pi scei  (New England Biolabs)


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    New England Biolabs pi scei
    Plasmid toolbox for the construction of CRISPR/Cas9-HCAdV genomes. ( A ) Schematic presentation of intermediate CRISPR/Cas9 shuttle plasmids for simple <t>gRNA</t> manipulation and multiplexing and subsequent transfer of the customized CRISPR/Cas9 machinery into the HCAdV genome. Option 1: pShV-CBh-Cas9-gRNA for constitutive Cas9 expression. Option 2: pShV-TRE-Cas9-TeOn3G-gRNA for inducible Cas9 expression utilizing the TetOn3G system. Black arrowheads indicate unique restriction enzyme sites for insertion of further gRNA expression units. ( B ) Workflow for gRNA customization and multiplexing of the CRISPR/Cas9 machinery. Step1: Complementary annealed gRNA oligonucleotides are separately inserted between the Bsa I restriction enzyme sites resulting in pShV-CBh-Cas9-gRNA1, pShV-CBh-Cas9-gRNA2 and pShV-CBh-Cas9- gRNA3. Step 2: Customized gRNA expression units gRNA1 and gRNA2 are amplified by PCR using primers generating desired restriction enzyme sites. Step 3: gRNA1 and 2 are inserted into the respective restriction enzyme site within pShV-CBh-Cas9-gRNA1 resulting in pShV-CBh-Cas9-CBh-gRNA1-gRNA2-gRNA3. ( C ) Transfer of customized CRISPR/Cas9 transgenes into the HCAdV genomes. Option 1: Released CRISPR/Cas9 transgene cassettes flanked by homology arms are inserted into pHCAdV-HOM-CcdB-AMP-HOM replacing the CcdB-Amp R cassette. Option 2: Endonuclease guided cloning into pAd-FTC utilizing PI- <t>Sce</t> I and I- Ceu I. HOM, homology arms for homologous recombination into pHCAdV-HOM-CCBD-AMP-HOM; CBh-P, constitutive hybrid CMV enhancer/chicken β-actin promotor; TRE-P, inducible tetracycline responsible element promotor; TetOn3G, TetOn3G transactivator; Ef1-α-P, Ef1-α-Promotor; Cas9, Streptococcus pyogenes Cas9, gRNA, guide RNA expression unit; U6-P, U6 RNA polymerase III promotor, Kan R , Kanamycin resistance cassette; Amp R ; Ampicillin resistance cassette, Chl R , Chloramphenicol resistance cassette; CcdB, control of cell death B expression cassette; ITR, adenovirus serotype 5 inverted terminal repeat; Ψ, adenovirus serotype 5 packaging signal.
    Pi Scei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pi scei/product/New England Biolabs
    Average 93 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    pi scei - by Bioz Stars, 2022-12
    93/100 stars

    Images

    1) Product Images from "CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes"

