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RNAi载体pSIREN-DNR Vector

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2658

Note: The vector sequence file has been compiled from information in the sequence database, published literature, and other sources, together with partial sequences obtained by BD Biosciences Clontech. This vector has not been completely sequenced.

Description

The RNAi-Ready pSIREN-DNR Donor Vector, a component in the BD Knockout™ Adenoviral System 2 (Cat. No. 631529), is designed to express a small interfering ds RNA (siRNA) driven by the human U6 promoter (PU6; RNA Pol III-dependent). RNAi-Ready pSIREN-DNR is provided as a linearized vector digested with BamH I and EcoR I. It is used for targeted gene silencing when an oligonuceotide encoding an appropriate siRNA is ligated into the vector. pSIREN-DNR has been designed as a Donor Vector to transfer the siRNA cassette to the adenoviral Acceptor Vector pLP-Adeno-X-PRLS Viral DNA by Cre/loxP mediated recombination. In addition, recombinant pSIREN-DNR can be used directly in transient transfection experiments to screen siRNA constructs for functionality.

Cre, a 38-kDa recombinase protein from bacteriophage P1, mediates recombination between DNA sequences at specific locations called loxP sites (1, 2). The pSIREN-DNR Vector contains two loxP sites, which flank the 5' end of the siRNA expression cassette and the 5' end of the open reading frame encoding the chloramphenicol resistance gene (Cmr). Once your oligonucleotide is inserted, the resulting Donor Vector construct can be combined with any promoterless Acceptor Vector and Cre recombinase; Cre mediates the transfer of the siRNA cassette to the Acceptor Vector. As a result, a recombinant plasmid is created that expresses your siRNA in the desired host system. The siRNA cassette, once transferred, will become linked to the specific expression elements for which the Acceptor Vector was designed. For a complete list of Acceptor Vectors, visit our web site at localhost/clontech.

This vector also contains an ampicillin resistance gene, which is the marker for propagation and selection of the Donor Vector in E. coli. In addition, pSIREN-DNR contains the sucrase gene from B. subtillis (SacB), which provides negative selection against incorrect recombinants and the parental Donor Vector following recombination.

Use

RNAi-Ready pSIREN-DNR is used for targeted gene silencing when an oligonuceotide encoding an appropriate siRNA is inserted. To construct recombinant pSIREN-DNR, first design, generate, and anneal complementary siRNA oligonucleotides using the protocols in the BD™ Knockout RNAi Systems User Manual (PT3739-1). The annealed oligonucleotide should contain 5'-BamH I and 3'-EcoR I sites. Then ligate the annealed oligonucleotide into RNAi-Ready pSIREN-DNR (provided in the BD Knockout Adenoviral RNAi System 2).

The resulting siRNA cassette can be transferred from pSIREN-DNR to pLP-Adeno-X-PRLS Viral DNA (or any Acceptor Vector) by Cre-mediated recombination as described in the BD Adeno-X Expression Systems 2 User Manual (PT3674-1). Recombinant clones can be selected by growth on media containing chloramphenicol and sucrose. After selection, recombinant plasmids can be further propagated in chloramphenicol or the antibiotic that is appropriate for the resistance marker of the Acceptor Vector.

To introduce the adenoviral siRNA construct into cells, refer to the BD Adeno-X™ Expression System User Manual (PT3414-1).

Location of Features

  • loxP recombination sites: 4834–4867; 902–935
  • Chloramphenicol (Cmr) open reading frame (ORF): 873–214
  • SacB negative selection marker: 944–2854
  • Ampicillin-resistance gene:
  • Start codon: 2990–2992; stop codon: 3848–3850
  • pUC origin of replication: 3998–4641
  • T7 RNA polymerase promoter: 4752–4770
  • Human U6 promoter (PU6): 4877–5133

Primer Locations

  • M13-reverse sequencing primer site: 65–50
    • 5'-AACAGCTATGACCATG-3'
  • M13-forward sequencing primer site: 4726–4742
    • 5'-GTAAAACGACGGCCAGT-3'


