LOCUS       sequence-482012-        4164 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Middle entry vector for mini-Golden subcloning platform; 
            acry-mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4164)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 4164)
  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: FRT-acrymCherry-FRT
FEATURES             Location/Qualifiers
     source          1..4164
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..22
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(181..208)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(300..386)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     435..457
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     449..466
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     450..466
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    503..536
                     /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    537..658
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(548..567)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     602..621
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(781..1491)
                     /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(861..884)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(1059..1078)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1328..1346
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      1488..1497
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2133..2166
                     /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             2543..3334
                     /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      3427..4015
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3916..3935
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
       61 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
      121 cagtgagcgc aacgcaatta atacgcgtac cgctagccag gaagagtttg tagaaacgca
      181 aaaaggccat ccgtcaggat ggccttctgc ttagtttgat gcctggcagt ttatggcggg
      241 cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt tcaaatccgc tcccggcgga
      301 tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct
      361 tccgactgag cctttcgttt tatttgatgc ctggcagttc cctactctcg cgttaacgct
      421 agcatggatg ttttcccagt cacgacgttg taaaacgacg gccagtctta agctcgggcc
      481 cggtctcttc gccgatatcg atgaagttcc tattctctag aaagtatagg aacttctaag
      541 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
      601 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
      661 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
      721 aagcaagtaa aacctctaca aatgtggtat ggctgattat gatctagagt cgcggccgct
      781 ttacttgtac agctcgtcca tgccgccggt ggagtggtgg ccctcggcgc gctcgtactg
      841 ttccacgatg gtgtagtcct cgttgtggga ggtgatgtcc agcttgatgt tgacgttgta
      901 ggcgccgggc agctgcacgg gcttcttggc cttgtaggtg gtcttgacct cggcgtcgta
      961 gtggccgccg tccttcagct tcagcctctg cttgatctcg cccttcaggg cgccgtcctc
     1021 ggggtacatc cgctcggagg aggcctccca gcccatggtc ttcttctgca ttacggggcc
     1081 gtcggagggg aagttggtgc cgcgcagctt caccttgtag atgaactcgc cgtcctgcag
     1141 ggaggagtcc tgggtcacgg tcaccacgcc gccgtcctcg aagttcatca cgcgctccca
     1201 cttgaagccc tcggggaagg acagcttcaa gtagtcgggg atgtcggcgg ggtgcttcac
     1261 gtaggccttg gagccgtaca tgaactgagg ggacaggatg tcccaggcga agggcagggg
     1321 gccgcccttg gtcaccttca gcttggcggt ctgggtgccc tcgtaggggc ggccctcgcc
     1381 ctcgccctcg atctcgaact cgtggccgtt cacggagccc tccatgtgca ccttgaagcg
     1441 catgaactcc ttgatgatgg ccatgttgtc atcctcgccc ttgctcacca tggtggcgac
     1501 cggtaatgtc agacctggta actccttact gtaacttatt gcacctgatg aacaaaagat
     1561 gtgaactgaa cgcaagtagg aggctaatag aggctagtac agtacaaaga tccccaaatg
     1621 atgtgtgccg gccttagtgg aggcagaccc ctggttatat acctgtgctg cagaaagagg
     1681 ggcttacaaa tggatctctc tggaatggca ttatctcatg gtgggaacag tgttgtcagc
     1741 agaatcagcg gggtggactc tgacctgaga atgacagtct agcactgaat ggctcagaca
     1801 atactcttgt ggccaactat caccggaaaa taagtgcctg ccaaaacaaa ccagaccaga
     1861 tgaagctaca tggctgtaga acgtcagtat ttaatgtgtt gtggccactt ggttcaccag
     1921 atttaagagc taattttttt aaatatcaca gataatcaat cgcttttttt tttcttgact
     1981 gaaaagctta gactggaaaa ttctttgaaa aagtttgcac tacagaaaat aaatacataa
     2041 catcctgcac tgaatgcaca ctattaatag aaaagagagc attattgtag gaaatgtatt
     2101 gtatatgtac attctgtgca tttcacaatt ttgaagttcc tattctctag aaagtatagg
     2161 aacttcggat ccctcaagag acctctagac cagccaggac agaaatgcct cgacttcgct
     2221 gctacccaag gttgccgggt gacgcacacc gtggaaacgg atgaaggcac gaacccagtg
     2281 gacataagcc tgttcggttc gtaagctgta atgcaagtag cgtatgcgct cacgcaactg
     2341 gtccagaacc ttgaccgaac gcagcggtgg taacggcgca gtggcggttt tcatggcttg
     2401 ttatgactgt ttttttgggg tacagtctat gcctcgggca tccaagcagc aagcgcgtta
     2461 cgccgtgggt cgatgtttga tgttatggag cagcaacgat gttacgcagc agggcagtcg
     2521 ccctaaaaca aagttaaaca ttatgaggga agcggtgatc gccgaagtat cgactcaact
     2581 atcagaggta gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg ccgtacattt
     2641 gtacggctcc gcagtggatg gcggcctgaa gccacacagt gatattgatt tgctggttac
     2701 ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg atcaacgacc ttttggaaac
     2761 ttcggcttcc cctggagaga gcgagattct ccgcgctgta gaagtcacca ttgttgtgca
     2821 cgacgacatc attccgtggc gttatccagc taagcgcgaa ctgcaatttg gagaatggca
     2881 gcgcaatgac attcttgcag gtatcttcga gccagccacg atcgacattg atctggctat
     2941 cttgctgaca aaagcaagag aacatagcgt tgccttggta ggtccagcgg cggaggaact
     3001 ctttgatccg gttcctgaac aggatctatt tgaggcgcta aatgaaacct taacgctatg
     3061 gaactcgccg cccgactggg ctggcgatga gcgaaatgta gtgcttacgt tgtcccgcat
     3121 ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg actgggcaat
     3181 ggagcgcctg ccggcccagt atcagcccgt catacttgaa gctagacagg cttatcttgg
     3241 acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg tccactacgt
     3301 gaaaggcgag atcaccaagg tagtcggcaa ataaccctcg agccacccat gaccaaaatc
     3361 ccttaacgtg agttacgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3421 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3481 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3541 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     3601 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3661 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3721 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3781 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3841 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3901 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3961 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4021 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4081 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4141 cgctcgccgc agccgaacga ccga
//