    Article Title: CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17180-w

    Plasmid toolbox for the construction of CRISPR/Cas9-HCAdV genomes. ( A ) Schematic presentation of intermediate CRISPR/Cas9 shuttle plasmids for simple gRNA manipulation and multiplexing and subsequent transfer of the customized CRISPR/Cas9 machinery into the HCAdV genome. Option 1: pShV-CBh-Cas9-gRNA for constitutive Cas9 expression. Option 2: pShV-TRE-Cas9-TeOn3G-gRNA for inducible Cas9 expression utilizing the TetOn3G system. Black arrowheads indicate unique restriction enzyme sites for insertion of further gRNA expression units. ( B ) Workflow for gRNA customization and multiplexing of the CRISPR/Cas9 machinery. Step1: Complementary annealed gRNA oligonucleotides are separately inserted between the Bsa I restriction enzyme sites resulting in pShV-CBh-Cas9-gRNA1, pShV-CBh-Cas9-gRNA2 and pShV-CBh-Cas9- gRNA3. Step 2: Customized gRNA expression units gRNA1 and gRNA2 are amplified by PCR using primers generating desired restriction enzyme sites. Step 3: gRNA1 and 2 are inserted into the respective restriction enzyme site within pShV-CBh-Cas9-gRNA1 resulting in pShV-CBh-Cas9-CBh-gRNA1-gRNA2-gRNA3. ( C ) Transfer of customized CRISPR/Cas9 transgenes into the HCAdV genomes. Option 1: Released CRISPR/Cas9 transgene cassettes flanked by homology arms are inserted into pHCAdV-HOM-CcdB-AMP-HOM replacing the CcdB-Amp R cassette. Option 2: Endonuclease guided cloning into pAd-FTC utilizing PI- Sce I and I- Ceu I. HOM, homology arms for homologous recombination into pHCAdV-HOM-CCBD-AMP-HOM; CBh-P, constitutive hybrid CMV enhancer/chicken β-actin promotor; TRE-P, inducible tetracycline responsible element promotor; TetOn3G, TetOn3G transactivator; Ef1-α-P, Ef1-α-Promotor; Cas9, Streptococcus pyogenes Cas9, gRNA, guide RNA expression unit; U6-P, U6 RNA polymerase III promotor, Kan R , Kanamycin resistance cassette; Amp R ; Ampicillin resistance cassette, Chl R , Chloramphenicol resistance cassette; CcdB, control of cell death B expression cassette; ITR, adenovirus serotype 5 inverted terminal repeat; Ψ, adenovirus serotype 5 packaging signal.
    Figure Legend Snippet: Plasmid toolbox for the construction of CRISPR/Cas9-HCAdV genomes. ( A ) Schematic presentation of intermediate CRISPR/Cas9 shuttle plasmids for simple gRNA manipulation and multiplexing and subsequent transfer of the customized CRISPR/Cas9 machinery into the HCAdV genome. Option 1: pShV-CBh-Cas9-gRNA for constitutive Cas9 expression. Option 2: pShV-TRE-Cas9-TeOn3G-gRNA for inducible Cas9 expression utilizing the TetOn3G system. Black arrowheads indicate unique restriction enzyme sites for insertion of further gRNA expression units. ( B ) Workflow for gRNA customization and multiplexing of the CRISPR/Cas9 machinery. Step1: Complementary annealed gRNA oligonucleotides are separately inserted between the Bsa I restriction enzyme sites resulting in pShV-CBh-Cas9-gRNA1, pShV-CBh-Cas9-gRNA2 and pShV-CBh-Cas9- gRNA3. Step 2: Customized gRNA expression units gRNA1 and gRNA2 are amplified by PCR using primers generating desired restriction enzyme sites. Step 3: gRNA1 and 2 are inserted into the respective restriction enzyme site within pShV-CBh-Cas9-gRNA1 resulting in pShV-CBh-Cas9-CBh-gRNA1-gRNA2-gRNA3. ( C ) Transfer of customized CRISPR/Cas9 transgenes into the HCAdV genomes. Option 1: Released CRISPR/Cas9 transgene cassettes flanked by homology arms are inserted into pHCAdV-HOM-CcdB-AMP-HOM replacing the CcdB-Amp R cassette. Option 2: Endonuclease guided cloning into pAd-FTC utilizing PI- Sce I and I- Ceu I. HOM, homology arms for homologous recombination into pHCAdV-HOM-CCBD-AMP-HOM; CBh-P, constitutive hybrid CMV enhancer/chicken β-actin promotor; TRE-P, inducible tetracycline responsible element promotor; TetOn3G, TetOn3G transactivator; Ef1-α-P, Ef1-α-Promotor; Cas9, Streptococcus pyogenes Cas9, gRNA, guide RNA expression unit; U6-P, U6 RNA polymerase III promotor, Kan R , Kanamycin resistance cassette; Amp R ; Ampicillin resistance cassette, Chl R , Chloramphenicol resistance cassette; CcdB, control of cell death B expression cassette; ITR, adenovirus serotype 5 inverted terminal repeat; Ψ, adenovirus serotype 5 packaging signal.

    Techniques Used: Plasmid Preparation, CRISPR, Multiplexing, Expressing, Amplification, Polymerase Chain Reaction, Clone Assay, Homologous Recombination, RNA Expression

    2) Product Images from "CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes"