  • U6-forward sequencing primer site: 4876–4894
    • 5'-GGGCAGGAAGAGGGCCTAT-3'
      GAATTCTCTA GACCATTCGT TTGGCGCGCG GGCCCAGTAG GTAAGTGAAC ATGGTCATAG
      CTGTTTCCTA GGAGATCCTG GTCATGACTA GTGCTTGGAT TCTCACCAAT AAAAAACGCC
      CGGCGGCAAC CGAGCGTTCT GAACAAATCC AGATGGAGTT CTGAGGTCAT TACTGGATCT
      ATCAACAGGA GTCCAAGCGA GCTCGATATC AAATTACGCC CCGCCCTGCC ACTCATCGCA
      GTACTGTTGT AATTCATTAA GCATTCTGCC GACATGGAAG CCATCACAAA CGGCATGATG
      AACCTGAATC GCCAGCGGCA TCAGCACCTT GTCGCCTTGC GTATAATATT TGCCCATGGT
      GAAAACGGGG GCGAAGAAGT TGTCCATATT GGCCACGTTT AAATCAAAAC TGGTGAAACT
      CACCCAGGGA TTGGCTGAGA CGAAAAACAT ATTCTCAATA AACCCTTTAG GGAAATAGGC
      CAGGTTTTCA CCGTAACACG CCACATCTTG CGAATATATG TGTAGAAACT GCCGGAAATC
      GTCGTGGTAT TCACTCCAGA GCGATGAAAA CGTTTCAGTT TGCTCATGGA AAACGGTGTA
      ACAAGGGTGA ACACTATCCC ATATCACCAG CTCACCGTCT TTCATTGCCA TACGAAATTC
      CGGATGAGCA TTCATCAGGC GGGCAAGAAT GTGAATAAAG GCCGGATAAA ACTTGTGCTT
      ATTTTTCTTT ACGGTCTTTA AAAAGGCCGT AATATCCAGC TGAACGGTCT GGTTATAGGT
      ACATTGAGCA ACTGACTGAA ATGCCTCAAA ATGTTCTTTA CGATGCCATT GGGATATATC
      AACGGTGGTA TATCCAGTGA TTTTTTTCTC CATTTTAGCT TCCTTAGCTC CTGAAAGATC
      CATAACTTCG TATAGCATAC ATTATACGAA GTTATGCGGC CGCGACGTCC ACATATACCT
      GCCGTTCACT ATTATTTAGT GAAATGAGAT ATTATGATAT TTTCTGAATT GTGATTAAAA
      AGGCAACTTT ATGCCCATGC AACAGAAACT ATAAAAAATA CAGAGAATGA AAAGAAACAG
      ATAGATTTTT TAGTTCTTTA GGCCCGTAGT CTGCAAATCC TTTTATGATT TTCTATCAAA
      CAAAAGAGGA AAATAGACCA GTTGCAATCC AAACGAGAGT CTAATAGAAT GAGGTCGAAA
      AGTAAATCGC GCGGGTTTGT TACTGATAAA GCAGGCAAGA CCTAAAATGT GTAAAGGGCA
      AAGTGTATAC TTTGGCGTCA CCCCTTACAT ATTTTAGGTC TTTTTTTATT GTGCGTAACT
      AACTTGCCAT CTTCAAACAG GAGGGCTGGA AGAAGCAGAC CGCTAACACA GTACATAAAA
      AAGGAGACAT GAACGATGAA CATCAAAAAG TTTGCAAAAC AAGCAACAGT ATTAACCTTT
      ACTACCGCAC TGCTGGCAGG AGGCGCAACT CAAGCGTTTG CGAAAGAAAC GAACCAAAAG
      CCATATAAGG AAACATACGG CATTTCCCAT ATTACACGCC ATGATATGCT GCAAATCCCT
      GAACAGCAAA AAAATGAAAA ATATCAAGTT CCTGAGTTCG ATTCGTCCAC AATTAAAAAT
      ATCTCTTCTG CAAAAGGCCT GGACGTTTGG GACAGCTGGC CATTACAAAA CGCTGACGGC
