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  • 99
    New England Biolabs bsmbi
    Construction of the mutant libraries. (A) A schematic representation of the fitness profiling experiment is shown. A 240 bp insert was generated by error-prone PCR and <t>BsaI</t> digestion. The corresponding vector was generated by high-fidelity PCR and <t>BsmBI</t> digestion. Each of the nine plasmid libraries in this study consist of ∼ 50,000 clones. Each viral mutant library was rescued by transfecting ∼ 35 million 293T cells. Each infection was performed with ∼ 10 million A549 cells. (B) A schematic representation of the sequencing library preparation is shown. DNA plasmid mutant library or viral cDNA was used for PCR. This PCR amplified the 240 bp randomized region. The amplicon product was then digested with BpmI, end-repaired, dA-tailed, ligated to sequencing adapters, and sequenced using the Illumina MiSeq platform. BpmI digestion removed the primer region in the amplicon PCR, resulting in sequencing reads covering only the barcode for multiplex sequencing and the 240 bp region that was randomized in the mutant library. With this experimental design, the number of mutations carried by individual genomes in the mutant libraries could be precisely determined.
    Bsmbi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Fisher Scientific bsmbi
    Construction of the mutant libraries. (A) A schematic representation of the fitness profiling experiment is shown. A 240 bp insert was generated by error-prone PCR and <t>BsaI</t> digestion. The corresponding vector was generated by high-fidelity PCR and <t>BsmBI</t> digestion. Each of the nine plasmid libraries in this study consist of ∼ 50,000 clones. Each viral mutant library was rescued by transfecting ∼ 35 million 293T cells. Each infection was performed with ∼ 10 million A549 cells. (B) A schematic representation of the sequencing library preparation is shown. DNA plasmid mutant library or viral cDNA was used for PCR. This PCR amplified the 240 bp randomized region. The amplicon product was then digested with BpmI, end-repaired, dA-tailed, ligated to sequencing adapters, and sequenced using the Illumina MiSeq platform. BpmI digestion removed the primer region in the amplicon PCR, resulting in sequencing reads covering only the barcode for multiplex sequencing and the 240 bp region that was randomized in the mutant library. With this experimental design, the number of mutations carried by individual genomes in the mutant libraries could be precisely determined.
    Bsmbi, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher bsmbi
    Targeted mutagenesis using the CRISPR/Cas9 system in transient expression in N. benthamiana. a Schematic representation of the structure of Niben101Scf04205Ctg025 (XT1) and Niben101Scf04551Ctg021 (XT2) (exons in grey , introns in white ) with the sequences of the target sites. Diagnostic restriction sites are underlined and the PAM sequence is shown in bold . b Comparison of the mutation efficiency of hCas9 and pcoCas9 targeting the XT2. Red arrow shows SpeI resistant PCR fragments only visible on the gRNA and hCas9 combination. c PCR/RE assay to detect simultaneous targeted mutations on XT1 and XT2. Red arrows show <t>BsmBI</t> and SpeI resistant PCR fragments amplified from N. benthamiana genomic <t>DNA.</t> d Alignment of XT1 and XT2 sequences obtained from different clones of uncleaved bands (see c ). XT1 target site appears in blue and XT2 target site in green . Red letters and dashes indicate insertions and deletions respectively
    Bsmbi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 419 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher bsmbi fastdigest
    Targeted mutagenesis using the CRISPR/Cas9 system in transient expression in N. benthamiana. a Schematic representation of the structure of Niben101Scf04205Ctg025 (XT1) and Niben101Scf04551Ctg021 (XT2) (exons in grey , introns in white ) with the sequences of the target sites. Diagnostic restriction sites are underlined and the PAM sequence is shown in bold . b Comparison of the mutation efficiency of hCas9 and pcoCas9 targeting the XT2. Red arrow shows SpeI resistant PCR fragments only visible on the gRNA and hCas9 combination. c PCR/RE assay to detect simultaneous targeted mutations on XT1 and XT2. Red arrows show <t>BsmBI</t> and SpeI resistant PCR fragments amplified from N. benthamiana genomic <t>DNA.</t> d Alignment of XT1 and XT2 sequences obtained from different clones of uncleaved bands (see c ). XT1 target site appears in blue and XT2 target site in green . Red letters and dashes indicate insertions and deletions respectively
    Bsmbi Fastdigest, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Addgene inc bsmbi site
    Targeted mutagenesis using the CRISPR/Cas9 system in transient expression in N. benthamiana. a Schematic representation of the structure of Niben101Scf04205Ctg025 (XT1) and Niben101Scf04551Ctg021 (XT2) (exons in grey , introns in white ) with the sequences of the target sites. Diagnostic restriction sites are underlined and the PAM sequence is shown in bold . b Comparison of the mutation efficiency of hCas9 and pcoCas9 targeting the XT2. Red arrow shows SpeI resistant PCR fragments only visible on the gRNA and hCas9 combination. c PCR/RE assay to detect simultaneous targeted mutations on XT1 and XT2. Red arrows show <t>BsmBI</t> and SpeI resistant PCR fragments amplified from N. benthamiana genomic <t>DNA.</t> d Alignment of XT1 and XT2 sequences obtained from different clones of uncleaved bands (see c ). XT1 target site appears in blue and XT2 target site in green . Red letters and dashes indicate insertions and deletions respectively
    Bsmbi Site, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 242 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Fisher Scientific bsmbi fastdigest
    Targeted mutagenesis using the CRISPR/Cas9 system in transient expression in N. benthamiana. a Schematic representation of the structure of Niben101Scf04205Ctg025 (XT1) and Niben101Scf04551Ctg021 (XT2) (exons in grey , introns in white ) with the sequences of the target sites. Diagnostic restriction sites are underlined and the PAM sequence is shown in bold . b Comparison of the mutation efficiency of hCas9 and pcoCas9 targeting the XT2. Red arrow shows SpeI resistant PCR fragments only visible on the gRNA and hCas9 combination. c PCR/RE assay to detect simultaneous targeted mutations on XT1 and XT2. Red arrows show <t>BsmBI</t> and SpeI resistant PCR fragments amplified from N. benthamiana genomic <t>DNA.</t> d Alignment of XT1 and XT2 sequences obtained from different clones of uncleaved bands (see c ). XT1 target site appears in blue and XT2 target site in green . Red letters and dashes indicate insertions and deletions respectively
    Bsmbi Fastdigest, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 87/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Addgene inc bsmbi bsmbi sites
    Procedure to make a template clone ( a ) In the original clone, two unique sites recognized by two different type II restriction enzymes (labeled as TII-e1 and TII-e2) are selected in the gene of interest. The number x amino acid encoded by N 2 N 3 N 4 (in red) is indicated by aa x , or next amino acid by aa x+1 (in green). A template fragment contains a TIIS DNA cassette (highlighted in blue box) in between aa x and aa x+1 . All the cutting positions are indicated by arrows and complementary bases by apostrophe (’). ( b ) <t>pBS-KSII</t> contains a multiple cloning site (MCS) and a BsaI in the ampicillin (Amp) coding gene, which was destroyed by site-directed mutagenesis to create pBS-KSII-4B. Wild-type pBS-KSII was linearized by BsaI and two overlapping fragments were released by BsaI and NaeI double digestion. pBS-KSII-4B does not have BsaI, BbsI, <t>BsmBI,</t> and BfuAI/BspMI and can be linearized by NaeI. Three brighter bands from GeneRuler 1kb plus DNA marker are labeled.
