LOCUS       sequence-482149-        3475 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Middle entry vector for mini-Golden subcloning platform; BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3475)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 3475)
  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-BFP-pA-ORF-3 without ATG
FEATURES             Location/Qualifiers
     source          1..3475
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(1..87)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     136..158
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     150..167
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     151..167
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             196..252
                     /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             253..951
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     polyA_signal    984..1118
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1008..1027)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1062..1081
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1555..2346
                     /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      2439..3027
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2928..2947
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3181..3198
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3357..3384)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc
       61 ttccgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc gcgttaacgc
      121 tagcatggat gttttcccag tcacgacgtt gtaaaacgac ggccagtctt aagctcgggc
      181 ccggtctctt cgcccgctac aaacttctct ctgctgaaac aagctggcga cgtggaggag
      241 aaccctggac ctagcgagct gattaaggag aacatgcaca tgaagctgta catggagggc
      301 accgtggaca accatcactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc
      361 acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc
      421 ctggctacta gcttcctcta cggcagcaag accttcatca accacaccca gggcatcccc
      481 gacttcttca agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa
      541 gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac
      601 aacgtcaaga tcagaggggt gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca
      661 ctcggctggg aggccttcac cgagacgctg taccccgctg acggcggcct ggaaggcaga
      721 aacgacatgg ccctgaagct cgtgggcggg agccatctga tcgcaaacat caagaccaca
      781 tatagatcca agaaacccgc taagaacctc aagatgcctg gcgtctacta tgtggactac
      841 agactggaaa gaatcaagga ggccaacaac gagacgtacg tcgagcagca cgaggtggca
      901 gtggccagat actgcgacct ccctagcaaa ctggggcaca agcttaatta ataagctaga
      961 actatagtga gtcgtattac gtagatccag acatgataag atacattgat gagtttggac
     1021 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     1081 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     1141 ttatgtttca ggttcagggg gaggtgtggg aggtttttta attcctcaag agacctctag
     1201 accagccagg acagaaatgc ctcgacttcg ctgctaccca aggttgccgg gtgacgcaca
     1261 ccgtggaaac ggatgaaggc acgaacccag tggacataag cctgttcggt tcgtaagctg
     1321 taatgcaagt agcgtatgcg ctcacgcaac tggtccagaa ccttgaccga acgcagcggt
     1381 ggtaacggcg cagtggcggt tttcatggct tgttatgact gtttttttgg ggtacagtct
     1441 atgcctcggg catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg
     1501 agcagcaacg atgttacgca gcagggcagt cgccctaaaa caaagttaaa cattatgagg
     1561 gaagcggtga tcgccgaagt atcgactcaa ctatcagagg tagttggcgt catcgagcgc
     1621 catctcgaac cgacgttgct ggccgtacat ttgtacggct ccgcagtgga tggcggcctg
     1681 aagccacaca gtgatattga tttgctggtt acggtgaccg taaggcttga tgaaacaacg
     1741 cggcgagctt tgatcaacga ccttttggaa acttcggctt cccctggaga gagcgagatt
     1801 ctccgcgctg tagaagtcac cattgttgtg cacgacgaca tcattccgtg gcgttatcca
     1861 gctaagcgcg aactgcaatt tggagaatgg cagcgcaatg acattcttgc aggtatcttc
     1921 gagccagcca cgatcgacat tgatctggct atcttgctga caaaagcaag agaacatagc
     1981 gttgccttgg taggtccagc ggcggaggaa ctctttgatc cggttcctga acaggatcta
     2041 tttgaggcgc taaatgaaac cttaacgcta tggaactcgc cgcccgactg ggctggcgat
     2101 gagcgaaatg tagtgcttac gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc
     2161 gcgccgaagg atgtcgctgc cgactgggca atggagcgcc tgccggccca gtatcagccc
     2221 gtcatacttg aagctagaca ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc
     2281 gcagatcagt tggaagaatt tgtccactac gtgaaaggcg agatcaccaa ggtagtcggc
     2341 aaataaccct cgagccaccc atgaccaaaa tcccttaacg tgagttacgc gtcgttccac
     2401 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2461 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2521 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     2581 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     2641 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     2701 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     2761 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2821 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2881 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2941 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3001 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3061 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     3121 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     3181 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     3241 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     3301 gagcgcaacg caattaatac gcgtaccgct agccaggaag agtttgtaga aacgcaaaaa
     3361 ggccatccgt caggatggcc ttctgcttag tttgatgcct ggcagtttat ggcgggcgtc
     3421 ctgcccgcca ccctccgggc cgttgcttca caacgttcaa atccgctccc ggcgg
//