      ACTGTCGCAA ACTATCACGG CTACCACATC GTCTTTGCAT TAGCCGGAGA TCCTAAAAAT
      GCGGATGACA CATCGATTTA CATGTTCTAT CAAAAAGTCG GCGAAACTTC TATTGACAGC
      TGGAAAAACG CTGGCCGCGT CTTTAAAGAC AGCGACAAAT TCGATGCAAA TGATTCTATC
      CTAAAAGACC AAACACAAGA ATGGTCAGGT TCAGCCACAT TTACATCTGA CGGAAAAATC
      CGTTTATTCT ACACTGATTT CTCCGGTAAA CATTACGGCA AACAAACACT GACAACTGCA
      CAAGTTAACG TATCAGCATC AGACAGCTCT TTGAACATCA ACGGTGTAGA GGATTATAAA
      TCAATCTTTG ACGGTGACGG AAAAACGTAT CAAAATGTAC AGCAGTTCAT CGATGAAGGC
      AACTACAGCT CAGGCGACAA CCATACGCTG AGAGATCCTC ACTACGTAGA AGATAAAGGC
      CACAAATACT TAGTATTTGA AGCAAACACT GGAACTGAAG ATGGCTACCA AGGCGAAGAA
      TCTTTATTTA ACAAAGCATA CTATGGCAAA AGCACATCAT TCTTCCGTCA AGAAAGTCAA
      AAACTTCTGC AAAGCGATAA AAAACGCACG GCTGAGTTAG CAAACGGCGC TCTCGGTATG
      ATTGAGCTAA ACGATGATTA CACACTGAAA AAAGTGATGA AACCGCTGAT TGCATCTAAC
      ACAGTAACAG ATGAAATTGA ACGCGCGAAC GTCTTTAAAA TGAACGGCAA ATGGTACCTG
      TTCACTGACT CCCGCGGATC AAAAATGACG ATTGACGGCA TTACGTCTAA CGATATTTAC
      ATGCTTGGTT ATGTTTCTAA TTCTTTAACT GGCCCATACA AGCCGCTGAA CAAAACTGGC
      CTTGTGTTAA AAATGGATCT TGATCCTAAC GATGTAACCT TTACTTACTC ACACTTCGCT
      GTACCTCAAG CGAAAGGAAA CAATGTCGTG ATTACAAGCT ATATGACAAA CAGAGGATTC
      TACGCAGACA AACAATCAAC GTTTGCGCCT AGCTTCCTGC TGAACATCAA AGGCAAGAAA
      ACATCTGTTG TCAAAGACAG CATCCTTGAA CAAGGACAAT TAACAGTTAA CAAATAAAAA
      CGCAAAAGAA AATGCCGATA TCCTATTGGC ATTGACGTCA GGTGGCACTT TTCGGGGAAA
      TGTGCGCGGA ACCCCTATTT GTTTATTTTT CTAAATACAT TCAAATATGT ATCCGCTCAT
      GAGACAATAA CCCTGATAAA TGCTTCAATA ATATTGAAAA AGGAAGAGTA TGAGTATTCA
      ACATTTCCGT GTCGCCCTTA TTCCCTTTTT TGCGGCATTT TGCCTTCCTG TTTTTGCTCA
      CCCAGAAACG CTGGTGAAAG TAAAAGATGC TGAAGATCAG TTGGGTGCAC GAGTGGGTTA
      CATCGAACTG GATCTCAACA GCGGTAAGAT CCTTGAGAGT TTTCGCCCCG AAGAACGTTT
      TCCAATGATG AGCACTTTTA AAGTTCTGCT ATGTGGCGCG GTATTATCCC GTATTGACGC
      CGGGCAAGAG CAACTCGGTC GCCGCATACA CTATTCTCAG AATGACTTGG TTGAGTACTC
      ACCAGTCACA GAAAAGCATC TTACGGATGG CATGACAGTA AGAGAATTAT GCAGTGCTGC
      CATAACCATG AGTGATAACA CTGCGGCCAA CTTACTTCTG ACAACGATCG GAGGACCGAA