    Bsmbi Bsmbi Sites, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher bsmbi restriction enzyme
    Procedure to make a template clone ( a ) In the original clone, two unique sites recognized by two different type II restriction enzymes (labeled as TII-e1 and TII-e2) are selected in the gene of interest. The number x amino acid encoded by N 2 N 3 N 4 (in red) is indicated by aa x , or next amino acid by aa x+1 (in green). A template fragment contains a TIIS DNA cassette (highlighted in blue box) in between aa x and aa x+1 . All the cutting positions are indicated by arrows and complementary bases by apostrophe (’). ( b ) <t>pBS-KSII</t> contains a multiple cloning site (MCS) and a BsaI in the ampicillin (Amp) coding gene, which was destroyed by site-directed mutagenesis to create pBS-KSII-4B. Wild-type pBS-KSII was linearized by BsaI and two overlapping fragments were released by BsaI and NaeI double digestion. pBS-KSII-4B does not have BsaI, BbsI, <t>BsmBI,</t> and BfuAI/BspMI and can be linearized by NaeI. Three brighter bands from GeneRuler 1kb plus DNA marker are labeled.
    Bsmbi Restriction Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs esp3i bsmbi
    Procedure to make a template clone ( a ) In the original clone, two unique sites recognized by two different type II restriction enzymes (labeled as TII-e1 and TII-e2) are selected in the gene of interest. The number x amino acid encoded by N 2 N 3 N 4 (in red) is indicated by aa x , or next amino acid by aa x+1 (in green). A template fragment contains a TIIS DNA cassette (highlighted in blue box) in between aa x and aa x+1 . All the cutting positions are indicated by arrows and complementary bases by apostrophe (’). ( b ) <t>pBS-KSII</t> contains a multiple cloning site (MCS) and a BsaI in the ampicillin (Amp) coding gene, which was destroyed by site-directed mutagenesis to create pBS-KSII-4B. Wild-type pBS-KSII was linearized by BsaI and two overlapping fragments were released by BsaI and NaeI double digestion. pBS-KSII-4B does not have BsaI, BbsI, <t>BsmBI,</t> and BfuAI/BspMI and can be linearized by NaeI. Three brighter bands from GeneRuler 1kb plus DNA marker are labeled.
    Esp3i Bsmbi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Addgene inc bsmbi digested lentiguide puro
    Procedure to make a template clone ( a ) In the original clone, two unique sites recognized by two different type II restriction enzymes (labeled as TII-e1 and TII-e2) are selected in the gene of interest. The number x amino acid encoded by N 2 N 3 N 4 (in red) is indicated by aa x , or next amino acid by aa x+1 (in green). A template fragment contains a TIIS DNA cassette (highlighted in blue box) in between aa x and aa x+1 . All the cutting positions are indicated by arrows and complementary bases by apostrophe (’). ( b ) <t>pBS-KSII</t> contains a multiple cloning site (MCS) and a BsaI in the ampicillin (Amp) coding gene, which was destroyed by site-directed mutagenesis to create pBS-KSII-4B. Wild-type pBS-KSII was linearized by BsaI and two overlapping fragments were released by BsaI and NaeI double digestion. pBS-KSII-4B does not have BsaI, BbsI, <t>BsmBI,</t> and BfuAI/BspMI and can be linearized by NaeI. Three brighter bands from GeneRuler 1kb plus DNA marker are labeled.
    Bsmbi Digested Lentiguide Puro, supplied by Addgene inc, used in various techniques. Bioz Stars score: 95/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega bsmbi ligation strategy
    Procedure to make a template clone ( a ) In the original clone, two unique sites recognized by two different type II restriction enzymes (labeled as TII-e1 and TII-e2) are selected in the gene of interest. The number x amino acid encoded by N 2 N 3 N 4 (in red) is indicated by aa x , or next amino acid by aa x+1 (in green). A template fragment contains a TIIS DNA cassette (highlighted in blue box) in between aa x and aa x+1 . All the cutting positions are indicated by arrows and complementary bases by apostrophe (’). ( b ) <t>pBS-KSII</t> contains a multiple cloning site (MCS) and a BsaI in the ampicillin (Amp) coding gene, which was destroyed by site-directed mutagenesis to create pBS-KSII-4B. Wild-type pBS-KSII was linearized by BsaI and two overlapping fragments were released by BsaI and NaeI double digestion. pBS-KSII-4B does not have BsaI, BbsI, <t>BsmBI,</t> and BfuAI/BspMI and can be linearized by NaeI. Three brighter bands from GeneRuler 1kb plus DNA marker are labeled.
    Bsmbi Ligation Strategy, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher fastdigest bsmbi restriction enzyme
    Procedure to make a template clone ( a ) In the original clone, two unique sites recognized by two different type II restriction enzymes (labeled as TII-e1 and TII-e2) are selected in the gene of interest. The number x amino acid encoded by N 2 N 3 N 4 (in red) is indicated by aa x , or next amino acid by aa x+1 (in green). A template fragment contains a TIIS DNA cassette (highlighted in blue box) in between aa x and aa x+1 . All the cutting positions are indicated by arrows and complementary bases by apostrophe (’). ( b ) <t>pBS-KSII</t> contains a multiple cloning site (MCS) and a BsaI in the ampicillin (Amp) coding gene, which was destroyed by site-directed mutagenesis to create pBS-KSII-4B. Wild-type pBS-KSII was linearized by BsaI and two overlapping fragments were released by BsaI and NaeI double digestion. pBS-KSII-4B does not have BsaI, BbsI, <t>BsmBI,</t> and BfuAI/BspMI and can be linearized by NaeI. Three brighter bands from GeneRuler 1kb plus DNA marker are labeled.
    Fastdigest Bsmbi Restriction Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Addgene inc bsmbi linearized pt7 grna
    Procedure to make a template clone ( a ) In the original clone, two unique sites recognized by two different type II restriction enzymes (labeled as TII-e1 and TII-e2) are selected in the gene of interest. The number x amino acid encoded by N 2 N 3 N 4 (in red) is indicated by aa x , or next amino acid by aa x+1 (in green). A template fragment contains a TIIS DNA cassette (highlighted in blue box) in between aa x and aa x+1 . All the cutting positions are indicated by arrows and complementary bases by apostrophe (’). ( b ) <t>pBS-KSII</t> contains a multiple cloning site (MCS) and a BsaI in the ampicillin (Amp) coding gene, which was destroyed by site-directed mutagenesis to create pBS-KSII-4B. Wild-type pBS-KSII was linearized by BsaI and two overlapping fragments were released by BsaI and NaeI double digestion. pBS-KSII-4B does not have BsaI, BbsI, <t>BsmBI,</t> and BfuAI/BspMI and can be linearized by NaeI. Three brighter bands from GeneRuler 1kb plus DNA marker are labeled.
    Bsmbi Linearized Pt7 Grna, supplied by Addgene inc, used in various techniques. Bioz Stars score: 95/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Addgene inc plenti sp bsmbi sgrna hygro
    Procedure to make a template clone ( a ) In the original clone, two unique sites recognized by two different type II restriction enzymes (labeled as TII-e1 and TII-e2) are selected in the gene of interest. The number x amino acid encoded by N 2 N 3 N 4 (in red) is indicated by aa x , or next amino acid by aa x+1 (in green). A template fragment contains a TIIS DNA cassette (highlighted in blue box) in between aa x and aa x+1 . All the cutting positions are indicated by arrows and complementary bases by apostrophe (’). ( b ) <t>pBS-KSII</t> contains a multiple cloning site (MCS) and a BsaI in the ampicillin (Amp) coding gene, which was destroyed by site-directed mutagenesis to create pBS-KSII-4B. Wild-type pBS-KSII was linearized by BsaI and two overlapping fragments were released by BsaI and NaeI double digestion. pBS-KSII-4B does not have BsaI, BbsI, <t>BsmBI,</t> and BfuAI/BspMI and can be linearized by NaeI. Three brighter bands from GeneRuler 1kb plus DNA marker are labeled.
