaggg aggagtcctg ggtcacggtc accacgccgc cgtcctcgaa
     2941 gttcatcacg cgctcccact tgaagccctc ggggaaggac agcttcaagt agtcggggat
     3001 gtcggcgggg tgcttcacgt aggccttgga gccgtacatg aactgagggg acaggatgtc
     3061 ccaggcgaag ggcagggggc cgcccttggt caccttcagc ttggcggtct gggtgccctc
     3121 gtaggggcgg ccctcgccct cgccctcgat ctcgaactcg tggccgttca cggagccctc
     3181 catgtgcacc ttgaagcgca tgaactcctt gatgatggcc atgttgtcat cctcgccctt
     3241 gctcaccatg gtggcgaccg gtaatgtcag acctggtaac tccttactgt aacttattgc
     3301 acctgatgaa caaaagatgt gaactgaacg caagtaggag gctaatagag gctagtacag
     3361 tacaaagatc cccaaatgat gtgtgccggc cttagtggag gcagacccct ggttatatac
     3421 ctgtgctgca gaaagagggg cttacaaatg gatctctctg gaatggcatt atctcatggt
     3481 gggaacagtg ttgtcagcag aatcagcggg gtggactctg acctgagaat gacagtctag
     3541 cactgaatgg ctcagacaat actcttgtgg ccaactatca ccggaaaata agtgcctgcc
     3601 aaaacaaacc agaccagatg aagctacatg gctgtagaac gtcagtattt aatgtgttgt
     3661 ggccacttgg ttcaccagat ttaagagcta atttttttaa atatcacaga taatcaatcg
     3721 cttttttttt tcttgactga aaagcttaga ctggaaaatt ctttgaaaaa gtttgcacta
     3781 cagaaaataa atacataaca tcctgcactg aatgcacact attaatagaa aagagagcat
     3841 tattgtagga aatgtattgt atatgtacat tctgtgcatt tcacaatttt gaagttccta
     3901 ttctctagaa agtataggaa cttcgaattg cattcatttt atgtttcagg ttcaggggga
     3961 ggtgtgggag gttttttaat tcctcaagag acctctagac cagccaggac agaaatgcct
     4021 cgacttcgct gctacccaag gttgccgggt gacgcacacc gtggaaacgg atgaaggcac
     4081 gaacccagtg gacataagcc tgttcggttc gtaagctgta atgcaagtag cgtatgcgct
     4141 cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg taacggcgca gtggcggttt
     4201 tcatggcttg ttatgactgt ttttttgggg tacagtctat gcctcgggca tccaagcagc
     4261 aagcgcgtta cgccgtgggt cgatgtttga tgttatggag cagcaacgat gttacgcagc
     4321 agggcagtcg ccctaaaaca aagttaaaca ttatgaggga agcggtgatc gccgaagtat
     4381 cgactcaact atcagaggta gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg
     4441 ccgtacattt gtacggctcc gcagtggatg gcggcctgaa gccacacagt gatattgatt
     4501 tgctggttac ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg atcaacgacc
     4561 ttttggaaac ttcggcttcc cctggagaga gcgagattct ccgcgctgta gaagtcacca
     4621 ttgttgtgca cgacgacatc attccgtggc gttatccagc taagcgcgaa ctgcaatttg
     4681 gagaatggca gcgcaatgac attcttgcag gtatcttcga gccagccacg atcgacattg
     4741 atctggctat cttgctgaca aaagcaagag aacatagcgt tgccttggta ggtccagcgg
     4801 cggaggaact ctttgatccg gttcctgaac aggatctatt tgaggcgcta aatgaaacct
     4861 taacgctatg gaactcgccg cccgactggg ctggcgatga gcgaaatgta gtgcttacgt
     4921 tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg
     4981 actgggcaat ggagcgcctg ccggcccagt atcagcccgt catacttgaa gctagacagg
     5041 cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg
     5101 tccactacgt gaaaggcgag atcaccaagg tagtcggcaa ataaccctcg agccacccat
     5161 gaccaaaatc ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa
     5221 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     5281 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     5341 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     5401 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     5461 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     5521 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     5581 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     5641 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     5701 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     5761 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     5821 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     5881 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     5941 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     6001 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     6061 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatacgc
     6121 gtaccgctag ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt
     6181 ctgcttagtt tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctccgggccg
     6241 ttgcttcaca acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac
     6301 cgacaaacaa cagataaaac gaaaggccca gtcttccgac tgagcctttc gttttatttg
     6361 atgcctggca gttccctact ctcgcgttaa cgctagcatg gatgttttcc cagtcacgac
     6421 gtt
//
