LOCUS       sequence-482029-        4242 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 4242)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 4242)
  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-CAAX ORF2
FEATURES             Location/Qualifiers
     source          1..4242
                     /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             204..257
                     /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             918..974
                     /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             975..1679
                     /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      1227..1236
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1751..1885
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1775..1794)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1829..1848
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2322..3113
                     /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      3206..3794
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3695..3714
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3948..3965
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4124..4151)
                     /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 cgccggctct ggggagggca gaggcagtct gctgacatgc ggtgacgtgg
      241 aagagaatcc cggccctatg actattgttc aagctgccca atcccgctac tccaccaaag
      301 cctttgatgc ttcgcgcaaa ttgcctgaag agaaagtcgc ggcagtgaaa gagttaatcc
      361 gcatgagtgc gtccagtgtc aactcgcaac cttggcattt tattgtcgcg agcagtgaag
      421 agggaaaagc gcgcatcgcc aaagcaacac aaggtggttt tgctgctaat gagcgcaaga
      481 ttttggatgc ttctcacgtt gtggtgttct gcgccaaaac ggcgattgat gaagcgtacc
      541 tactcgacct tttggaaagc gaagacaaag atggccgcta cgccgatgtc gaagcaaaaa
      601 atggcatgca cgctggtcgt tcatttttcg tcaacatgca ccgctttgac ttgaaagacg
      661 cgcatcactg gatggaaaag caagtttacc tcaatgtggg gacgctgcta ttgggtgctt
      721 ctgcgatgga gatcgacgcg gtgccaattg aaggcttcga tgccaaggtg cttgatgagg
      781 agtttggtct gcgtgagaag ggctttacca gcgtggtgat tgtgccgctg ggttaccata
      841 gcgaagacga ttttaatgct aagctgccaa aatcacgttg gtcagcagag actgttttta
      901 ccgaaatcgg ctctggggct acaaacttct ctctgctgaa acaagctggc gacgtggagg
      961 agaaccctgg acctgtgagc aagggcgagg aggataacat ggcctctctc ccagcgacac
     1021 atgagttaca catctttggc tccatcaacg gtgtggactt tgacatggtg ggtcagggca
     1081 ccggcaatcc aaatgatggt tatgaggagt taaacctgaa gtccaccaag ggtgacctcc
     1141 agttctcccc ctggattctg gtccctcata tcgggtatgg cttccatcag tacctgccct
     1201 accctgacgg gatgtcgcct ttccaggccg ccatggtaga tggctccggc taccaagtcc
     1261 atcgcacaat gcagtttgaa gatggtgcct cccttactgt taactaccgc tacacctacg
     1321 agggaagcca catcaaagga gaggcccagg tgaaggggac tggtttccct gctgacggtc
     1381 ctgtgatgac caactcgctg accgctgcgg actggtgcag gtcgaagaag acttacccca
     1441 acgacaaaac catcatcagt acctttaagt ggagttacac cactggaaat ggcaagcgct
     1501 accggagcac tgcgcggacc acctacacct ttgccaagcc aatggcggct aactatctga
     1561 agaaccagcc gatgtacgtg ttccgtaaga cggagctcaa gcactccaag accgagctca
     1621 acttcaagga gtggcaaaag gcctttaccg atgtgatggg catggacgag ctgtacaaga
     1681 agaagaaaaa gaagtcaaag acaaagtgtg taattatgta agctagaact atagtgagtc
     1741 gtattacgta gatccagaca tgataagata cattgatgag tttggacaaa ccacaactag
     1801 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     1861 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     1921 tcagggggag gtgtgggagg ttttttaatt cctcaagaga cctctagacc agccaggaca
     1981 gaaatgcctc gacttcgctg ctacccaagg ttgccgggtg acgcacaccg tggaaacgga
     2041 tgaaggcacg aacccagtgg acataagcct gttcggttcg taagctgtaa tgcaagtagc
     2101 gtatgcgctc acgcaactgg tccagaacct tgaccgaacg cagcggtggt aacggcgcag
     2161 tggcggtttt catggcttgt tatgactgtt tttttggggt acagtctatg cctcgggcat
     2221 ccaagcagca agcgcgttac gccgtgggtc gatgtttgat gttatggagc agcaacgatg
     2281 ttacgcagca gggcagtcgc cctaaaacaa agttaaacat tatgagggaa gcggtgatcg
     2341 ccgaagtatc gactcaacta tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga
     2401 cgttgctggc cgtacatttg tacggctccg cagtggatgg cggcctgaag ccacacagtg
     2461 atattgattt gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga
     2521 tcaacgacct tttggaaact tcggcttccc ctggagagag cgagattctc cgcgctgtag
     2581 aagtcaccat tgttgtgcac gacgacatca ttccgtggcg ttatccagct aagcgcgaac
     2641 tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg tatcttcgag ccagccacga
     2701 tcgacattga tctggctatc ttgctgacaa aagcaagaga acatagcgtt gccttggtag
     2761 gtccagcggc ggaggaactc tttgatccgg ttcctgaaca ggatctattt gaggcgctaa
     2821 atgaaacctt aacgctatgg aactcgccgc ccgactgggc tggcgatgag cgaaatgtag
     2881 tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg
     2941 tcgctgccga ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc atacttgaag
     3001 ctagacaggc ttatcttgga caagaagaag atcgcttggc ctcgcgcgca gatcagttgg
     3061 aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt agtcggcaaa taaccctcga
     3121 gccacccatg accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc
     3181 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3241 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3301 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     3361 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3421 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3481 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3541 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3601 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3661 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3721 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3781 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3841 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3901 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3961 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     4021 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     4081 ttaatacgcg taccgctagc caggaagagt ttgtagaaac gcaaaaaggc catccgtcag
     4141 gatggccttc tgcttagttt gatgcctggc agtttatggc gggcgtcctg cccgccaccc
     4201 tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc gg
//
