LOCUS       sequence-484600-        3550 bp    DNA     circular SYN 26-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 3550)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 3550)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 26, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: SA-linker-P2A-mNeongreen-ORF3
FEATURES             Location/Qualifiers
     source          1..3550
                     /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             265..321
                     /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             322..1029
                     /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      574..583
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1059..1193
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1083..1102)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1137..1156
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1630..2421
                     /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      2514..3102
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3003..3022
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3256..3273
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3432..3459)
                     /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 cgccactaat ccggagtgtg tgtgtgtttc tgacgtgtct ctctcttccc
      241 tttttttttt cagccggctc tggggctaca aacttctctc tgctgaaaca agctggcgac
      301 gtggaggaga accctggacc tgtgagcaag ggcgaggagg ataacatggc ctctctccca
      361 gcgacacatg agttacacat ctttggctcc atcaacggtg tggactttga catggtgggt
      421 cagggcaccg gcaatccaaa tgatggttat gaggagttaa acctgaagtc caccaagggt
      481 gacctccagt tctccccctg gattctggtc cctcatatcg ggtatggctt ccatcagtac
      541 ctgccctacc ctgacgggat gtcgcctttc caggccgcca tggtagatgg ctccggctac
      601 caagtccatc gcacaatgca gtttgaagat ggtgcctccc ttactgttaa ctaccgctac
      661 acctacgagg gaagccacat caaaggagag gcccaggtga aggggactgg tttccctgct
      721 gacggtcctg tgatgaccaa ctcgctgacc gctgcggact ggtgcaggtc gaagaagact
      781 taccccaacg acaaaaccat catcagtacc tttaagtgga gttacaccac tggaaatggc
      841 aagcgctacc ggagcactgc gcggaccacc tacacctttg ccaagccaat ggcggctaac
      901 tatctgaaga accagccgat gtacgtgttc cgtaagacgg agctcaagca ctccaagacc
      961 gagctcaact tcaaggagtg gcaaaaggcc tttaccgatg tgatgggcat ggacgagctg
     1021 tacaagtaag ctagaactat agtgagtcgt attacgtaga tccagacatg ataagataca
     1081 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     1141 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
     1201 acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaattcc
     1261 tcaagagacc tctagaccag ccaggacaga aatgcctcga cttcgctgct acccaaggtt
     1321 gccgggtgac gcacaccgtg gaaacggatg aaggcacgaa cccagtggac ataagcctgt
     1381 tcggttcgta agctgtaatg caagtagcgt atgcgctcac gcaactggtc cagaaccttg
     1441 accgaacgca gcggtggtaa cggcgcagtg gcggttttca tggcttgtta tgactgtttt
     1501 tttggggtac agtctatgcc tcgggcatcc aagcagcaag cgcgttacgc cgtgggtcga
     1561 tgtttgatgt tatggagcag caacgatgtt acgcagcagg gcagtcgccc taaaacaaag
     1621 ttaaacatta tgagggaagc ggtgatcgcc gaagtatcga ctcaactatc agaggtagtt
     1681 ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg tacatttgta cggctccgca
     1741 gtggatggcg gcctgaagcc acacagtgat attgatttgc tggttacggt gaccgtaagg
     1801 cttgatgaaa caacgcggcg agctttgatc aacgaccttt tggaaacttc ggcttcccct
     1861 ggagagagcg agattctccg cgctgtagaa gtcaccattg ttgtgcacga cgacatcatt
     1921 ccgtggcgtt atccagctaa gcgcgaactg caatttggag aatggcagcg caatgacatt
     1981 cttgcaggta tcttcgagcc agccacgatc gacattgatc tggctatctt gctgacaaaa
     2041 gcaagagaac atagcgttgc cttggtaggt ccagcggcgg aggaactctt tgatccggtt
     2101 cctgaacagg atctatttga ggcgctaaat gaaaccttaa cgctatggaa ctcgccgccc
     2161 gactgggctg gcgatgagcg aaatgtagtg cttacgttgt cccgcatttg gtacagcgca
     2221 gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact gggcaatgga gcgcctgccg
     2281 gcccagtatc agcccgtcat acttgaagct agacaggctt atcttggaca agaagaagat
     2341 cgcttggcct cgcgcgcaga tcagttggaa gaatttgtcc actacgtgaa aggcgagatc
     2401 accaaggtag tcggcaaata accctcgagc cacccatgac caaaatccct taacgtgagt
     2461 tacgcgtcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2521 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     2581 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     2641 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     2701 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     2761 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     2821 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     2881 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     2941 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3001 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3061 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3121 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3181 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3241 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     3301 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     3361 tggaaagcgg gcagtgagcg caacgcaatt aatacgcgta ccgctagcca ggaagagttt
     3421 gtagaaacgc aaaaaggcca tccgtcagga tggccttctg cttagtttga tgcctggcag
     3481 tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg cttcacaacg ttcaaatccg
     3541 ctcccggcgg
//