      GGAGCTAACC GCTTTTTTGC ACAACATGGG GGATCATGTA ACTCGCCTTG ATCGTTGGGA
      ACCGGAGCTG AATGAAGCCA TACCAAACGA CGAGCGTGAC ACCACGATGC CTGTAGCAAT
      GGCAACAACG TTGCGCAAAC TATTAACTGG CGAACTACTT ACTCTAGCTT CCCGGCAACA
      ATTAATAGAC TGGATGGAGG CGGATAAAGT TGCAGGACCA CTTCTGCGCT CGGCCCTTCC
      GGCTGGCTGG TTTATTGCTG ATAAATCTGG AGCCGGTGAG CGTGGGTCTC GCGGTATCAT
      TGCAGCACTG GGGCCAGATG GTAAGCCCTC CCGTATCGTA GTTATCTACA CGACGGGGAG
      TCAGGCAACT ATGGATGAAC GAAATAGACA GATCGCTGAG ATAGGTGCCT CACTGATTAA
      GCATTGGTAA CTGTCAGACC AAGTTTACTC ATATATACTT TAGATTGATT TAAAACTTCA
      TTTTTAATTT AAAAGGATCT AGGTGAAGAT CCTTTTTGAT AATCTCATGA CCAAAATCCC
      TTAACGTGAG TTTTCGTTCC ACTGAGCGTC AGACCCCGTA GAAAAGATCA AAGGATCTTC
      TTGAGATCCT TTTTTTCTGC GCGTAATCTG CTGCTTGCAA ACAAAAAAAC CACCGCTACC
      AGCGGTGGTT TGTTTGCCGG ATCAAGAGCT ACCAACTCTT TTTCCGAAGG TAACTGGCTT
      CAGCAGAGCG CAGATACCAA ATACTGTTCT TCTAGTGTAG CCGTAGTTAG GCCACCACTT
      CAAGAACTCT GTAGCACCGC CTACATACCT CGCTCTGCTA ATCCTGTTAC CAGTGGCTGC
      TGCCAGTGGC GATAAGTCGT GTCTTACCGG GTTGGACTCA AGACGATAGT TACCGGATAA
      GGCGCAGCGG TCGGGCTGAA CGGGGGGTTC GTGCACACAG CCCAGCTTGG AGCGAACGAC
      CTACACCGAA CTGAGATACC TACAGCGTGA GCTATGAGAA AGCGCCACGC TTCCCGAAGG
      GAGAAAGGCG GACAGGTATC CGGTAAGCGG CAGGGTCGGA ACAGGAGAGC GCACGAGGGA
      GCTTCCAGGG GGAAACGCCT GGTATCTTTA TAGTCCTGTC GGGTTTCGCC ACCTCTGACT
      TGAGCGTCGA TTTTTGTGAT GCTCGTCAGG GGGGCGGAGC CTATGGAAAA ACGCCAGCAA
      CGCGGCCTTT TTACGGTTCC TGGCCTTTTG CTGGCCTTTT GCTCACATGT TCTTTCCTGC
      GTTATCCCCT GATTCTGTGG ATAACCGTAT TACCGCCTTA CGCGTGTAAA ACGACGGCCA
      GTAGATCTGT AATACGACTC ACTATAGGGC GCTAGCTGCT CGCCGCAGCC GAACGACCGA
      GCGCAGCGAG TCAGTGAGCG AGGAAGCGGC CGCATAACTT CGTATAGCAT ACATTATACG
      AAGTTATCAG TCGACGGGCA GGAAGAGGGC CTATTTCCCA TGATTCCTTC ATATTTGCAT
      ATACGATACA AGGCTGTTAG AGAGATAATT AGAATTAATT TGACTGTAAA CACAAAGATA
      TTAGTACAAA ATACGTGACG TAGAAAGTAA TAATTTCTTG GGTAGTTTGC AGTTTTAAAA
      TTATGTTTTA AAATGGACTA TCATATGCTT ACCGTAACTT GAAAGTATTT CGATTTCTTG
      GCTTTATATA TCTTGTGGAA AGGACGAGGA TCC
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