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