    Plenti Sp Bsmbi Sgrna Hygro, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Addgene inc bsmbi digested lenticrisprv2 plasmid
    Procedure to make a template clone ( a ) In the original clone, two unique sites recognized by two different type II restriction enzymes (labeled as TII-e1 and TII-e2) are selected in the gene of interest. The number x amino acid encoded by N 2 N 3 N 4 (in red) is indicated by aa x , or next amino acid by aa x+1 (in green). A template fragment contains a TIIS DNA cassette (highlighted in blue box) in between aa x and aa x+1 . All the cutting positions are indicated by arrows and complementary bases by apostrophe (’). ( b ) <t>pBS-KSII</t> contains a multiple cloning site (MCS) and a BsaI in the ampicillin (Amp) coding gene, which was destroyed by site-directed mutagenesis to create pBS-KSII-4B. Wild-type pBS-KSII was linearized by BsaI and two overlapping fragments were released by BsaI and NaeI double digestion. pBS-KSII-4B does not have BsaI, BbsI, <t>BsmBI,</t> and BfuAI/BspMI and can be linearized by NaeI. Three brighter bands from GeneRuler 1kb plus DNA marker are labeled.
    Bsmbi Digested Lenticrisprv2 Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher bsmbi digestion enzyme
    CRISPR vector design for knockout of A 2A R in K562 Cells. ( a ) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A 2A R gene. Additional arrowheads indicate genes with similar regions to the A 2A R gene, including the A 2B R gene on chromosome 17. ( b ) Left, guide-RNAs found for the A 2A R gene. Right, guide sequence #1 was selected for insertion into the <t>plentiCRISPR</t> loxP vector plasmid DNA. <t>BsmBI</t> cut sites were added to gRNA #1 to facilitate insertion. ( c ) Top, Cas9 cut site in the A 2A R gene. Bottom, map of the plentiCRISPR construct. ( d ) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.
    Bsmbi Digestion Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Addgene inc bsmbi restricted lenticrisprv1 plasmid
    CRISPR vector design for knockout of A 2A R in K562 Cells. ( a ) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A 2A R gene. Additional arrowheads indicate genes with similar regions to the A 2A R gene, including the A 2B R gene on chromosome 17. ( b ) Left, guide-RNAs found for the A 2A R gene. Right, guide sequence #1 was selected for insertion into the <t>plentiCRISPR</t> loxP vector plasmid DNA. <t>BsmBI</t> cut sites were added to gRNA #1 to facilitate insertion. ( c ) Top, Cas9 cut site in the A 2A R gene. Bottom, map of the plentiCRISPR construct. ( d ) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.
    Bsmbi Restricted Lenticrisprv1 Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher type ii restriction enzyme bsmbi
    CRISPR vector design for knockout of A 2A R in K562 Cells. ( a ) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A 2A R gene. Additional arrowheads indicate genes with similar regions to the A 2A R gene, including the A 2B R gene on chromosome 17. ( b ) Left, guide-RNAs found for the A 2A R gene. Right, guide sequence #1 was selected for insertion into the <t>plentiCRISPR</t> loxP vector plasmid DNA. <t>BsmBI</t> cut sites were added to gRNA #1 to facilitate insertion. ( c ) Top, Cas9 cut site in the A 2A R gene. Bottom, map of the plentiCRISPR construct. ( d ) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.
    Type Ii Restriction Enzyme Bsmbi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Addgene inc bsmbi digested lenticrispr v2 vector
    CRISPR vector design for knockout of A 2A R in K562 Cells. ( a ) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A 2A R gene. Additional arrowheads indicate genes with similar regions to the A 2A R gene, including the A 2B R gene on chromosome 17. ( b ) Left, guide-RNAs found for the A 2A R gene. Right, guide sequence #1 was selected for insertion into the <t>plentiCRISPR</t> loxP vector plasmid DNA. <t>BsmBI</t> cut sites were added to gRNA #1 to facilitate insertion. ( c ) Top, Cas9 cut site in the A 2A R gene. Bottom, map of the plentiCRISPR construct. ( d ) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.
    Bsmbi Digested Lenticrispr V2 Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc bsmbi digested plenti crisprv2 vector
    CRISPR vector design for knockout of A 2A R in K562 Cells. ( a ) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A 2A R gene. Additional arrowheads indicate genes with similar regions to the A 2A R gene, including the A 2B R gene on chromosome 17. ( b ) Left, guide-RNAs found for the A 2A R gene. Right, guide sequence #1 was selected for insertion into the <t>plentiCRISPR</t> loxP vector plasmid DNA. <t>BsmBI</t> cut sites were added to gRNA #1 to facilitate insertion. ( c ) Top, Cas9 cut site in the A 2A R gene. Bottom, map of the plentiCRISPR construct. ( d ) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.
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    CRISPR vector design for knockout of A 2A R in K562 Cells. ( a ) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A 2A R gene. Additional arrowheads indicate genes with similar regions to the A 2A R gene, including the A 2B R gene on chromosome 17. ( b ) Left, guide-RNAs found for the A 2A R gene. Right, guide sequence #1 was selected for insertion into the <t>plentiCRISPR</t> loxP vector plasmid DNA. <t>BsmBI</t> cut sites were added to gRNA #1 to facilitate insertion. ( c ) Top, Cas9 cut site in the A 2A R gene. Bottom, map of the plentiCRISPR construct. ( d ) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.
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    CRISPR vector design for knockout of A 2A R in K562 Cells. ( a ) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A 2A R gene. Additional arrowheads indicate genes with similar regions to the A 2A R gene, including the A 2B R gene on chromosome 17. ( b ) Left, guide-RNAs found for the A 2A R gene. Right, guide sequence #1 was selected for insertion into the <t>plentiCRISPR</t> loxP vector plasmid DNA. <t>BsmBI</t> cut sites were added to gRNA #1 to facilitate insertion. ( c ) Top, Cas9 cut site in the A 2A R gene. Bottom, map of the plentiCRISPR construct. ( d ) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.
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    CRISPR vector design for knockout of A 2A R in K562 Cells. ( a ) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A 2A R gene. Additional arrowheads indicate genes with similar regions to the A 2A R gene, including the A 2B R gene on chromosome 17. ( b ) Left, guide-RNAs found for the A 2A R gene. Right, guide sequence #1 was selected for insertion into the <t>plentiCRISPR</t> loxP vector plasmid DNA. <t>BsmBI</t> cut sites were added to gRNA #1 to facilitate insertion. ( c ) Top, Cas9 cut site in the A 2A R gene. Bottom, map of the plentiCRISPR construct. ( d ) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.
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    CRISPR vector design for knockout of A 2A R in K562 Cells. ( a ) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A 2A R gene. Additional arrowheads indicate genes with similar regions to the A 2A R gene, including the A 2B R gene on chromosome 17. ( b ) Left, guide-RNAs found for the A 2A R gene. Right, guide sequence #1 was selected for insertion into the <t>plentiCRISPR</t> loxP vector plasmid DNA. <t>BsmBI</t> cut sites were added to gRNA #1 to facilitate insertion. ( c ) Top, Cas9 cut site in the A 2A R gene. Bottom, map of the plentiCRISPR construct. ( d ) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.
