LOCUS       sequence-482031-        3496 bp    DNA     circular SYN 05-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 3496)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 3496)
  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: lifeact-mNeongreen-pA
FEATURES             Location/Qualifiers
     source          1..3496
                     /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"
     regulatory      194..203
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             200..250
                     /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             254..961
                     /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      506..515
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1084..1205
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1121..1140)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1175..1194
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             1576..2367
                     /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      2460..3048
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2949..2968
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3202..3219
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3378..3405)
                     /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 cgcgccacca tgggcgtggc cgacctgatc aagaagttcg agagcatcag
      241 caaggaggag ggcgtgagca agggcgagga ggataacatg gcctctctcc cagcgacaca
      301 tgagttacac atctttggct ccatcaacgg tgtggacttt gacatggtgg gtcagggcac
      361 cggcaatcca aatgatggtt atgaggagtt aaacctgaag tccaccaagg gtgacctcca
      421 gttctccccc tggattctgg tccctcatat cgggtatggc ttccatcagt acctgcccta
      481 ccctgacggg atgtcgcctt tccaggccgc catggtagat ggctccggct accaagtcca
      541 tcgcacaatg cagtttgaag atggtgcctc ccttactgtt aactaccgct acacctacga
      601 gggaagccac atcaaaggag aggcccaggt gaaggggact ggtttccctg ctgacggtcc
      661 tgtgatgacc aactcgctga ccgctgcgga ctggtgcagg tcgaagaaga cttaccccaa
      721 cgacaaaacc atcatcagta cctttaagtg gagttacacc actggaaatg gcaagcgcta
      781 ccggagcact gcgcggacca cctacacctt tgccaagcca atggcggcta actatctgaa
      841 gaaccagccg atgtacgtgt tccgtaagac ggagctcaag cactccaaga ccgagctcaa
      901 cttcaaggag tggcaaaagg cctttaccga tgtgatgggc atggacgagc tgtacaagta
      961 aagcggccgc gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct
     1021 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     1081 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     1141 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     1201 tcttactcaa gagacctcta gaccagccag gacagaaatg cctcgacttc gctgctaccc
     1261 aaggttgccg ggtgacgcac accgtggaaa cggatgaagg cacgaaccca gtggacataa
     1321 gcctgttcgg ttcgtaagct gtaatgcaag tagcgtatgc gctcacgcaa ctggtccaga
     1381 accttgaccg aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc ttgttatgac
     1441 tgtttttttg gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg
     1501 ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc agcagggcag tcgccctaaa
     1561 acaaagttaa acattatgag ggaagcggtg atcgccgaag tatcgactca actatcagag
     1621 gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc
     1681 tccgcagtgg atggcggcct gaagccacac agtgatattg atttgctggt tacggtgacc
     1741 gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg accttttgga aacttcggct
     1801 tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt gcacgacgac
     1861 atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg gcagcgcaat
     1921 gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc tatcttgctg
     1981 acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga actctttgat
     2041 ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct atggaactcg
     2101 ccgcccgact gggctggcga tgagcgaaat gtagtgctta cgttgtcccg catttggtac
     2161 agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc
     2221 ctgccggccc agtatcagcc cgtcatactt gaagctagac aggcttatct tggacaagaa
     2281 gaagatcgct tggcctcgcg cgcagatcag ttggaagaat ttgtccacta cgtgaaaggc
     2341 gagatcacca aggtagtcgg caaataaccc tcgagccacc catgaccaaa atcccttaac
     2401 gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     2461 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     2521 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     2581 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     2641 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     2701 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     2761 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     2821 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     2881 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     2941 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3001 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3061 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3121 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     3181 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     3241 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     3301 cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc tagccaggaa
     3361 gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta gtttgatgcc
     3421 tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc acaacgttca
     3481 aatccgctcc cggcgg
//
