Structured Review

Addgene inc 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.
Bsmbi, supplied by Addgene inc, used in various techniques. Bioz Stars score: 99/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/bsmbi/product/Addgene inc
Average 99 stars, based on 33 article reviews
Price from $9.99 to $1999.99
bsmbi - by Bioz Stars, 2022-10
99/100 stars

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1) Product Images from "Highly Efficient One-Step Scarless Protein Tagging by Type IIS Restriction Endonuclease-Mediated Precision Cloning"

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

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2017.05.153

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.
Figure Legend 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.

Techniques Used: Labeling, Clone Assay, Mutagenesis, Marker

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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.
    Bsmbi Digested Csy4 Flanked Grna Backbone, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc 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.
    Bsmbi, supplied by Addgene inc, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsmbi/product/Addgene inc
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    Addgene inc rtta3 ires neo cassette
    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.
    Rtta3 Ires Neo Cassette, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    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