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    CRISPR vector design for knockout of A 2A R in K562 Cells. ( a ) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A 2A R gene. Additional arrowheads indicate genes with similar regions to the A 2A R gene, including the A 2B R gene on chromosome 17. ( b ) Left, guide-RNAs found for the A 2A R gene. Right, guide sequence #1 was selected for insertion into the <t>plentiCRISPR</t> loxP vector plasmid DNA. <t>BsmBI</t> cut sites were added to gRNA #1 to facilitate insertion. ( c ) Top, Cas9 cut site in the A 2A R gene. Bottom, map of the plentiCRISPR construct. ( d ) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.
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    Addgene inc bsmbi digested pmlm3636 vector
    CRISPR vector design for knockout of A 2A R in K562 Cells. ( a ) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A 2A R gene. Additional arrowheads indicate genes with similar regions to the A 2A R gene, including the A 2B R gene on chromosome 17. ( b ) Left, guide-RNAs found for the A 2A R gene. Right, guide sequence #1 was selected for insertion into the <t>plentiCRISPR</t> loxP vector plasmid DNA. <t>BsmBI</t> cut sites were added to gRNA #1 to facilitate insertion. ( c ) Top, Cas9 cut site in the A 2A R gene. Bottom, map of the plentiCRISPR construct. ( d ) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.
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    CRISPR vector design for knockout of A 2A R in K562 Cells. ( a ) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A 2A R gene. Additional arrowheads indicate genes with similar regions to the A 2A R gene, including the A 2B R gene on chromosome 17. ( b ) Left, guide-RNAs found for the A 2A R gene. Right, guide sequence #1 was selected for insertion into the <t>plentiCRISPR</t> loxP vector plasmid DNA. <t>BsmBI</t> cut sites were added to gRNA #1 to facilitate insertion. ( c ) Top, Cas9 cut site in the A 2A R gene. Bottom, map of the plentiCRISPR construct. ( d ) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.
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    Addgene inc plenti u6 sp sgrna bsmbi hygro cloning vector
    CRISPR vector design for knockout of A 2A R in K562 Cells. ( a ) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A 2A R gene. Additional arrowheads indicate genes with similar regions to the A 2A R gene, including the A 2B R gene on chromosome 17. ( b ) Left, guide-RNAs found for the A 2A R gene. Right, guide sequence #1 was selected for insertion into the <t>plentiCRISPR</t> loxP vector plasmid DNA. <t>BsmBI</t> cut sites were added to gRNA #1 to facilitate insertion. ( c ) Top, Cas9 cut site in the A 2A R gene. Bottom, map of the plentiCRISPR construct. ( d ) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.
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    Addgene inc bsmbi digested csy4 flanked grna backbone
    RNA-guided FokI nucleases and a <t>Csy4-based</t> multiplex <t>gRNA</t> expression system (a) Schematic overview of RNA-guided FokI nucleases. Two FokI-dCas9 fusion proteins are recruited to adjacent target sites by two different gRNAs in order to facilitate FokI dimerization and DNA cleavage. (b) Schematic overview of a Csy4-based multiplex gRNA expression system. Two gRNAs (with any 5′ end nucleotide) are co-expressed in a single transcript from a U6 promoter with each gRNA flanked by Csy4 recognition sites. Csy4 cleaves and releases gRNAs from the transcript. The Csy4 recognition site remains at the 3′ end of the gRNA with a Csy4 nuclease bound to that site. (c) Comparison of the activities of Cas9 co-expressed with either Csy4-processed single gRNAs (blue bars) or single gRNAs expressed from a standard U6 promoter (red bars) in the U2OS.EGFP disruption assay. The target sequences in EGFP gene for the three different single gRNAs tested are provided in the table to the right of the graph. Error bars represent s.e.m, n= 3. (d) Validation of the multiplex, Csy4-based system. Two gRNAs targeted to adjacent sites in EGFP were expressed in a single RNA transcript using the Csy4-based system in human U2OS.EGFP cells together with Csy4 and wild-type Cas9 nuclease. Sequences of indel mutations induced in these cells are shown. The wild-type sequence is shown at the top with both target sites highlighted in yellow and PAM sequences shown as red, underlined text. Deletions are indicated by red dashes against gray background and insertions by lowercase letters against a light blue background. To the right of each sequence, the sizes of insertions (+) or deletions (Δ) are specified.
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    Addgene inc nme cas9 expressing plasmid psimple u6 tracr u6 bsmbi nls nmcas9 ha nls
    RNA-guided FokI nucleases and a <t>Csy4-based</t> multiplex <t>gRNA</t> expression system (a) Schematic overview of RNA-guided FokI nucleases. Two FokI-dCas9 fusion proteins are recruited to adjacent target sites by two different gRNAs in order to facilitate FokI dimerization and DNA cleavage. (b) Schematic overview of a Csy4-based multiplex gRNA expression system. Two gRNAs (with any 5′ end nucleotide) are co-expressed in a single transcript from a U6 promoter with each gRNA flanked by Csy4 recognition sites. Csy4 cleaves and releases gRNAs from the transcript. The Csy4 recognition site remains at the 3′ end of the gRNA with a Csy4 nuclease bound to that site. (c) Comparison of the activities of Cas9 co-expressed with either Csy4-processed single gRNAs (blue bars) or single gRNAs expressed from a standard U6 promoter (red bars) in the U2OS.EGFP disruption assay. The target sequences in EGFP gene for the three different single gRNAs tested are provided in the table to the right of the graph. Error bars represent s.e.m, n= 3. (d) Validation of the multiplex, Csy4-based system. Two gRNAs targeted to adjacent sites in EGFP were expressed in a single RNA transcript using the Csy4-based system in human U2OS.EGFP cells together with Csy4 and wild-type Cas9 nuclease. Sequences of indel mutations induced in these cells are shown. The wild-type sequence is shown at the top with both target sites highlighted in yellow and PAM sequences shown as red, underlined text. Deletions are indicated by red dashes against gray background and insertions by lowercase letters against a light blue background. To the right of each sequence, the sizes of insertions (+) or deletions (Δ) are specified.
    Nme Cas9 Expressing Plasmid Psimple U6 Tracr U6 Bsmbi Nls Nmcas9 Ha Nls, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Construction of the mutant libraries. (A) A schematic representation of the fitness profiling experiment is shown. A 240 bp insert was generated by error-prone PCR and BsaI digestion. The corresponding vector was generated by high-fidelity PCR and BsmBI digestion. Each of the nine plasmid libraries in this study consist of ∼ 50,000 clones. Each viral mutant library was rescued by transfecting ∼ 35 million 293T cells. Each infection was performed with ∼ 10 million A549 cells. (B) A schematic representation of the sequencing library preparation is shown. DNA plasmid mutant library or viral cDNA was used for PCR. This PCR amplified the 240 bp randomized region. The amplicon product was then digested with BpmI, end-repaired, dA-tailed, ligated to sequencing adapters, and sequenced using the Illumina MiSeq platform. BpmI digestion removed the primer region in the amplicon PCR, resulting in sequencing reads covering only the barcode for multiplex sequencing and the 240 bp region that was randomized in the mutant library. With this experimental design, the number of mutations carried by individual genomes in the mutant libraries could be precisely determined.

