LOCUS       sequence-8566-ex        3043 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry vector for miRNA expression under U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3043)
  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 3043)
  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(U6) (w51-1)
FEATURES             Location/Qualifiers
     source          1..3043
                     /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"
     promoter        717..966
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     717..737
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     889..908
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     ncRNA           1021..1114
                     /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           1128..1240
                     /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(1272..1371)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1483..1502)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1494..2303
                     /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(1561..1580)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2393..2981
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2882..2901
                     /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 aaggaaccaa ttcagtcgac
      481 tggatccccc cgagtccaac acccgtggga atcccatggg caccatggcc cctcgctcca
      541 aaaatgcttt cgcgtctcgc agacactgct cggtagtttc ggggatcagc gtttgagtaa
      601 gagcccgcgt ctgaaccctc cgcgccgccc cggcccagtg gaaagacgcg caggcaaaac
      661 gcaccacgtg acggagcgtg accgcgcgcc gagcgcgcgc caaggtcggg caggaagagg
      721 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
      781 ttagaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
      841 aataatttct tgggtagttt gcagttttta aaattatgtt ttaaaatgga ctatcatatg
      901 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
      961 aacaccgtgc tcgcttcggc agcacatata ctagtcgact agggataaca gggtaattgt
     1021 ttgaatgagg cttcagtact ttacagaatc gttgcctgca catcttggaa acacttgctg
     1081 ggattacttc ttcaggttaa cccaacagaa ggctcgagca accagaattc aaggggctac
     1141 tttaggagca attatcttgt ttactaaaac tgaatacctt gctatctctt tgatacattt
     1201 ttacaaagct gaattaaaat ggtataaatt aaatcacttt tttcaattgg aagactaatg
     1261 cgtttatcta gacccagctt tcttgtacaa agttggcatt ataagaaagc attgcttatc
     1321 aatttgttgc aacgaacagg tcactatcag tcaaaataaa atcattattt gccatccagc
     1381 tgcagctctg gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat
     1441 catcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc
     1501 atattcaacg ggaaacgtcg aggccgcgat taaattccaa catggatgct gatttatatg
     1561 ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg
     1621 ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg
     1681 ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca
     1741 agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccggaaaaa
     1801 cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg
     1861 cagtgtccct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc
     1921 gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg
     1981 attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac
     2041 ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta
     2101 tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
     2161 gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
     2221 aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt
     2281 tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacat tattcagatt
     2341 gggccccgtt ccactgagcg tcagacccgg tagaaaagat caaaggatct tcttgagatc
     2401 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2461 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2521 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2581 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2641 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     2701 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     2761 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     2821 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     2881 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     2941 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3001 ttttacggtt cctggccttt tgctggcctt ttgctcacat gtt
//
