LOCUS       sequence-482204-        4192 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Middle entry vector for mini-Golden subcloning platform; BFP; 
            NTR-v2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4192)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 4192)
  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: T2A-BFP-P2A-NTRv2-pA-ORF-3
FEATURES             Location/Qualifiers
     source          1..4192
                     /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..249
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             250..945
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     CDS             955..1011
                     /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"
     polyA_signal    1701..1835
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1725..1744)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1779..1798
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2272..3063
                     /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      3156..3744
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3645..3664
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3898..3915
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4074..4101)
                     /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 cgcccgaggg cagaggcagt ctgctgacat gcggtgacgt ggaagagaat
      241 cccggcccta gcgagctgat taaggagaac atgcacatga agctgtacat ggagggcacc
      301 gtggacaacc atcacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc
      361 cagaccatga gaatcaaggt ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg
      421 gctactagct tcctctacgg cagcaagacc ttcatcaacc acacccaggg catccccgac
      481 ttcttcaagc agtccttccc tgagggcttc acatgggaga gagtcaccac atacgaagac
      541 gggggcgtgc tgaccgctac ccaggacacc agcctccagg acggctgcct catctacaac
      601 gtcaagatca gaggggtgaa cttcacatcc aacggccctg tgatgcagaa gaaaacactc
      661 ggctgggagg ccttcaccga gacgctgtac cccgctgacg gcggcctgga aggcagaaac
      721 gacatggccc tgaagctcgt gggcgggagc catctgatcg caaacatcaa gaccacatat
      781 agatccaaga aacccgctaa gaacctcaag atgcctggcg tctactatgt ggactacaga
      841 ctggaaagaa tcaaggaggc caacaacgag acgtacgtcg agcagcacga ggtggcagtg
      901 gccagatact gcgacctccc tagcaaactg gggcacaagc ttaatggaag cggagctact
      961 aacttcagcc tgctgaagca ggctggagac gtggaggaga accccgggcc tatgactatt
     1021 gttcaagctg cccaatcccg ctactccacc aaagcctttg atgcttcgcg caaattgcct
     1081 gaagagaaag tcgcggcagt gaaagagtta atccgcatga gtgcgtccag tgtcaactcg
     1141 caaccttggc attttattgt cgcgagcagt gaagagggaa aagcgcgcat cgccaaagca
     1201 acacaaggtg gttttgctgc taatgagcgc aagattttgg atgcttctca cgttgtggtg
     1261 ttctgcgcca aaacggcgat tgatgaagcg tacctactcg accttttgga aagcgaagac
     1321 aaagatggcc gctacgccga tgtcgaagca aaaaatggca tgcacgctgg tcgttcattt
     1381 ttcgtcaaca tgcaccgctt tgacttgaaa gacgcgcatc actggatgga aaagcaagtt
     1441 tacctcaatg tggggacgct gctattgggt gcttctgcga tggagatcga cgcggtgcca
     1501 attgaaggct tcgatgccaa ggtgcttgat gaggagtttg gtctgcgtga gaagggcttt
     1561 accagcgtgg tgattgtgcc gctgggttac catagcgaag acgattttaa tgctaagctg
     1621 ccaaaatcac gttggtcagc agagactgtt tttaccgaaa tctaactcga gtctagaact
     1681 atagtgagtc gtattacgta gatccagaca tgataagata cattgatgag tttggacaaa
     1741 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     1801 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     1861 tgtttcaggt tcagggggag gtgtgggagg ttttttaatt cctcaagaga cctctagacc
     1921 agccaggaca gaaatgcctc gacttcgctg ctacccaagg ttgccgggtg acgcacaccg
     1981 tggaaacgga tgaaggcacg aacccagtgg acataagcct gttcggttcg taagctgtaa
     2041 tgcaagtagc gtatgcgctc acgcaactgg tccagaacct tgaccgaacg cagcggtggt
     2101 aacggcgcag tggcggtttt catggcttgt tatgactgtt tttttggggt acagtctatg
     2161 cctcgggcat ccaagcagca agcgcgttac gccgtgggtc gatgtttgat gttatggagc
     2221 agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa agttaaacat tatgagggaa
     2281 gcggtgatcg ccgaagtatc gactcaacta tcagaggtag ttggcgtcat cgagcgccat
     2341 ctcgaaccga cgttgctggc cgtacatttg tacggctccg cagtggatgg cggcctgaag
     2401 ccacacagtg atattgattt gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg
     2461 cgagctttga tcaacgacct tttggaaact tcggcttccc ctggagagag cgagattctc
     2521 cgcgctgtag aagtcaccat tgttgtgcac gacgacatca ttccgtggcg ttatccagct
     2581 aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg tatcttcgag
     2641 ccagccacga tcgacattga tctggctatc ttgctgacaa aagcaagaga acatagcgtt
     2701 gccttggtag gtccagcggc ggaggaactc tttgatccgg ttcctgaaca ggatctattt
     2761 gaggcgctaa atgaaacctt aacgctatgg aactcgccgc ccgactgggc tggcgatgag
     2821 cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg
     2881 ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc
     2941 atacttgaag ctagacaggc ttatcttgga caagaagaag atcgcttggc ctcgcgcgca
     3001 gatcagttgg aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt agtcggcaaa
     3061 taaccctcga gccacccatg accaaaatcc cttaacgtga gttacgcgtc gttccactga
     3121 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3181 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3241 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3301 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3361 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3421 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3481 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3541 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3601 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3661 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3721 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3781 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     3841 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     3901 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     3961 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     4021 cgcaacgcaa ttaatacgcg taccgctagc caggaagagt ttgtagaaac gcaaaaaggc
     4081 catccgtcag gatggccttc tgcttagttt gatgcctggc agtttatggc gggcgtcctg
     4141 cccgccaccc tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc gg
//
