Restriction Map and Cloning Site of the RNAi-Ready pSIREN-DNR Vector. Unique restriction sites are in bold. RNAi-Ready pSIREN-DNR is provided as a linearized vector digested with BamH I and EcoR I. Nucleotides in gray were removed during linearization. This linearized vector is ready for ligation of an appropriate siRNA containing BamH I and EcoR I overhangs. RNAi-Ready pSIREN-DNR is provided as a component of the BD Knockout Adenoviral System 2 (Cat. No. 631529).
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
