LOCUS       sequence-482028-        4241 bp    DNA     circular SYN 15-DEC-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 4241)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 4241)
  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: linker-T2A-NTRv2-linker-P2A-mNeonGreen-CAAX ORF1
FEATURES             Location/Qualifiers
     source          1..4241
                     /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..1678
                     /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    1750..1884
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1774..1793)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1828..1847
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2321..3112
                     /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      3205..3793
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3694..3713
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3947..3964
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4123..4150)
                     /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 tgtacaagaa
     1681 gaagaaaaag aagtcaaaga caaagtgtgt aattatgtaa gctagaacta tagtgagtcg
     1741 tattacgtag atccagacat gataagatac attgatgagt ttggacaaac cacaactaga
     1801 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     1861 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     1921 cagggggagg tgtgggaggt tttttaattc ctcaagagac ctctagacca gccaggacag
     1981 aaatgcctcg acttcgctgc tacccaaggt tgccgggtga cgcacaccgt ggaaacggat
     2041 gaaggcacga acccagtgga cataagcctg ttcggttcgt aagctgtaat gcaagtagcg
     2101 tatgcgctca cgcaactggt ccagaacctt gaccgaacgc agcggtggta acggcgcagt
     2161 ggcggttttc atggcttgtt atgactgttt ttttggggta cagtctatgc ctcgggcatc
     2221 caagcagcaa gcgcgttacg ccgtgggtcg atgtttgatg ttatggagca gcaacgatgt
     2281 tacgcagcag ggcagtcgcc ctaaaacaaa gttaaacatt atgagggaag cggtgatcgc
     2341 cgaagtatcg actcaactat cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac
     2401 gttgctggcc gtacatttgt acggctccgc agtggatggc ggcctgaagc cacacagtga
     2461 tattgatttg ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc gagctttgat
     2521 caacgacctt ttggaaactt cggcttcccc tggagagagc gagattctcc gcgctgtaga
     2581 agtcaccatt gttgtgcacg acgacatcat tccgtggcgt tatccagcta agcgcgaact
     2641 gcaatttgga gaatggcagc gcaatgacat tcttgcaggt atcttcgagc cagccacgat
     2701 cgacattgat ctggctatct tgctgacaaa agcaagagaa catagcgttg ccttggtagg
     2761 tccagcggcg gaggaactct ttgatccggt tcctgaacag gatctatttg aggcgctaaa
     2821 tgaaacctta acgctatgga actcgccgcc cgactgggct ggcgatgagc gaaatgtagt
     2881 gcttacgttg tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc cgaaggatgt
     2941 cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca tacttgaagc
     3001 tagacaggct tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag atcagttgga
     3061 agaatttgtc cactacgtga aaggcgagat caccaaggta gtcggcaaat aaccctcgag
     3121 ccacccatga ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc
     3181 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3241 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3301 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     3361 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3421 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3481 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3541 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     3601 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3661 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3721 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3781 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3841 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3901 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3961 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     4021 ttaatgcagc tggcacgaca ggtttcccga ctggaaagtg ggcagtgagc gcaacgcaat
     4081 taatacgcgt accgctagcc aggaagagtt tgtagaaacg caaaaaggcc atccgtcagg
     4141 atggccttct gcttagtttg atgcctggca gtttatggcg ggcgtcctgc ccgccaccct
     4201 ccgggccgtt gcttcacaac gttcaaatcc gctcccggcg g
//