    Journal: PLoS Genetics

    Article Title: Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality

    doi: 10.1371/journal.pgen.1005310

    Figure Lengend Snippet: Construction of the mutant libraries. (A) A schematic representation of the fitness profiling experiment is shown. A 240 bp insert was generated by error-prone PCR and BsaI digestion. The corresponding vector was generated by high-fidelity PCR and BsmBI digestion. Each of the nine plasmid libraries in this study consist of ∼ 50,000 clones. Each viral mutant library was rescued by transfecting ∼ 35 million 293T cells. Each infection was performed with ∼ 10 million A549 cells. (B) A schematic representation of the sequencing library preparation is shown. DNA plasmid mutant library or viral cDNA was used for PCR. This PCR amplified the 240 bp randomized region. The amplicon product was then digested with BpmI, end-repaired, dA-tailed, ligated to sequencing adapters, and sequenced using the Illumina MiSeq platform. BpmI digestion removed the primer region in the amplicon PCR, resulting in sequencing reads covering only the barcode for multiplex sequencing and the 240 bp region that was randomized in the mutant library. With this experimental design, the number of mutations carried by individual genomes in the mutant libraries could be precisely determined.

    Article Snippet: The insert was then digested by BsaI (New England Biolabs, Ipswich, MA), whereas the vector was digested by BsmBI (New England Biolabs).

    Techniques: Mutagenesis, Generated, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay, Infection, Sequencing, Amplification, Multiplex Assay

    gRNA library construction using a semi-random primer. ( A ) Semi-random primer. Poly(A), polyadenylate. ( B ) Type III and type IIS restriction sites to cut out the 20-bp guide sequence. Ec, Eco P15I; Ac, Acu I. ( C ) Scheme of gRNA library construction. Bg, Bgl II; Xb, Xba I; Bs, Bsm BI; Aa, Aat II. PCR, polymerase chain reaction; lentiCRISPR v2, lentiCRISPR version 2. ( D ) Short-range PCR for PCR cycle optimization and size fractionation of the guide sequence. PCR products were run on 20% polyacrylamide gels. A 10-bp ladder was used as the size marker. Bands of the expected sizes are marked by triangles.

    Journal: Science Advances

    Article Title: A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism

    doi: 10.1126/sciadv.1600699

    Figure Lengend Snippet: gRNA library construction using a semi-random primer. ( A ) Semi-random primer. Poly(A), polyadenylate. ( B ) Type III and type IIS restriction sites to cut out the 20-bp guide sequence. Ec, Eco P15I; Ac, Acu I. ( C ) Scheme of gRNA library construction. Bg, Bgl II; Xb, Xba I; Bs, Bsm BI; Aa, Aat II. PCR, polymerase chain reaction; lentiCRISPR v2, lentiCRISPR version 2. ( D ) Short-range PCR for PCR cycle optimization and size fractionation of the guide sequence. PCR products were run on 20% polyacrylamide gels. A 10-bp ladder was used as the size marker. Bands of the expected sizes are marked by triangles.

    Article Snippet: Bsm BI/Aat II digestion The PCR product was digested with 10 μl of Bsm BI (10 U/μl; NEB) in 1× NEBuffer 3.1 in a 100-μl volume at 55°C for 6 hours, and then 5 μl of Aat II (20 U/μl; NEB) was added to the solution, which was left at 37°C overnight.

    Techniques: Sequencing, Polymerase Chain Reaction, Fractionation, Marker

    Schematic diagram of the RNA interference expression vector pRNAi-U6H1/Neo. The vector contains a multiple cloning sites with two BsmB I restriction sites to permit cloning of Fok I-digested sRNA fragments and an Sfi I site to assist in screening for recombinants.

    Journal: Nucleic Acid Therapeutics

    Article Title: Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens

    doi: 10.1089/nat.2014.0514

    Figure Lengend Snippet: Schematic diagram of the RNA interference expression vector pRNAi-U6H1/Neo. The vector contains a multiple cloning sites with two BsmB I restriction sites to permit cloning of Fok I-digested sRNA fragments and an Sfi I site to assist in screening for recombinants.

