LOCUS       sequence-482089-        4204 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 4204)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 4204)
  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 ORF3
FEATURES             Location/Qualifiers
     source          1..4204
                     /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..1683
                     /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    1713..1847
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1737..1756)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1791..1810
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2284..3075
                     /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      3168..3756
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3657..3676
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3910..3927
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4086..4113)
                     /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 taagctagaa ctatagtgag tcgtattacg tagatccaga catgataaga tacattgatg
     1741 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     1801 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     1861 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa ttcctcaaga
     1921 gacctctaga ccagccagga cagaaatgcc tcgacttcgc tgctacccaa ggttgccggg
     1981 tgacgcacac cgtggaaacg gatgaaggca cgaacccagt ggacataagc ctgttcggtt
     2041 cgtaagctgt aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac cttgaccgaa
     2101 cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg tttttttggg
     2161 gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg tcgatgtttg
     2221 atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac aaagttaaac
     2281 attatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt agttggcgtc
     2341 atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat
     2401 ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat
     2461 gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag
     2521 agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg
     2581 cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca
     2641 ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga
     2701 gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa
     2761 caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg
     2821 gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc
     2881 ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag
     2941 tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg
     3001 gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcaccaag
     3061 gtagtcggca aataaccctc gagccaccca tgaccaaaat cccttaacgt gagttacgcg
     3121 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3181 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3241 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     3301 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     3361 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     3421 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     3481 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     3541 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     3601 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     3661 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     3721 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     3781 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     3841 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     3901 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     3961 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     4021 gcgggcagtg agcgcaacgc aattaatacg cgtaccgcta gccaggaaga gtttgtagaa
     4081 acgcaaaaag gccatccgtc aggatggcct tctgcttagt ttgatgcctg gcagtttatg
     4141 gcgggcgtcc tgcccgccac cctccgggcc gttgcttcac aacgttcaaa tccgctcccg
     4201 gcgg
//