    Article Title: CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17180-w

    Plasmid toolbox for the construction of CRISPR/Cas9-HCAdV genomes. ( A ) Schematic presentation of intermediate CRISPR/Cas9 shuttle plasmids for simple gRNA manipulation and multiplexing and subsequent transfer of the customized CRISPR/Cas9 machinery into the HCAdV genome. Option 1: pShV-CBh-Cas9-gRNA for constitutive Cas9 expression. Option 2: pShV-TRE-Cas9-TeOn3G-gRNA for inducible Cas9 expression utilizing the TetOn3G system. Black arrowheads indicate unique restriction enzyme sites for insertion of further gRNA expression units. ( B ) Workflow for gRNA customization and multiplexing of the CRISPR/Cas9 machinery. Step1: Complementary annealed gRNA oligonucleotides are separately inserted between the Bsa I restriction enzyme sites resulting in pShV-CBh-Cas9-gRNA1, pShV-CBh-Cas9-gRNA2 and pShV-CBh-Cas9- gRNA3. Step 2: Customized gRNA expression units gRNA1 and gRNA2 are amplified by PCR using primers generating desired restriction enzyme sites. Step 3: gRNA1 and 2 are inserted into the respective restriction enzyme site within pShV-CBh-Cas9-gRNA1 resulting in pShV-CBh-Cas9-CBh-gRNA1-gRNA2-gRNA3. ( C ) Transfer of customized CRISPR/Cas9 transgenes into the HCAdV genomes. Option 1: Released CRISPR/Cas9 transgene cassettes flanked by homology arms are inserted into pHCAdV-HOM-CcdB-AMP-HOM replacing the CcdB-Amp R cassette. Option 2: Endonuclease guided cloning into pAd-FTC utilizing PI- Sce I and I- Ceu I. HOM, homology arms for homologous recombination into pHCAdV-HOM-CCBD-AMP-HOM; CBh-P, constitutive hybrid CMV enhancer/chicken β-actin promotor; TRE-P, inducible tetracycline responsible element promotor; TetOn3G, TetOn3G transactivator; Ef1-α-P, Ef1-α-Promotor; Cas9, Streptococcus pyogenes Cas9, gRNA, guide RNA expression unit; U6-P, U6 RNA polymerase III promotor, Kan R , Kanamycin resistance cassette; Amp R ; Ampicillin resistance cassette, Chl R , Chloramphenicol resistance cassette; CcdB, control of cell death B expression cassette; ITR, adenovirus serotype 5 inverted terminal repeat; Ψ, adenovirus serotype 5 packaging signal.
    Figure Legend Snippet: Plasmid toolbox for the construction of CRISPR/Cas9-HCAdV genomes. ( A ) Schematic presentation of intermediate CRISPR/Cas9 shuttle plasmids for simple gRNA manipulation and multiplexing and subsequent transfer of the customized CRISPR/Cas9 machinery into the HCAdV genome. Option 1: pShV-CBh-Cas9-gRNA for constitutive Cas9 expression. Option 2: pShV-TRE-Cas9-TeOn3G-gRNA for inducible Cas9 expression utilizing the TetOn3G system. Black arrowheads indicate unique restriction enzyme sites for insertion of further gRNA expression units. ( B ) Workflow for gRNA customization and multiplexing of the CRISPR/Cas9 machinery. Step1: Complementary annealed gRNA oligonucleotides are separately inserted between the Bsa I restriction enzyme sites resulting in pShV-CBh-Cas9-gRNA1, pShV-CBh-Cas9-gRNA2 and pShV-CBh-Cas9- gRNA3. Step 2: Customized gRNA expression units gRNA1 and gRNA2 are amplified by PCR using primers generating desired restriction enzyme sites. Step 3: gRNA1 and 2 are inserted into the respective restriction enzyme site within pShV-CBh-Cas9-gRNA1 resulting in pShV-CBh-Cas9-CBh-gRNA1-gRNA2-gRNA3. ( C ) Transfer of customized CRISPR/Cas9 transgenes into the HCAdV genomes. Option 1: Released CRISPR/Cas9 transgene cassettes flanked by homology arms are inserted into pHCAdV-HOM-CcdB-AMP-HOM replacing the CcdB-Amp R cassette. Option 2: Endonuclease guided cloning into pAd-FTC utilizing PI- Sce I and I- Ceu I. HOM, homology arms for homologous recombination into pHCAdV-HOM-CCBD-AMP-HOM; CBh-P, constitutive hybrid CMV enhancer/chicken β-actin promotor; TRE-P, inducible tetracycline responsible element promotor; TetOn3G, TetOn3G transactivator; Ef1-α-P, Ef1-α-Promotor; Cas9, Streptococcus pyogenes Cas9, gRNA, guide RNA expression unit; U6-P, U6 RNA polymerase III promotor, Kan R , Kanamycin resistance cassette; Amp R ; Ampicillin resistance cassette, Chl R , Chloramphenicol resistance cassette; CcdB, control of cell death B expression cassette; ITR, adenovirus serotype 5 inverted terminal repeat; Ψ, adenovirus serotype 5 packaging signal.