    Article Snippet: Fok I-digested fragments were ligated into linearized pRNAi-U6H1/Neo. pRNAi-U6H1/Neo was digested with BsmB I and dephosphorylated with calf intestinal alkaline phosphatase according to manufacturer's (NEB) protocols.

    Techniques: Expressing, Plasmid Preparation, Clone Assay

    Targeted mutagenesis using the CRISPR/Cas9 system in transient expression in N. benthamiana. a Schematic representation of the structure of Niben101Scf04205Ctg025 (XT1) and Niben101Scf04551Ctg021 (XT2) (exons in grey , introns in white ) with the sequences of the target sites. Diagnostic restriction sites are underlined and the PAM sequence is shown in bold . b Comparison of the mutation efficiency of hCas9 and pcoCas9 targeting the XT2. Red arrow shows SpeI resistant PCR fragments only visible on the gRNA and hCas9 combination. c PCR/RE assay to detect simultaneous targeted mutations on XT1 and XT2. Red arrows show BsmBI and SpeI resistant PCR fragments amplified from N. benthamiana genomic DNA. d Alignment of XT1 and XT2 sequences obtained from different clones of uncleaved bands (see c ). XT1 target site appears in blue and XT2 target site in green . Red letters and dashes indicate insertions and deletions respectively

    Journal: Plant Methods

    Article Title: A modular toolbox for gRNA–Cas9 genome engineering in plants based on the GoldenBraid standard

    doi: 10.1186/s13007-016-0101-2

    Figure Lengend Snippet: Targeted mutagenesis using the CRISPR/Cas9 system in transient expression in N. benthamiana. a Schematic representation of the structure of Niben101Scf04205Ctg025 (XT1) and Niben101Scf04551Ctg021 (XT2) (exons in grey , introns in white ) with the sequences of the target sites. Diagnostic restriction sites are underlined and the PAM sequence is shown in bold . b Comparison of the mutation efficiency of hCas9 and pcoCas9 targeting the XT2. Red arrow shows SpeI resistant PCR fragments only visible on the gRNA and hCas9 combination. c PCR/RE assay to detect simultaneous targeted mutations on XT1 and XT2. Red arrows show BsmBI and SpeI resistant PCR fragments amplified from N. benthamiana genomic DNA. d Alignment of XT1 and XT2 sequences obtained from different clones of uncleaved bands (see c ). XT1 target site appears in blue and XT2 target site in green . Red letters and dashes indicate insertions and deletions respectively

    Article Snippet: The resulting PCR products were purified with the QIAquick PCR purification kit (QIAGEN) following the manufacturer’s protocol and restriction reactions were set up with 500 ng of purified DNA and the corresponding restriction enzyme; BsmBI (Fermentas) for XT1 and SpeI (Fermentas) for XT2.

    Techniques: Mutagenesis, CRISPR, Expressing, Diagnostic Assay, Sequencing, Polymerase Chain Reaction, Amplification, Clone Assay

    Schematic overview of the subcloning procedure . The upper box contains the two vectors the reaction starts with, i.e. the entry vector with its two key elements flanking an insert and the destination vector with its NheI recognition site. By the enzymatic action (arrows) of Esp3I and NheI these vectors are linearized to form linear intermediate products as shown in the central box. These intermediates are subject to T4 ligase activity and can be ligated to yield a range of products: the initial vectors (upper box), circular intermediate products (lower box) and the desired product vector. Note that all circular intermediate products are again substrate for Esp3I and thus again linearized. There is a sole stable product in this reaction system, which is the desired product vector (circular products only shown if carrying at least one resistance marker). Shaded boxes termed KEY: key element as shown in Fig. 1.

    Journal: Journal of Biological Engineering

    Article Title: Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase

    doi: 10.1186/1754-1611-1-7

    Figure Lengend Snippet: Schematic overview of the subcloning procedure . The upper box contains the two vectors the reaction starts with, i.e. the entry vector with its two key elements flanking an insert and the destination vector with its NheI recognition site. By the enzymatic action (arrows) of Esp3I and NheI these vectors are linearized to form linear intermediate products as shown in the central box. These intermediates are subject to T4 ligase activity and can be ligated to yield a range of products: the initial vectors (upper box), circular intermediate products (lower box) and the desired product vector. Note that all circular intermediate products are again substrate for Esp3I and thus again linearized. There is a sole stable product in this reaction system, which is the desired product vector (circular products only shown if carrying at least one resistance marker). Shaded boxes termed KEY: key element as shown in Fig. 1.

    Article Snippet: We combined entry vector, destination vector, Esp3I (Fermentas), NheI (Fermentas) and T4 ligase (Promega) in a buffer allowing all three enzyme actions (**) and incubated at room temperature for 1 hour (Figure ).

    Techniques: Subcloning, Plasmid Preparation, Activity Assay, Marker

    Sequence elements facilitating the subcloning procedure . (A) A double stranded oligonucleotide was cloned into pGEM-T easy to provide two key elements flanking a BglII site. We inserted a 1166 bp PCR amplified fragment into this restriction site. In future applications this step may as well be achieved by TA cloning or other methods. (B) A key element consists of a Esp3I recognition site and a restriction site generating a NheI compatible overhang, that is not recognized by NheI. (C) We used NheI as target recognition site in the destination vector.

    Journal: Journal of Biological Engineering

    Article Title: Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase

    doi: 10.1186/1754-1611-1-7

    Figure Lengend Snippet: Sequence elements facilitating the subcloning procedure . (A) A double stranded oligonucleotide was cloned into pGEM-T easy to provide two key elements flanking a BglII site. We inserted a 1166 bp PCR amplified fragment into this restriction site. In future applications this step may as well be achieved by TA cloning or other methods. (B) A key element consists of a Esp3I recognition site and a restriction site generating a NheI compatible overhang, that is not recognized by NheI. (C) We used NheI as target recognition site in the destination vector.

    Article Snippet: We combined entry vector, destination vector, Esp3I (Fermentas), NheI (Fermentas) and T4 ligase (Promega) in a buffer allowing all three enzyme actions (**) and incubated at room temperature for 1 hour (Figure ).

