LOCUS       sequence-481985-        4202 bp    DNA     circular SYN 05-AUG-2025
DEFINITION  Middle entry vector for mini-Golden subcloning platform; NTRv2; 
            mNeonGreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4202)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 4202)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 5, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: linker-T2A-NTRv2-linker-P2A-mNeonGreen ORF1
FEATURES             Location/Qualifiers
     source          1..4202
                     /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             203..256
                     /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             917..973
                     /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             974..1681
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEE
                     LNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDG
                     ASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIST
                     FKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQK
                     AFTDVMGMDELYK"
     regulatory      1226..1235
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1711..1845
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1735..1754)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1789..1808
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2282..3073
                     /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      3166..3754
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3655..3674
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3908..3925
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4084..4111)
                     /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 cgcggctctg gggagggcag aggcagtctg ctgacatgcg gtgacgtgga
      241 agagaatccc ggccctatga ctattgttca agctgcccaa tcccgctact ccaccaaagc
      301 ctttgatgct tcgcgcaaat tgcctgaaga gaaagtcgcg gcagtgaaag agttaatccg
      361 catgagtgcg tccagtgtca actcgcaacc ttggcatttt attgtcgcga gcagtgaaga
      421 gggaaaagcg cgcatcgcca aagcaacaca aggtggtttt gctgctaatg agcgcaagat
      481 tttggatgct tctcacgttg tggtgttctg cgccaaaacg gcgattgatg aagcgtacct
      541 actcgacctt ttggaaagcg aagacaaaga tggccgctac gccgatgtcg aagcaaaaaa
      601 tggcatgcac gctggtcgtt catttttcgt caacatgcac cgctttgact tgaaagacgc
      661 gcatcactgg atggaaaagc aagtttacct caatgtgggg acgctgctat tgggtgcttc
      721 tgcgatggag atcgacgcgg tgccaattga aggcttcgat gccaaggtgc ttgatgagga
      781 gtttggtctg cgtgagaagg gctttaccag cgtggtgatt gtgccgctgg gttaccatag
      841 cgaagacgat tttaatgcta agctgccaaa atcacgttgg tcagcagaga ctgtttttac
      901 cgaaatcggc tctggggcta caaacttctc tctgctgaaa caagctggcg acgtggagga
      961 gaaccctgga cctgtgagca agggcgagga ggataacatg gcctctctcc cagcgacaca
     1021 tgagttacac atctttggct ccatcaacgg tgtggacttt gacatggtgg gtcagggcac
     1081 cggcaatcca aatgatggtt atgaggagtt aaacctgaag tccaccaagg gtgacctcca
     1141 gttctccccc tggattctgg tccctcatat cgggtatggc ttccatcagt acctgcccta
     1201 ccctgacggg atgtcgcctt tccaggccgc catggtagat ggctccggct accaagtcca
     1261 tcgcacaatg cagtttgaag atggtgcctc ccttactgtt aactaccgct acacctacga
     1321 gggaagccac atcaaaggag aggcccaggt gaaggggact ggtttccctg ctgacggtcc
     1381 tgtgatgacc aactcgctga ccgctgcgga ctggtgcagg tcgaagaaga cttaccccaa
     1441 cgacaaaacc atcatcagta cctttaagtg gagttacacc actggaaatg gcaagcgcta
     1501 ccggagcact gcgcggacca cctacacctt tgccaagcca atggcggcta actatctgaa
     1561 gaaccagccg atgtacgtgt tccgtaagac ggagctcaag cactccaaga ccgagctcaa
     1621 cttcaaggag tggcaaaagg cctttaccga tgtgatgggc atggacgagc tgtacaagta
     1681 agctagaact atagtgagtc gtattacgta gatccagaca tgataagata cattgatgag
     1741 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     1801 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     1861 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaatt cctcaagaga
     1921 cctctagacc agccaggaca gaaatgcctc gacttcgctg ctacccaagg ttgccgggtg
     1981 acgcacaccg tggaaacgga tgaaggcacg aacccagtgg acataagcct gttcggttcg
     2041 taagctgtaa tgcaagtagc gtatgcgctc acgcaactgg tccagaacct tgaccgaacg
     2101 cagcggtggt aacggcgcag tggcggtttt catggcttgt tatgactgtt tttttggggt
     2161 acagtctatg cctcgggcat ccaagcagca agcgcgttac gccgtgggtc gatgtttgat
     2221 gttatggagc agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa agttaaacat
     2281 tatgagggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag ttggcgtcat
     2341 cgagcgccat ctcgaaccga cgttgctggc cgtacatttg tacggctccg cagtggatgg
     2401 cggcctgaag ccacacagtg atattgattt gctggttacg gtgaccgtaa ggcttgatga
     2461 aacaacgcgg cgagctttga tcaacgacct tttggaaact tcggcttccc ctggagagag
     2521 cgagattctc cgcgctgtag aagtcaccat tgttgtgcac gacgacatca ttccgtggcg
     2581 ttatccagct aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg
     2641 tatcttcgag ccagccacga tcgacattga tctggctatc ttgctgacaa aagcaagaga
     2701 acatagcgtt gccttggtag gtccagcggc ggaggaactc tttgatccgg ttcctgaaca
     2761 ggatctattt gaggcgctaa atgaaacctt aacgctatgg aactcgccgc ccgactgggc
     2821 tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg
     2881 caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc cggcccagta
     2941 tcagcccgtc atacttgaag ctagacaggc ttatcttgga caagaagaag atcgcttggc
     3001 ctcgcgcgca gatcagttgg aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt
     3061 agtcggcaaa taaccctcga gccacccatg accaaaatcc cttaacgtga gttacgcgtc
     3121 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3181 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3241 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3301 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3361 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3421 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3481 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3541 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3601 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3661 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3721 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3781 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3841 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3901 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     3961 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagt
     4021 gggcagtgag cgcaacgcaa ttaatacgcg taccgctagc caggaagagt ttgtagaaac
     4081 gcaaaaaggc catccgtcag gatggccttc tgcttagttt gatgcctggc agtttatggc
     4141 gggcgtcctg cccgccaccc tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc
     4201 gg
//