    Techniques Used: Plasmid Preparation, CRISPR, Multiplexing, Expressing, Amplification, Polymerase Chain Reaction, Clone Assay, Homologous Recombination, RNA Expression

    3) Product Images from "Native homing endonucleases can target conserved genes in humans and in animal models"

    Article Title: Native homing endonucleases can target conserved genes in humans and in animal models

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr242

    Characterization of HEase plasticity in target recognition and its use for finding HEase targets in the human and other genomes. ( a ) and ( b ): HEase cleavage is tolerant of concomitant mutations at all wobble positions along the target site. The cleavage efficiency of the HEases PI-SceI (from S. cerevisiae) (a) and PI-PspI (from Pyrococcus species GB-D) (b) was assayed on different targets cloned on a bacterial plasmid. The cloned vectors were then fragmented by a restriction enzyme for the sake of visual clarity. Both HEases cleave targets mutated at all wobble positions (top DNA sequence) with higher efficiency than they cleave their native targets (bottom DNA sequence). Conversely, a single non-synonymous mutation (red arrow) is sometimes sufficient to abolish cleavage by PI-SceI and to reduce cleavage by PI-PspI. ( c ) and ( d ) : PI-SceI can cleave its predicted targets from the human ATP6V1A1 gene and its homologs in the genomes of animal models. (c) Alignment of the native target of the PI-SceI HEase from S. cerevisiae with the predicted targets in the human ATP6V1A1 gene and its homologs in the genomes of animal models. (d) Results of an in vitro cleavage assay demonstrating that PI-SceI can cleave its predicted targets from the genomes of diverse organisms. UC, uncut; RE, cut by a restriction enzyme (XbaI); HEase, cut by an HEase, RE + HEase, cut by XbaI AND by PI-SceI. S. cerevisiae, Saccharomyces cerevisiae; H. sapiens, Homo sapiens; C. familiaris, Canis familiaris; C. jacchus, Callithrix jacchus; M. musculus, Mus musculus; G. gallus, Gallus Gallus; R. norvegicus, Rattus norvegicus; D. rerio, Danio rerio .
    Figure Legend Snippet: Characterization of HEase plasticity in target recognition and its use for finding HEase targets in the human and other genomes. ( a ) and ( b ): HEase cleavage is tolerant of concomitant mutations at all wobble positions along the target site. The cleavage efficiency of the HEases PI-SceI (from S. cerevisiae) (a) and PI-PspI (from Pyrococcus species GB-D) (b) was assayed on different targets cloned on a bacterial plasmid. The cloned vectors were then fragmented by a restriction enzyme for the sake of visual clarity. Both HEases cleave targets mutated at all wobble positions (top DNA sequence) with higher efficiency than they cleave their native targets (bottom DNA sequence). Conversely, a single non-synonymous mutation (red arrow) is sometimes sufficient to abolish cleavage by PI-SceI and to reduce cleavage by PI-PspI. ( c ) and ( d ) : PI-SceI can cleave its predicted targets from the human ATP6V1A1 gene and its homologs in the genomes of animal models. (c) Alignment of the native target of the PI-SceI HEase from S. cerevisiae with the predicted targets in the human ATP6V1A1 gene and its homologs in the genomes of animal models. (d) Results of an in vitro cleavage assay demonstrating that PI-SceI can cleave its predicted targets from the genomes of diverse organisms. UC, uncut; RE, cut by a restriction enzyme (XbaI); HEase, cut by an HEase, RE + HEase, cut by XbaI AND by PI-SceI. S. cerevisiae, Saccharomyces cerevisiae; H. sapiens, Homo sapiens; C. familiaris, Canis familiaris; C. jacchus, Callithrix jacchus; M. musculus, Mus musculus; G. gallus, Gallus Gallus; R. norvegicus, Rattus norvegicus; D. rerio, Danio rerio .