    Techniques: Sequencing, Subcloning, Clone Assay, Polymerase Chain Reaction, Amplification, TA Cloning, Plasmid Preparation

    Cpf1–Flip–Cre-inducible sequential mutagenesis by a single crRNA FlipArray. a Schematic of vectors used in the study. The Cpf1-Flip construct contains an EFS promoter driving expression of Cpf1 and puromycin resistance, and a U6 expression cassette containing two inverted BsmbI restriction sites, flanked by a lox66 sequence and an inverted lox71 sequence. After BsmbI digestion, a crRNA FlipArray is cloned in. The FlipArray inverts upon Cre recombination, thereby switching the crRNA that is expressed. b Schematic of experimental design. Cells were first infected with lentivirus containing EFS-Cpf1-puro; U6-FlipArray. After 7 days, cells were then infected with lentivirus containing EFS-Cre to induce inversion of the FlipArray. Prior to Cre recombination, only crNf1 is expressed; following Cre recombination, crPten becomes expressed. c Sequences of the FlipArray construct before and after Cre recombination. Red boxes denote mutants from wild-type loxP . Prior to Cre, single mutant lox66 and lox71 sites are present. After Cre recombination, a wild-type loxP site and a double mutant lox72 site are generated

    Journal: Nature Communications

    Article Title: Programmable sequential mutagenesis by inducible Cpf1 crRNA array inversion

    doi: 10.1038/s41467-018-04158-z

    Figure Lengend Snippet: Cpf1–Flip–Cre-inducible sequential mutagenesis by a single crRNA FlipArray. a Schematic of vectors used in the study. The Cpf1-Flip construct contains an EFS promoter driving expression of Cpf1 and puromycin resistance, and a U6 expression cassette containing two inverted BsmbI restriction sites, flanked by a lox66 sequence and an inverted lox71 sequence. After BsmbI digestion, a crRNA FlipArray is cloned in. The FlipArray inverts upon Cre recombination, thereby switching the crRNA that is expressed. b Schematic of experimental design. Cells were first infected with lentivirus containing EFS-Cpf1-puro; U6-FlipArray. After 7 days, cells were then infected with lentivirus containing EFS-Cre to induce inversion of the FlipArray. Prior to Cre recombination, only crNf1 is expressed; following Cre recombination, crPten becomes expressed. c Sequences of the FlipArray construct before and after Cre recombination. Red boxes denote mutants from wild-type loxP . Prior to Cre, single mutant lox66 and lox71 sites are present. After Cre recombination, a wild-type loxP site and a double mutant lox72 site are generated

    Article Snippet: After BsmbI digestion (FastDigest Esp3I, ThermoScientific) to linearize the U6 crRNA expression cassette, oligo cloning was performed to insert a lox66 sequence, a DR, two BsmbI sites, and an inverted lox71 .

    Techniques: Mutagenesis, Construct, Expressing, Sequencing, Clone Assay, Infection, Generated

    Golden Gate assembly of custom TAL effector and TALEN constructs using module, array, last repeat and backbone plasmids. By using the type IIS restriction endonucleases BsaI and Esp3I, modules containing the desired RVDs can be released with unique cohesive ends for ordered, single-reaction assembly into array plasmids in a first step, and those arrays subsequently released and assembled in order in a second step into a backbone plasmid to create full length constructs with custom repeat arrays (see text for details). NLS, nuclear localization signal(s); AD, transcriptional activation domain; tet , tetracycline resistance; spec , spectinomycin resistance; amp , ampicillin resistance; attL1 and attL2, recombination sites for Gateway cloning; B, BamHI, and S, SphI, useful for subcloning custom repeat arrays. Unique restriction enzyme sites flanking the coding sequences, useful for subcloning the entire constructs into other vectors, are not shown but can be found in the sequence files ( Supplementary Data ).

    Journal: Nucleic Acids Research

    Article Title: Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

    doi: 10.1093/nar/gkr218

    Figure Lengend Snippet: Golden Gate assembly of custom TAL effector and TALEN constructs using module, array, last repeat and backbone plasmids. By using the type IIS restriction endonucleases BsaI and Esp3I, modules containing the desired RVDs can be released with unique cohesive ends for ordered, single-reaction assembly into array plasmids in a first step, and those arrays subsequently released and assembled in order in a second step into a backbone plasmid to create full length constructs with custom repeat arrays (see text for details). NLS, nuclear localization signal(s); AD, transcriptional activation domain; tet , tetracycline resistance; spec , spectinomycin resistance; amp , ampicillin resistance; attL1 and attL2, recombination sites for Gateway cloning; B, BamHI, and S, SphI, useful for subcloning custom repeat arrays. Unique restriction enzyme sites flanking the coding sequences, useful for subcloning the entire constructs into other vectors, are not shown but can be found in the sequence files ( Supplementary Data ).

    Article Snippet: Finally, into this plasmid, the XbaI–SacI fragment of pTAL3 containing the TALEN backbone construct was introduced at the corresponding sites. pTAL1 was created by replacing the SphI fragment of tal1C in pCS691 with the corresponding SphI fragment of pTAL3, containing the lacZ gene and the Esp3I sites and flanking sequences for accepting final arrays. pCS691 is a derivative of Gateway entry vector pENTR-D (Invitrogen) containing between the attL sites, the complete tal1c gene preceded by both Kozak and Shine–Dalgarno consensus sequences for efficient translation in eukaryotic or bacterial cells, respectively.

    Techniques: Construct, Plasmid Preparation, Activation Assay, Clone Assay, Subcloning, Sequencing

    Procedure to make a template clone ( a ) In the original clone, two unique sites recognized by two different type II restriction enzymes (labeled as TII-e1 and TII-e2) are selected in the gene of interest. The number x amino acid encoded by N 2 N 3 N 4 (in red) is indicated by aa x , or next amino acid by aa x+1 (in green). A template fragment contains a TIIS DNA cassette (highlighted in blue box) in between aa x and aa x+1 . All the cutting positions are indicated by arrows and complementary bases by apostrophe (’). ( b ) pBS-KSII contains a multiple cloning site (MCS) and a BsaI in the ampicillin (Amp) coding gene, which was destroyed by site-directed mutagenesis to create pBS-KSII-4B. Wild-type pBS-KSII was linearized by BsaI and two overlapping fragments were released by BsaI and NaeI double digestion. pBS-KSII-4B does not have BsaI, BbsI, BsmBI, and BfuAI/BspMI and can be linearized by NaeI. Three brighter bands from GeneRuler 1kb plus DNA marker are labeled.

    Journal: Biochemical and biophysical research communications

    Article Title: Highly Efficient One-Step Scarless Protein Tagging by Type IIS Restriction Endonuclease-Mediated Precision Cloning

    doi: 10.1016/j.bbrc.2017.05.153

    Figure Lengend Snippet: Procedure to make a template clone ( a ) In the original clone, two unique sites recognized by two different type II restriction enzymes (labeled as TII-e1 and TII-e2) are selected in the gene of interest. The number x amino acid encoded by N 2 N 3 N 4 (in red) is indicated by aa x , or next amino acid by aa x+1 (in green). A template fragment contains a TIIS DNA cassette (highlighted in blue box) in between aa x and aa x+1 . All the cutting positions are indicated by arrows and complementary bases by apostrophe (’). ( b ) pBS-KSII contains a multiple cloning site (MCS) and a BsaI in the ampicillin (Amp) coding gene, which was destroyed by site-directed mutagenesis to create pBS-KSII-4B. Wild-type pBS-KSII was linearized by BsaI and two overlapping fragments were released by BsaI and NaeI double digestion. pBS-KSII-4B does not have BsaI, BbsI, BsmBI, and BfuAI/BspMI and can be linearized by NaeI. Three brighter bands from GeneRuler 1kb plus DNA marker are labeled.

