LOCUS       sequence-482084-        3542 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 3542)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 3542)
  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-lifeact-mNeongreen-ORF-1
FEATURES             Location/Qualifiers
     source          1..3542
                     /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             203..259
                     /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             260..310
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     CDS             314..1021
                     /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      566..575
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1051..1185
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1075..1094)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1129..1148
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1622..2413
                     /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      2506..3094
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2995..3014
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3248..3265
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3424..3451)
                     /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 cgcggctctg gggctacaaa cttctctctg ctgaaacaag ctggcgacgt
      241 ggaggagaac cctggaccta tgggcgtggc cgacctgatc aagaagttcg agagcatcag
      301 caaggaggag ggcgtgagca agggcgagga ggataacatg gcctctctcc cagcgacaca
      361 tgagttacac atctttggct ccatcaacgg tgtggacttt gacatggtgg gtcagggcac
      421 cggcaatcca aatgatggtt atgaggagtt aaacctgaag tccaccaagg gtgacctcca
      481 gttctccccc tggattctgg tccctcatat cgggtatggc ttccatcagt acctgcccta
      541 ccctgacggg atgtcgcctt tccaggccgc catggtagat ggctccggct accaagtcca
      601 tcgcacaatg cagtttgaag atggtgcctc ccttactgtt aactaccgct acacctacga
      661 gggaagccac atcaaaggag aggcccaggt gaaggggact ggtttccctg ctgacggtcc
      721 tgtgatgacc aactcgctga ccgctgcgga ctggtgcagg tcgaagaaga cttaccccaa
      781 cgacaaaacc atcatcagta cctttaagtg gagttacacc actggaaatg gcaagcgcta
      841 ccggagcact gcgcggacca cctacacctt tgccaagcca atggcggcta actatctgaa
      901 gaaccagccg atgtacgtgt tccgtaagac ggagctcaag cactccaaga ccgagctcaa
      961 cttcaaggag tggcaaaagg cctttaccga tgtgatgggc atggacgagc tgtacaagta
     1021 agctagaact atagtgagtc gtattacgta gatccagaca tgataagata cattgatgag
     1081 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     1141 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     1201 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaatt cctcaagaga
     1261 cctctagacc agccaggaca gaaatgcctc gacttcgctg ctacccaagg ttgccgggtg
     1321 acgcacaccg tggaaacgga tgaaggcacg aacccagtgg acataagcct gttcggttcg
     1381 taagctgtaa tgcaagtagc gtatgcgctc acgcaactgg tccagaacct tgaccgaacg
     1441 cagcggtggt aacggcgcag tggcggtttt catggcttgt tatgactgtt tttttggggt
     1501 acagtctatg cctcgggcat ccaagcagca agcgcgttac gccgtgggtc gatgtttgat
     1561 gttatggagc agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa agttaaacat
     1621 tatgagggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag ttggcgtcat
     1681 cgagcgccat ctcgaaccga cgttgctggc cgtacatttg tacggctccg cagtggatgg
     1741 cggcctgaag ccacacagtg atattgattt gctggttacg gtgaccgtaa ggcttgatga
     1801 aacaacgcgg cgagctttga tcaacgacct tttggaaact tcggcttccc ctggagagag
     1861 cgagattctc cgcgctgtag aagtcaccat tgttgtgcac gacgacatca ttccgtggcg
     1921 ttatccagct aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg
     1981 tatcttcgag ccagccacga tcgacattga tctggctatc ttgctgacaa aagcaagaga
     2041 acatagcgtt gccttggtag gtccagcggc ggaggaactc tttgatccgg ttcctgaaca
     2101 ggatctattt gaggcgctaa atgaaacctt aacgctatgg aactcgccgc ccgactgggc
     2161 tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg
     2221 caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc cggcccagta
     2281 tcagcccgtc atacttgaag ctagacaggc ttatcttgga caagaagaag atcgcttggc
     2341 ctcgcgcgca gatcagttgg aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt
     2401 agtcggcaaa taaccctcga gccacccatg accaaaatcc cttaacgtga gttacgcgtc
     2461 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2521 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2581 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2641 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2701 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2761 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2821 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2881 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     2941 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3001 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3061 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3121 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3181 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3241 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     3301 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     3361 gggcagtgag cgcaacgcaa ttaatacgcg taccgctagc caggaagagt ttgtagaaac
     3421 gcaaaaaggc catccgtcag gatggccttc tgcttagttt gatgcctggc agtttatggc
     3481 gggcgtcctg cccgccaccc tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc
     3541 gg
//