    Techniques Used: Clone Assay, Plasmid Preparation, Sequencing, Mutagenesis, In Vitro, Cleavage Assay

    4) Product Images from "Efficient assembly of de novo human artificial chromosomes from large genomic loci"

    Article Title: Efficient assembly of de novo human artificial chromosomes from large genomic loci

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-5-21

    Strategy for construction of a bimolecular, prefabricated, linear HAC vector. Digestion of BAC-CEN and BAC-GEN vectors with the ultra-rare homing endonucleases I-CeuI and PI-SceI permits directional ligation of both
    Figure Legend Snippet: Strategy for construction of a bimolecular, prefabricated, linear HAC vector. Digestion of BAC-CEN and BAC-GEN vectors with the ultra-rare homing endonucleases I-CeuI and PI-SceI permits directional ligation of both "arms" to form a linear HAC vector.

    Techniques Used: HAC Assay, Plasmid Preparation, BAC Assay, Ligation

    5) Product Images from "Efficient assembly of de novo human artificial chromosomes from large genomic loci"

    Article Title: Efficient assembly of de novo human artificial chromosomes from large genomic loci

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-5-21

    Strategy for construction of a bimolecular, prefabricated, linear HAC vector. Digestion of BAC-CEN and BAC-GEN vectors with the ultra-rare homing endonucleases I-CeuI and PI-SceI permits directional ligation of both
    Figure Legend Snippet: Strategy for construction of a bimolecular, prefabricated, linear HAC vector. Digestion of BAC-CEN and BAC-GEN vectors with the ultra-rare homing endonucleases I-CeuI and PI-SceI permits directional ligation of both "arms" to form a linear HAC vector.

    Techniques Used: HAC Assay, Plasmid Preparation, BAC Assay, Ligation

    6) Product Images from "Efficient assembly of de novo human artificial chromosomes from large genomic loci"

    Article Title: Efficient assembly of de novo human artificial chromosomes from large genomic loci

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-5-21

    Strategy for construction of a bimolecular, prefabricated, linear HAC vector. Digestion of BAC-CEN and BAC-GEN vectors with the ultra-rare homing endonucleases I-CeuI and PI-SceI permits directional ligation of both
    Figure Legend Snippet: Strategy for construction of a bimolecular, prefabricated, linear HAC vector. Digestion of BAC-CEN and BAC-GEN vectors with the ultra-rare homing endonucleases I-CeuI and PI-SceI permits directional ligation of both "arms" to form a linear HAC vector.

    Techniques Used: HAC Assay, Plasmid Preparation, BAC Assay, Ligation

    7) Product Images from "Efficient linking and transfer of multiple genes by a multigene assembly and transformation vector system"

    Article Title: Efficient linking and transfer of multiple genes by a multigene assembly and transformation vector system

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0931425100

    Physical maps of the acceptor vector pYLTAC747 and donor vectors pYLVS and pYLSV. ( A ) Structural features of the pYLTAC747 and sequence between the T-DNA LB and RB. Four unique restriction sites, a loxP site, and a recognition site for I- Sce I were placed between LB and RB. The plasmid backbone contains a kanamycin-resistance gene (Kan r , modified NPTI ), a P1 plasmid replicon for replication in E. coli , and an Ri replicon for replication in Agrobacterium . ( B ) Structural features of the donor vectors and a part of sequence of pYLVS showing the key sites. The orientation of the segment between the two Bgl II sites is reversed in the two constructs. Cm r denotes the chloramphenicol-resistance gene and Ori denotes the ColE1 replicon.
    Figure Legend Snippet: Physical maps of the acceptor vector pYLTAC747 and donor vectors pYLVS and pYLSV. ( A ) Structural features of the pYLTAC747 and sequence between the T-DNA LB and RB. Four unique restriction sites, a loxP site, and a recognition site for I- Sce I were placed between LB and RB. The plasmid backbone contains a kanamycin-resistance gene (Kan r , modified NPTI ), a P1 plasmid replicon for replication in E. coli , and an Ri replicon for replication in Agrobacterium . ( B ) Structural features of the donor vectors and a part of sequence of pYLVS showing the key sites. The orientation of the segment between the two Bgl II sites is reversed in the two constructs. Cm r denotes the chloramphenicol-resistance gene and Ori denotes the ColE1 replicon.

    Techniques Used: Plasmid Preparation, Sequencing, Modification, Construct

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    New England Biolabs pi scei
    Pi Scei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pi scei/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pi scei - by Bioz Stars, 2022-12
    93/100 stars
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