LOCUS       sequence-8936-ex        3861 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry vector for miRNA expression under CMV promoter and GFP
            reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3861)
  AUTHORS   Campeau E, Ruhl VE, Rodier F, Smith CL, Rahmberg BL, Fuss JO, 
            Campisi J, Yaswen P, Cooper PK, Kaufman PD.
  TITLE     A versatile viral system for expression and depletion of proteins in
            mammalian cells.
  JOURNAL   PLoS One. 2009 Aug 6;4(8):e6529.
  PUBMED    19657394
REFERENCE   2  (bases 1 to 3861)
  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"; journalName: "PLoS One.";
            date: "2009-08-6"; volume: "4(8)"; pages: "e6529"
COMMENT     Sequence Label: pENTR/pSM2(CMV) GFP (w513-1)
FEATURES             Location/Qualifiers
     source          1..3861
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      103..189
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      281..308
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     316..339
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    358..457
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     enhancer        480..783
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        784..987
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     938..958
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     984..1008
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1077..1086
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1083..1085,1086..1088,1089..1802)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1083..1085,1086..1088,1089..1799)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1128..1149)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1389..1408)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1736..1757
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           1839..1932
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           1946..2058
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     protein_bind    complement(2090..2189)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2301..2320)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2312..3121
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVSLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2379..2398)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3211..3799
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3700..3719
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgctagca tggatctcgg
       61 ggacgtctaa ctactaagcg agagtaggga actgccaggc atcaaataaa acgaaaggct
      121 cagtcggaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
      181 aggacaaatc cgccgggagc ggatttgaac gttgtgaagc aacggcccgg agggtggcgg
      241 gcaggacgcc cgccataaac tgccaggcat caaactaagc agaaggccat cctgacggat
      301 ggcctttttg cgtttctaca aactcttcct gttagttagt tacttaagct cgggccccaa
      361 ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga taagcaatgc
      421 ttttttataa tgccaacttt gtacaaaaaa gcaggcttta aaggaacctg ggagttccgc
      481 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      541 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      601 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      661 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      721 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      781 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
      841 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      901 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      961 cggtgggagg tctatataag cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc
     1021 catccacgct gttttgacct ccatagaaga caccgactct agaggatcca ccggtcgcca
     1081 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     1141 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     1201 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     1261 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     1321 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1381 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1441 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1501 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     1561 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     1621 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1681 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     1741 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     1801 aaagcggccg cgtcgactag ggataacagg gtaattgttt gaatgaggct tcagtacttt
     1861 acagaatcgt tgcctgcaca tcttggaaac acttgctggg attacttctt caggttaacc
     1921 caacagaagg ctcgagcaac cagaattcaa ggggctactt taggagcaat tatcttgttt
     1981 actaaaactg aataccttgc tatctctttg atacattttt acaaagctga attaaaatgg
     2041 tataaattaa atcacttttt tcaattggaa gactaatgcg tttatctaga cccagctttc
     2101 ttgtacaaag ttggcattat aagaaagcat tgcttatcaa tttgttgcaa cgaacaggtc
     2161 actatcagtc aaaataaaat cattatttgc catccagctg cagctctggc ccgtgtctca
     2221 aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc
     2281 tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag
     2341 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
     2401 tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt
     2461 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact
     2521 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
     2581 tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga
     2641 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgtccctgc gccggttgca
     2701 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc
     2761 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg
     2821 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc
     2881 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat
     2941 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct
     3001 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg
     3061 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta
     3121 atcagaattg gttaattggt tgtaacatta ttcagattgg gccccgttcc actgagcgtc
     3181 agacccggta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3241 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3301 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     3361 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3421 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     3481 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     3541 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     3601 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     3661 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     3721 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     3781 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     3841 ctggcctttt gctcacatgt t
//
