LOCUS       sequence-482034-        3543 bp    DNA     circular SYN 15-DEC-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 3543)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 3543)
  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-P2A-lifeact-mNeongreen-ORF-2
FEATURES             Location/Qualifiers
     source          1..3543
                     /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..311
                     /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             315..1022
                     /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      567..576
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1052..1186
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1076..1095)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1130..1149
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             1623..2414
                     /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      2507..3095
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2996..3015
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3249..3266
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3425..3452)
                     /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 atgggcgtgg ccgacctgat caagaagttc gagagcatca
      301 gcaaggagga gggcgtgagc aagggcgagg aggataacat ggcctctctc ccagcgacac
      361 atgagttaca catctttggc tccatcaacg gtgtggactt tgacatggtg ggtcagggca
      421 ccggcaatcc aaatgatggt tatgaggagt taaacctgaa gtccaccaag ggtgacctcc
      481 agttctcccc ctggattctg gtccctcata tcgggtatgg cttccatcag tacctgccct
      541 accctgacgg gatgtcgcct ttccaggccg ccatggtaga tggctccggc taccaagtcc
      601 atcgcacaat gcagtttgaa gatggtgcct cccttactgt taactaccgc tacacctacg
      661 agggaagcca catcaaagga gaggcccagg tgaaggggac tggtttccct gctgacggtc
      721 ctgtgatgac caactcgctg accgctgcgg actggtgcag gtcgaagaag acttacccca
      781 acgacaaaac catcatcagt acctttaagt ggagttacac cactggaaat ggcaagcgct
      841 accggagcac tgcgcggacc acctacacct ttgccaagcc aatggcggct aactatctga
      901 agaaccagcc gatgtacgtg ttccgtaaga cggagctcaa gcactccaag accgagctca
      961 acttcaagga gtggcaaaag gcctttaccg atgtgatggg catggacgag ctgtacaagt
     1021 aagctagaac tatagtgagt cgtattacgt agatccagac atgataagat acattgatga
     1081 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     1141 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     1201 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaat tcctcaagag
     1261 acctctagac cagccaggac agaaatgcct cgacttcgct gctacccaag gttgccgggt
     1321 gacgcacacc gtggaaacgg atgaaggcac gaacccagtg gacataagcc tgttcggttc
     1381 gtaagctgta atgcaagtag cgtatgcgct cacgcaactg gtccagaacc ttgaccgaac
     1441 gcagcggtgg taacggcgca gtggcggttt tcatggcttg ttatgactgt ttttttgggg
     1501 tacagtctat gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga
     1561 tgttatggag cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaaaca
     1621 ttatgaggga agcggtgatc gccgaagtat cgactcaact atcagaggta gttggcgtca
     1681 tcgagcgcca tctcgaaccg acgttgctgg ccgtacattt gtacggctcc gcagtggatg
     1741 gcggcctgaa gccacacagt gatattgatt tgctggttac ggtgaccgta aggcttgatg
     1801 aaacaacgcg gcgagctttg atcaacgacc ttttggaaac ttcggcttcc cctggagaga
     1861 gcgagattct ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc attccgtggc
     1921 gttatccagc taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac attcttgcag
     1981 gtatcttcga gccagccacg atcgacattg atctggctat cttgctgaca aaagcaagag
     2041 aacatagcgt tgccttggta ggtccagcgg cggaggaact ctttgatccg gttcctgaac
     2101 aggatctatt tgaggcgcta aatgaaacct taacgctatg gaactcgccg cccgactggg
     2161 ctggcgatga gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg
     2221 gcaaaatcgc gccgaaggat gtcgctgccg actgggcaat ggagcgcctg ccggcccagt
     2281 atcagcccgt catacttgaa gctagacagg cttatcttgg acaagaagaa gatcgcttgg
     2341 cctcgcgcgc agatcagttg gaagaatttg tccactacgt gaaaggcgag atcaccaagg
     2401 tagtcggcaa ataaccctcg agccacccat gaccaaaatc ccttaacgtg agttacgcgt
     2461 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2521 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2581 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2641 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2701 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2761 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     2821 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     2881 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     2941 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     3001 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     3061 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3121 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     3181 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     3241 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     3301 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     3361 cgggcagtga gcgcaacgca attaatacgc gtaccgctag ccaggaagag tttgtagaaa
     3421 cgcaaaaagg ccatccgtca ggatggcctt ctgcttagtt tgatgcctgg cagtttatgg
     3481 cgggcgtcct gcccgccacc ctccgggccg ttgcttcaca acgttcaaat ccgctcccgg
     3541 cgg
//
