LOCUS       sequence-482016-        3489 bp    DNA     circular SYN 05-AUG-2025
DEFINITION  Middle entry vector for mini-Golden subcloning platform; mNeongreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3489)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 3489)
  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-P2A-mNeongreen-ORF2
FEATURES             Location/Qualifiers
     source          1..3489
                     /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..260
                     /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             261..968
                     /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      513..522
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    998..1132
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1022..1041)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1076..1095
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1569..2360
                     /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      2453..3041
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2942..2961
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3195..3212
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3371..3398)
                     /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 ggggctacaa acttctctct gctgaaacaa gctggcgacg
      241 tggaggagaa ccctggacct gtgagcaagg gcgaggagga taacatggcc tctctcccag
      301 cgacacatga gttacacatc tttggctcca tcaacggtgt ggactttgac atggtgggtc
      361 agggcaccgg caatccaaat gatggttatg aggagttaaa cctgaagtcc accaagggtg
      421 acctccagtt ctccccctgg attctggtcc ctcatatcgg gtatggcttc catcagtacc
      481 tgccctaccc tgacgggatg tcgcctttcc aggccgccat ggtagatggc tccggctacc
      541 aagtccatcg cacaatgcag tttgaagatg gtgcctccct tactgttaac taccgctaca
      601 cctacgaggg aagccacatc aaaggagagg cccaggtgaa ggggactggt ttccctgctg
      661 acggtcctgt gatgaccaac tcgctgaccg ctgcggactg gtgcaggtcg aagaagactt
      721 accccaacga caaaaccatc atcagtacct ttaagtggag ttacaccact ggaaatggca
      781 agcgctaccg gagcactgcg cggaccacct acacctttgc caagccaatg gcggctaact
      841 atctgaagaa ccagccgatg tacgtgttcc gtaagacgga gctcaagcac tccaagaccg
      901 agctcaactt caaggagtgg caaaaggcct ttaccgatgt gatgggcatg gacgagctgt
      961 acaagtaagc tagaactata gtgagtcgta ttacgtagat ccagacatga taagatacat
     1021 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     1081 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     1141 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaattcct
     1201 caagagacct ctagaccagc caggacagaa atgcctcgac ttcgctgcta cccaaggttg
     1261 ccgggtgacg cacaccgtgg aaacggatga aggcacgaac ccagtggaca taagcctgtt
     1321 cggttcgtaa gctgtaatgc aagtagcgta tgcgctcacg caactggtcc agaaccttga
     1381 ccgaacgcag cggtggtaac ggcgcagtgg cggttttcat ggcttgttat gactgttttt
     1441 ttggggtaca gtctatgcct cgggcatcca agcagcaagc gcgttacgcc gtgggtcgat
     1501 gtttgatgtt atggagcagc aacgatgtta cgcagcaggg cagtcgccct aaaacaaagt
     1561 taaacattat gagggaagcg gtgatcgccg aagtatcgac tcaactatca gaggtagttg
     1621 gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt acatttgtac ggctccgcag
     1681 tggatggcgg cctgaagcca cacagtgata ttgatttgct ggttacggtg accgtaaggc
     1741 ttgatgaaac aacgcggcga gctttgatca acgacctttt ggaaacttcg gcttcccctg
     1801 gagagagcga gattctccgc gctgtagaag tcaccattgt tgtgcacgac gacatcattc
     1861 cgtggcgtta tccagctaag cgcgaactgc aatttggaga atggcagcgc aatgacattc
     1921 ttgcaggtat cttcgagcca gccacgatcg acattgatct ggctatcttg ctgacaaaag
     1981 caagagaaca tagcgttgcc ttggtaggtc cagcggcgga ggaactcttt gatccggttc
     2041 ctgaacagga tctatttgag gcgctaaatg aaaccttaac gctatggaac tcgccgcccg
     2101 actgggctgg cgatgagcga aatgtagtgc ttacgttgtc ccgcatttgg tacagcgcag
     2161 taaccggcaa aatcgcgccg aaggatgtcg ctgccgactg ggcaatggag cgcctgccgg
     2221 cccagtatca gcccgtcata cttgaagcta gacaggctta tcttggacaa gaagaagatc
     2281 gcttggcctc gcgcgcagat cagttggaag aatttgtcca ctacgtgaaa ggcgagatca
     2341 ccaaggtagt cggcaaataa ccctcgagcc acccatgacc aaaatccctt aacgtgagtt
     2401 acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2461 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2521 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2581 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2641 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2701 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     2761 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     2821 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     2881 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     2941 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3001 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3061 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3121 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3181 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     3241 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     3301 ggaaagcggg cagtgagcgc aacgcaatta atacgcgtac cgctagccag gaagagtttg
     3361 tagaaacgca aaaaggccat ccgtcaggat ggccttctgc ttagtttgat gcctggcagt
     3421 ttatggcggg cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt tcaaatccgc
     3481 tcccggcgg
//
