LOCUS       sequence-482465-        4191 bp    DNA     circular SYN 08-AUG-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 4191)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 4191)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 8, 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-2
FEATURES             Location/Qualifiers
     source          1..4191
                     /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             195..248
                     /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             249..944
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     CDS             954..1010
                     /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    1700..1834
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1724..1743)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1778..1797
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2271..3062
                     /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      3155..3743
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3644..3663
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3897..3914
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4073..4100)
                     /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 cgccgagggc agaggcagtc tgctgacatg cggtgacgtg gaagagaatc
      241 ccggccctag cgagctgatt aaggagaaca tgcacatgaa gctgtacatg gagggcaccg
      301 tggacaacca tcacttcaag tgcacatccg agggcgaagg caagccctac gagggcaccc
      361 agaccatgag aatcaaggtg gtcgagggcg gccctctccc cttcgccttc gacatcctgg
      421 ctactagctt cctctacggc agcaagacct tcatcaacca cacccagggc atccccgact
      481 tcttcaagca gtccttccct gagggcttca catgggagag agtcaccaca tacgaagacg
      541 ggggcgtgct gaccgctacc caggacacca gcctccagga cggctgcctc atctacaacg
      601 tcaagatcag aggggtgaac ttcacatcca acggccctgt gatgcagaag aaaacactcg
      661 gctgggaggc cttcaccgag acgctgtacc ccgctgacgg cggcctggaa ggcagaaacg
      721 acatggccct gaagctcgtg ggcgggagcc atctgatcgc aaacatcaag accacatata
      781 gatccaagaa acccgctaag aacctcaaga tgcctggcgt ctactatgtg gactacagac
      841 tggaaagaat caaggaggcc aacaacgaga cgtacgtcga gcagcacgag gtggcagtgg
      901 ccagatactg cgacctccct agcaaactgg ggcacaagct taatggaagc ggagctacta
      961 acttcagcct gctgaagcag gctggagacg tggaggagaa ccccgggcct atgactattg
     1021 ttcaagctgc ccaatcccgc tactccacca aagcctttga tgcttcgcgc aaattgcctg
     1081 aagagaaagt cgcggcagtg aaagagttaa tccgcatgag tgcgtccagt gtcaactcgc
     1141 aaccttggca ttttattgtc gcgagcagtg aagagggaaa agcgcgcatc gccaaagcaa
     1201 cacaaggtgg ttttgctgct aatgagcgca agattttgga tgcttctcac gttgtggtgt
     1261 tctgcgccaa aacggcgatt gatgaagcgt acctactcga ccttttggaa agcgaagaca
     1321 aagatggccg ctacgccgat gtcgaagcaa aaaatggcat gcacgctggt cgttcatttt
     1381 tcgtcaacat gcaccgcttt gacttgaaag acgcgcatca ctggatggaa aagcaagttt
     1441 acctcaatgt ggggacgctg ctattgggtg cttctgcgat ggagatcgac gcggtgccaa
     1501 ttgaaggctt cgatgccaag gtgcttgatg aggagtttgg tctgcgtgag aagggcttta
     1561 ccagcgtggt gattgtgccg ctgggttacc atagcgaaga cgattttaat gctaagctgc
     1621 caaaatcacg ttggtcagca gagactgttt ttaccgaaat ctaactcgag tctagaacta
     1681 tagtgagtcg tattacgtag atccagacat gataagatac attgatgagt ttggacaaac
     1741 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     1801 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     1861 gtttcaggtt cagggggagg tgtgggaggt tttttaattc ctcaagagac ctctagacca
     1921 gccaggacag aaatgcctcg acttcgctgc tacccaaggt tgccgggtga cgcacaccgt
     1981 ggaaacggat gaaggcacga acccagtgga cataagcctg ttcggttcgt aagctgtaat
     2041 gcaagtagcg tatgcgctca cgcaactggt ccagaacctt gaccgaacgc agcggtggta
     2101 acggcgcagt ggcggttttc atggcttgtt atgactgttt ttttggggta cagtctatgc
     2161 ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg atgtttgatg ttatggagca
     2221 gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa gttaaacatt atgagggaag
     2281 cggtgatcgc cgaagtatcg actcaactat cagaggtagt tggcgtcatc gagcgccatc
     2341 tcgaaccgac gttgctggcc gtacatttgt acggctccgc agtggatggc ggcctgaagc
     2401 cacacagtga tattgatttg ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc
     2461 gagctttgat caacgacctt ttggaaactt cggcttcccc tggagagagc gagattctcc
     2521 gcgctgtaga agtcaccatt gttgtgcacg acgacatcat tccgtggcgt tatccagcta
     2581 agcgcgaact gcaatttgga gaatggcagc gcaatgacat tcttgcaggt atcttcgagc
     2641 cagccacgat cgacattgat ctggctatct tgctgacaaa agcaagagaa catagcgttg
     2701 ccttggtagg tccagcggcg gaggaactct ttgatccggt tcctgaacag gatctatttg
     2761 aggcgctaaa tgaaacctta acgctatgga actcgccgcc cgactgggct ggcgatgagc
     2821 gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc
     2881 cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca
     2941 tacttgaagc tagacaggct tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag
     3001 atcagttgga agaatttgtc cactacgtga aaggcgagat caccaaggta gtcggcaaat
     3061 aaccctcgag ccacccatga ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag
     3121 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3181 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3241 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3301 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3361 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3421 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3481 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3541 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3601 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     3661 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     3721 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3781 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     3841 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     3901 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     3961 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     4021 gcaacgcaat taatacgcgt accgctagcc aggaagagtt tgtagaaacg caaaaaggcc
     4081 atccgtcagg atggccttct gcttagtttg atgcctggca gtttatggcg ggcgtcctgc
     4141 ccgccaccct ccgggccgtt gcttcacaac gttcaaatcc gctcccggcg g
//
