LOCUS       sequence-484612-        3602 bp    DNA     circular SYN 26-AUG-2025
DEFINITION  Middle entry vector for mini-Golden subcloning platform; 
            lifeact-mNeongreen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3602)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 3602)
  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-lifeact-mNeongreen-ORF-1
FEATURES             Location/Qualifiers
     source          1..3602
                     /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             263..319
                     /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             320..370
                     /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             374..1081
                     /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      626..635
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1111..1245
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1135..1154)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1189..1208
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1682..2473
                     /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      2566..3154
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3055..3074
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3308..3325
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3484..3511)
                     /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 cagggctctg gggctacaaa cttctctctg ctgaaacaag ctggcgacgt
      301 ggaggagaac cctggaccta tgggcgtggc cgacctgatc aagaagttcg agagcatcag
      361 caaggaggag ggcgtgagca agggcgagga ggataacatg gcctctctcc cagcgacaca
      421 tgagttacac atctttggct ccatcaacgg tgtggacttt gacatggtgg gtcagggcac
      481 cggcaatcca aatgatggtt atgaggagtt aaacctgaag tccaccaagg gtgacctcca
      541 gttctccccc tggattctgg tccctcatat cgggtatggc ttccatcagt acctgcccta
      601 ccctgacggg atgtcgcctt tccaggccgc catggtagat ggctccggct accaagtcca
      661 tcgcacaatg cagtttgaag atggtgcctc ccttactgtt aactaccgct acacctacga
      721 gggaagccac atcaaaggag aggcccaggt gaaggggact ggtttccctg ctgacggtcc
      781 tgtgatgacc aactcgctga ccgctgcgga ctggtgcagg tcgaagaaga cttaccccaa
      841 cgacaaaacc atcatcagta cctttaagtg gagttacacc actggaaatg gcaagcgcta
      901 ccggagcact gcgcggacca cctacacctt tgccaagcca atggcggcta actatctgaa
      961 gaaccagccg atgtacgtgt tccgtaagac ggagctcaag cactccaaga ccgagctcaa
     1021 cttcaaggag tggcaaaagg cctttaccga tgtgatgggc atggacgagc tgtacaagta
     1081 agctagaact atagtgagtc gtattacgta gatccagaca tgataagata cattgatgag
     1141 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     1201 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     1261 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaatt cctcaagaga
     1321 cctctagacc agccaggaca gaaatgcctc gacttcgctg ctacccaagg ttgccgggtg
     1381 acgcacaccg tggaaacgga tgaaggcacg aacccagtgg acataagcct gttcggttcg
     1441 taagctgtaa tgcaagtagc gtatgcgctc acgcaactgg tccagaacct tgaccgaacg
     1501 cagcggtggt aacggcgcag tggcggtttt catggcttgt tatgactgtt tttttggggt
     1561 acagtctatg cctcgggcat ccaagcagca agcgcgttac gccgtgggtc gatgtttgat
     1621 gttatggagc agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa agttaaacat
     1681 tatgagggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag ttggcgtcat
     1741 cgagcgccat ctcgaaccga cgttgctggc cgtacatttg tacggctccg cagtggatgg
     1801 cggcctgaag ccacacagtg atattgattt gctggttacg gtgaccgtaa ggcttgatga
     1861 aacaacgcgg cgagctttga tcaacgacct tttggaaact tcggcttccc ctggagagag
     1921 cgagattctc cgcgctgtag aagtcaccat tgttgtgcac gacgacatca ttccgtggcg
     1981 ttatccagct aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg
     2041 tatcttcgag ccagccacga tcgacattga tctggctatc ttgctgacaa aagcaagaga
     2101 acatagcgtt gccttggtag gtccagcggc ggaggaactc tttgatccgg ttcctgaaca
     2161 ggatctattt gaggcgctaa atgaaacctt aacgctatgg aactcgccgc ccgactgggc
     2221 tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg
     2281 caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc cggcccagta
     2341 tcagcccgtc atacttgaag ctagacaggc ttatcttgga caagaagaag atcgcttggc
     2401 ctcgcgcgca gatcagttgg aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt
     2461 agtcggcaaa taaccctcga gccacccatg accaaaatcc cttaacgtga gttacgcgtc
     2521 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2581 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2641 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2701 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2761 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2821 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2881 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2941 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3001 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3061 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3121 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3181 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3241 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3301 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     3361 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     3421 gggcagtgag cgcaacgcaa ttaatacgcg taccgctagc caggaagagt ttgtagaaac
     3481 gcaaaaaggc catccgtcag gatggccttc tgcttagttt gatgcctggc agtttatggc
     3541 gggcgtcctg cccgccaccc tccgggccgt tgcttcacaa cgttcaaatc cgctcccggc
     3601 gg
//