    Article Snippet: We confirmed that this modified pBS-KSII lacks BsaI, BbsI, BsmBI and BfuAI/BspMI (lanes 3–10 in lower panel in ) and named it pBS-KSII-4B, which is available immediately upon request and will be deposited at Addgene.

    Techniques: Labeling, Clone Assay, Mutagenesis, Marker

    CRISPR vector design for knockout of A 2A R in K562 Cells. ( a ) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A 2A R gene. Additional arrowheads indicate genes with similar regions to the A 2A R gene, including the A 2B R gene on chromosome 17. ( b ) Left, guide-RNAs found for the A 2A R gene. Right, guide sequence #1 was selected for insertion into the plentiCRISPR loxP vector plasmid DNA. BsmBI cut sites were added to gRNA #1 to facilitate insertion. ( c ) Top, Cas9 cut site in the A 2A R gene. Bottom, map of the plentiCRISPR construct. ( d ) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.

    Journal: Scientific Reports

    Article Title: A Novel Method for Screening Adenosine Receptor Specific Agonists for Use in Adenosine Drug Development

    doi: 10.1038/srep44816

    Figure Lengend Snippet: CRISPR vector design for knockout of A 2A R in K562 Cells. ( a ) Map of human chromosomes, with chromosome 22q11.23 highlighted, showing the location of the A 2A R gene. Additional arrowheads indicate genes with similar regions to the A 2A R gene, including the A 2B R gene on chromosome 17. ( b ) Left, guide-RNAs found for the A 2A R gene. Right, guide sequence #1 was selected for insertion into the plentiCRISPR loxP vector plasmid DNA. BsmBI cut sites were added to gRNA #1 to facilitate insertion. ( c ) Top, Cas9 cut site in the A 2A R gene. Bottom, map of the plentiCRISPR construct. ( d ) T7 E1 INDEL assay for CRISPR targeting efficiency. Lanes 1 and 5, universal marker. Lane 2, PCR product. Lanes 3 and 4, PCR product after denaturing and renaturing, without (3) and with (4) T7 EI enzyme. Arrowheads indicate T7 E1 cutting.

    Article Snippet: Using the BsmBI digestion enzyme (Thermo Fisher Scientific), the plentiCRISPR loxP plasmid was linearized and eluted for insertion of the A2A R guide RNA (gRNA).

    Techniques: CRISPR, Plasmid Preparation, Knock-Out, Sequencing, Construct, Marker, Polymerase Chain Reaction

    RNA-guided FokI nucleases and a Csy4-based multiplex gRNA expression system (a) Schematic overview of RNA-guided FokI nucleases. Two FokI-dCas9 fusion proteins are recruited to adjacent target sites by two different gRNAs in order to facilitate FokI dimerization and DNA cleavage. (b) Schematic overview of a Csy4-based multiplex gRNA expression system. Two gRNAs (with any 5′ end nucleotide) are co-expressed in a single transcript from a U6 promoter with each gRNA flanked by Csy4 recognition sites. Csy4 cleaves and releases gRNAs from the transcript. The Csy4 recognition site remains at the 3′ end of the gRNA with a Csy4 nuclease bound to that site. (c) Comparison of the activities of Cas9 co-expressed with either Csy4-processed single gRNAs (blue bars) or single gRNAs expressed from a standard U6 promoter (red bars) in the U2OS.EGFP disruption assay. The target sequences in EGFP gene for the three different single gRNAs tested are provided in the table to the right of the graph. Error bars represent s.e.m, n= 3. (d) Validation of the multiplex, Csy4-based system. Two gRNAs targeted to adjacent sites in EGFP were expressed in a single RNA transcript using the Csy4-based system in human U2OS.EGFP cells together with Csy4 and wild-type Cas9 nuclease. Sequences of indel mutations induced in these cells are shown. The wild-type sequence is shown at the top with both target sites highlighted in yellow and PAM sequences shown as red, underlined text. Deletions are indicated by red dashes against gray background and insertions by lowercase letters against a light blue background. To the right of each sequence, the sizes of insertions (+) or deletions (Δ) are specified.

    Journal: Nature biotechnology

    Article Title: Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing

    doi: 10.1038/nbt.2908

    Figure Lengend Snippet: RNA-guided FokI nucleases and a Csy4-based multiplex gRNA expression system (a) Schematic overview of RNA-guided FokI nucleases. Two FokI-dCas9 fusion proteins are recruited to adjacent target sites by two different gRNAs in order to facilitate FokI dimerization and DNA cleavage. (b) Schematic overview of a Csy4-based multiplex gRNA expression system. Two gRNAs (with any 5′ end nucleotide) are co-expressed in a single transcript from a U6 promoter with each gRNA flanked by Csy4 recognition sites. Csy4 cleaves and releases gRNAs from the transcript. The Csy4 recognition site remains at the 3′ end of the gRNA with a Csy4 nuclease bound to that site. (c) Comparison of the activities of Cas9 co-expressed with either Csy4-processed single gRNAs (blue bars) or single gRNAs expressed from a standard U6 promoter (red bars) in the U2OS.EGFP disruption assay. The target sequences in EGFP gene for the three different single gRNAs tested are provided in the table to the right of the graph. Error bars represent s.e.m, n= 3. (d) Validation of the multiplex, Csy4-based system. Two gRNAs targeted to adjacent sites in EGFP were expressed in a single RNA transcript using the Csy4-based system in human U2OS.EGFP cells together with Csy4 and wild-type Cas9 nuclease. Sequences of indel mutations induced in these cells are shown. The wild-type sequence is shown at the top with both target sites highlighted in yellow and PAM sequences shown as red, underlined text. Deletions are indicated by red dashes against gray background and insertions by lowercase letters against a light blue background. To the right of each sequence, the sizes of insertions (+) or deletions (Δ) are specified.

    Article Snippet: Plasmids encoding single or multiplex gRNAs were assembled in a single-step ligation of annealed target site oligosduplexes (Integrated DNA Technologies) ( and ) and a constant region oligoduplex (for multiplex gRNAs) with BsmBI-digested Csy4-flanked gRNA backbone (pSQT1313; to be deposited with Addgene http://www.addgene.org ) ( ).

    Techniques: Multiplex Assay, Expressing, Sequencing