LOCUS       sequence-482030-        4243 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 4243)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 4243)
  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 ORF3
FEATURES             Location/Qualifiers
     source          1..4243
                     /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             205..258
                     /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             919..975
                     /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             976..1680
                     /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      1228..1237
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1752..1886
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1776..1795)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1830..1849
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2323..3114
                     /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      3207..3795
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3696..3715
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3949..3966
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4125..4152)
                     /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 cgcccggctc tggggagggc agaggcagtc tgctgacatg cggtgacgtg
      241 gaagagaatc ccggccctat gactattgtt caagctgccc aatcccgcta ctccaccaaa
      301 gcctttgatg cttcgcgcaa attgcctgaa gagaaagtcg cggcagtgaa agagttaatc
      361 cgcatgagtg cgtccagtgt caactcgcaa ccttggcatt ttattgtcgc gagcagtgaa
      421 gagggaaaag cgcgcatcgc caaagcaaca caaggtggtt ttgctgctaa tgagcgcaag
      481 attttggatg cttctcacgt tgtggtgttc tgcgccaaaa cggcgattga tgaagcgtac
      541 ctactcgacc ttttggaaag cgaagacaaa gatggccgct acgccgatgt cgaagcaaaa
      601 aatggcatgc acgctggtcg ttcatttttc gtcaacatgc accgctttga cttgaaagac
      661 gcgcatcact ggatggaaaa gcaagtttac ctcaatgtgg ggacgctgct attgggtgct
      721 tctgcgatgg agatcgacgc ggtgccaatt gaaggcttcg atgccaaggt gcttgatgag
      781 gagtttggtc tgcgtgagaa gggctttacc agcgtggtga ttgtgccgct gggttaccat
      841 agcgaagacg attttaatgc taagctgcca aaatcacgtt ggtcagcaga gactgttttt
      901 accgaaatcg gctctggggc tacaaacttc tctctgctga aacaagctgg cgacgtggag
      961 gagaaccctg gacctgtgag caagggcgag gaggataaca tggcctctct cccagcgaca
     1021 catgagttac acatctttgg ctccatcaac ggtgtggact ttgacatggt gggtcagggc
     1081 accggcaatc caaatgatgg ttatgaggag ttaaacctga agtccaccaa gggtgacctc
     1141 cagttctccc cctggattct ggtccctcat atcgggtatg gcttccatca gtacctgccc
     1201 taccctgacg ggatgtcgcc tttccaggcc gccatggtag atggctccgg ctaccaagtc
     1261 catcgcacaa tgcagtttga agatggtgcc tcccttactg ttaactaccg ctacacctac
     1321 gagggaagcc acatcaaagg agaggcccag gtgaagggga ctggtttccc tgctgacggt
     1381 cctgtgatga ccaactcgct gaccgctgcg gactggtgca ggtcgaagaa gacttacccc
     1441 aacgacaaaa ccatcatcag tacctttaag tggagttaca ccactggaaa tggcaagcgc
     1501 taccggagca ctgcgcggac cacctacacc tttgccaagc caatggcggc taactatctg
     1561 aagaaccagc cgatgtacgt gttccgtaag acggagctca agcactccaa gaccgagctc
     1621 aacttcaagg agtggcaaaa ggcctttacc gatgtgatgg gcatggacga gctgtacaag
     1681 aagaagaaaa agaagtcaaa gacaaagtgt gtaattatgt aagctagaac tatagtgagt
     1741 cgtattacgt agatccagac atgataagat acattgatga gtttggacaa accacaacta
     1801 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     1861 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     1921 ttcaggggga ggtgtgggag gttttttaat tcctcaagag acctctagac cagccaggac
     1981 agaaatgcct cgacttcgct gctacccaag gttgccgggt gacgcacacc gtggaaacgg
     2041 atgaaggcac gaacccagtg gacataagcc tgttcggttc gtaagctgta atgcaagtag
     2101 cgtatgcgct cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg taacggcgca
     2161 gtggcggttt tcatggcttg ttatgactgt ttttttgggg tacagtctat gcctcgggca
     2221 tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga tgttatggag cagcaacgat
     2281 gttacgcagc agggcagtcg ccctaaaaca aagttaaaca ttatgaggga agcggtgatc
     2341 gccgaagtat cgactcaact atcagaggta gttggcgtca tcgagcgcca tctcgaaccg
     2401 acgttgctgg ccgtacattt gtacggctcc gcagtggatg gcggcctgaa gccacacagt
     2461 gatattgatt tgctggttac ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg
     2521 atcaacgacc ttttggaaac ttcggcttcc cctggagaga gcgagattct ccgcgctgta
     2581 gaagtcacca ttgttgtgca cgacgacatc attccgtggc gttatccagc taagcgcgaa
     2641 ctgcaatttg gagaatggca gcgcaatgac attcttgcag gtatcttcga gccagccacg
     2701 atcgacattg atctggctat cttgctgaca aaagcaagag aacatagcgt tgccttggta
     2761 ggtccagcgg cggaggaact ctttgatccg gttcctgaac aggatctatt tgaggcgcta
     2821 aatgaaacct taacgctatg gaactcgccg cccgactggg ctggcgatga gcgaaatgta
     2881 gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat
     2941 gtcgctgccg actgggcaat ggagcgcctg ccggcccagt atcagcccgt catacttgaa
     3001 gctagacagg cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg
     3061 gaagaatttg tccactacgt gaaaggcgag atcaccaagg tagtcggcaa ataaccctcg
     3121 agccacccat gaccaaaatc ccttaacgtg agttacgcgt cgttccactg agcgtcagac
     3181 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3241 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3301 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     3361 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3421 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3481 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3541 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3601 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3661 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3721 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3781 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3841 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     3901 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     3961 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     4021 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     4081 attaatacgc gtaccgctag ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca
     4141 ggatggcctt ctgcttagtt tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc
     4201 ctccgggccg ttgcttcaca acgttcaaat ccgctcccgg cgg
//
