LOCUS       sequence-482151-        4190 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 4190)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 4190)
  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-1
FEATURES             Location/Qualifiers
     source          1..4190
                     /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             194..247
                     /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             248..943
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     CDS             953..1009
                     /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    1699..1833
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1723..1742)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1777..1796
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2270..3061
                     /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      3154..3742
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3643..3662
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3896..3913
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4072..4099)
                     /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 cgcgagggca gaggcagtct gctgacatgc ggtgacgtgg aagagaatcc
      241 cggccctagc gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt
      301 ggacaaccat cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca
      361 gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc
      421 tactagcttc ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt
      481 cttcaagcag tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg
      541 gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt
      601 caagatcaga ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg
      661 ctgggaggcc ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga
      721 catggccctg aagctcgtgg gcgggagcca tctgatcgca aacatcaaga ccacatatag
      781 atccaagaaa cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact
      841 ggaaagaatc aaggaggcca acaacgagac gtacgtcgag cagcacgagg tggcagtggc
      901 cagatactgc gacctcccta gcaaactggg gcacaagctt aatggaagcg gagctactaa
      961 cttcagcctg ctgaagcagg ctggagacgt ggaggagaac cccgggccta tgactattgt
     1021 tcaagctgcc caatcccgct actccaccaa agcctttgat gcttcgcgca aattgcctga
     1081 agagaaagtc gcggcagtga aagagttaat ccgcatgagt gcgtccagtg tcaactcgca
     1141 accttggcat tttattgtcg cgagcagtga agagggaaaa gcgcgcatcg ccaaagcaac
     1201 acaaggtggt tttgctgcta atgagcgcaa gattttggat gcttctcacg ttgtggtgtt
     1261 ctgcgccaaa acggcgattg atgaagcgta cctactcgac cttttggaaa gcgaagacaa
     1321 agatggccgc tacgccgatg tcgaagcaaa aaatggcatg cacgctggtc gttcattttt
     1381 cgtcaacatg caccgctttg acttgaaaga cgcgcatcac tggatggaaa agcaagttta
     1441 cctcaatgtg gggacgctgc tattgggtgc ttctgcgatg gagatcgacg cggtgccaat
     1501 tgaaggcttc gatgccaagg tgcttgatga ggagtttggt ctgcgtgaga agggctttac
     1561 cagcgtggtg attgtgccgc tgggttacca tagcgaagac gattttaatg ctaagctgcc
     1621 aaaatcacgt tggtcagcag agactgtttt taccgaaatc taactcgagt ctagaactat
     1681 agtgagtcgt attacgtaga tccagacatg ataagataca ttgatgagtt tggacaaacc
     1741 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     1801 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     1861 tttcaggttc agggggaggt gtgggaggtt ttttaattcc tcaagagacc tctagaccag
     1921 ccaggacaga aatgcctcga cttcgctgct acccaaggtt gccgggtgac gcacaccgtg
     1981 gaaacggatg aaggcacgaa cccagtggac ataagcctgt tcggttcgta agctgtaatg
     2041 caagtagcgt atgcgctcac gcaactggtc cagaaccttg accgaacgca gcggtggtaa
     2101 cggcgcagtg gcggttttca tggcttgtta tgactgtttt tttggggtac agtctatgcc
     2161 tcgggcatcc aagcagcaag cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag
     2221 caacgatgtt acgcagcagg gcagtcgccc taaaacaaag ttaaacatta tgagggaagc
     2281 ggtgatcgcc gaagtatcga ctcaactatc agaggtagtt ggcgtcatcg agcgccatct
     2341 cgaaccgacg ttgctggccg tacatttgta cggctccgca gtggatggcg gcctgaagcc
     2401 acacagtgat attgatttgc tggttacggt gaccgtaagg cttgatgaaa caacgcggcg
     2461 agctttgatc aacgaccttt tggaaacttc ggcttcccct ggagagagcg agattctccg
     2521 cgctgtagaa gtcaccattg ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa
     2581 gcgcgaactg caatttggag aatggcagcg caatgacatt cttgcaggta tcttcgagcc
     2641 agccacgatc gacattgatc tggctatctt gctgacaaaa gcaagagaac atagcgttgc
     2701 cttggtaggt ccagcggcgg aggaactctt tgatccggtt cctgaacagg atctatttga
     2761 ggcgctaaat gaaaccttaa cgctatggaa ctcgccgccc gactgggctg gcgatgagcg
     2821 aaatgtagtg cttacgttgt cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc
     2881 gaaggatgtc gctgccgact gggcaatgga gcgcctgccg gcccagtatc agcccgtcat
     2941 acttgaagct agacaggctt atcttggaca agaagaagat cgcttggcct cgcgcgcaga
     3001 tcagttggaa gaatttgtcc actacgtgaa aggcgagatc accaaggtag tcggcaaata
     3061 accctcgagc cacccatgac caaaatccct taacgtgagt tacgcgtcgt tccactgagc
     3121 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     3181 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     3241 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     3301 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     3361 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     3421 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     3481 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     3541 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     3601 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     3661 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     3721 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     3781 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     3841 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     3901 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     3961 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     4021 caacgcaatt aatacgcgta ccgctagcca ggaagagttt gtagaaacgc aaaaaggcca
     4081 tccgtcagga tggccttctg cttagtttga tgcctggcag tttatggcgg gcgtcctgcc
     4141 cgccaccctc cgggccgttg cttcacaacg ttcaaatccg ctcccggcgg
//
