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  • 97
    New England Biolabs esp 3i
    Identification of marker mutations in recombinant virus. Various icMHV-A59#1 or wild-type virus amplicons were subcloned into Topo II vectors and sequenced. (A) icMHV-A59#1 sequence across the mutated 3512 <t>Esp</t> 3I site; (B) icMHV-A59#1 sequence across the mutated 48 Esp 3I site and the MHV A/B junction; (C) icMHV-A59#1 sequence across the mutated 17 Esp 3I site; (D) icMHV-A59#1 sequence across the Rsr II site and MHV F/G junction; (E) wild-type MHV-A59 sequence across the same domain as shown in panel D. Overlined sequences include the mutated restriction sites and the corresponding wild-type nucleotides at these positions. In panels B and D, the bracketed sequences represent the MHV A/B and F/G junction domains, respectively. Note that all evidence of the Esp 3I site, which had been engineered into the sequence, is gone.
    Esp 3i, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 194 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher fastdigest esp3 i
    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 <t>U6</t> expression cassette containing two inverted <t>BsmbI</t> 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
    Fastdigest Esp3 I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher esp 3i
    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 <t>Esp3I</t> 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.
    Esp 3i, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Identification of marker mutations in recombinant virus. Various icMHV-A59#1 or wild-type virus amplicons were subcloned into Topo II vectors and sequenced. (A) icMHV-A59#1 sequence across the mutated 3512 Esp 3I site; (B) icMHV-A59#1 sequence across the mutated 48 Esp 3I site and the MHV A/B junction; (C) icMHV-A59#1 sequence across the mutated 17 Esp 3I site; (D) icMHV-A59#1 sequence across the Rsr II site and MHV F/G junction; (E) wild-type MHV-A59 sequence across the same domain as shown in panel D. Overlined sequences include the mutated restriction sites and the corresponding wild-type nucleotides at these positions. In panels B and D, the bracketed sequences represent the MHV A/B and F/G junction domains, respectively. Note that all evidence of the Esp 3I site, which had been engineered into the sequence, is gone.

    Journal: Journal of Virology

    Article Title: Systematic Assembly of a Full-Length Infectious cDNA of Mouse Hepatitis Virus Strain A59

    doi: 10.1128/JVI.76.21.11065-11078.2002

    Figure Lengend Snippet: Identification of marker mutations in recombinant virus. Various icMHV-A59#1 or wild-type virus amplicons were subcloned into Topo II vectors and sequenced. (A) icMHV-A59#1 sequence across the mutated 3512 Esp 3I site; (B) icMHV-A59#1 sequence across the mutated 48 Esp 3I site and the MHV A/B junction; (C) icMHV-A59#1 sequence across the mutated 17 Esp 3I site; (D) icMHV-A59#1 sequence across the Rsr II site and MHV F/G junction; (E) wild-type MHV-A59 sequence across the same domain as shown in panel D. Overlined sequences include the mutated restriction sites and the corresponding wild-type nucleotides at these positions. In panels B and D, the bracketed sequences represent the MHV A/B and F/G junction domains, respectively. Note that all evidence of the Esp 3I site, which had been engineered into the sequence, is gone.

    Article Snippet: Each of the plasmids was grown to a high concentration, isolated, and digested, or double digested, with Esp 3I, Bgl I, or Not I according to the manufacturer's directions (New England Biolabs) (Fig. ).

    Techniques: Marker, Recombinant, Sequencing, End-sequence Profiling

    Restriction fragment length polymorphism analysis of molecularly cloned virus. Cultures were infected with MHV-A59 or icMHV-A59#1, and intracellular RNA was isolated at 8 h p.i. With primer pairs and RT-PCR, cDNA amplicons were isolated that contained the various marker mutations that had been inserted into the component clones. The purified wild-type MHV-A59 and icMHV-A59#1 amplicons were restricted with Esp 3I or Rsr II and separated in 0.8% agarose gels. Lane 1, wild-type A59 amplicon from nucleotides 2020 to 5031, uncut; lane 2, wild-type A59 amplicon from nucleotides 2020 to 5031 digested with Esp 3I; lane 3, icMHV-A59#1 amplicon from nucleotides 2020 to 5031, uncut; lane 4, icMHV-A59#1 amplicon from nucleotides 2020 to 5031, Esp 3I digested; lane 5, wild-type A59 amplicon from nucleotides 16351 to 17875, uncut; lane 6, wild-type A59 amplicon from nucleotides 16351 to 17875 restricted with Esp 3I; lane 7, icMHV-A59#1 amplicon from nucleotides 16351 to 17875, uncut; lane 8, icMHV-A59#1 amplicon from nucleotides 16351 to 17875 restricted with Esp 3I; lane 9, wild-type A59 amplicon from nucleotides 22060 to 25416, uncut; lane 10, wild-type A59 amplicon from nucleotides 22060 to 25416 restricted with Rsr II; lane 11, icMHV-A59#1 amplicon from nucleotides 22060 to 25416, uncut; lane 12, icMHV-A59#1 amplicon from nucleotides 22060 to 25416 restricted with Rsr II; lane 13, 1-kb ladder.

    Journal: Journal of Virology

    Article Title: Systematic Assembly of a Full-Length Infectious cDNA of Mouse Hepatitis Virus Strain A59

    doi: 10.1128/JVI.76.21.11065-11078.2002

    Figure Lengend Snippet: Restriction fragment length polymorphism analysis of molecularly cloned virus. Cultures were infected with MHV-A59 or icMHV-A59#1, and intracellular RNA was isolated at 8 h p.i. With primer pairs and RT-PCR, cDNA amplicons were isolated that contained the various marker mutations that had been inserted into the component clones. The purified wild-type MHV-A59 and icMHV-A59#1 amplicons were restricted with Esp 3I or Rsr II and separated in 0.8% agarose gels. Lane 1, wild-type A59 amplicon from nucleotides 2020 to 5031, uncut; lane 2, wild-type A59 amplicon from nucleotides 2020 to 5031 digested with Esp 3I; lane 3, icMHV-A59#1 amplicon from nucleotides 2020 to 5031, uncut; lane 4, icMHV-A59#1 amplicon from nucleotides 2020 to 5031, Esp 3I digested; lane 5, wild-type A59 amplicon from nucleotides 16351 to 17875, uncut; lane 6, wild-type A59 amplicon from nucleotides 16351 to 17875 restricted with Esp 3I; lane 7, icMHV-A59#1 amplicon from nucleotides 16351 to 17875, uncut; lane 8, icMHV-A59#1 amplicon from nucleotides 16351 to 17875 restricted with Esp 3I; lane 9, wild-type A59 amplicon from nucleotides 22060 to 25416, uncut; lane 10, wild-type A59 amplicon from nucleotides 22060 to 25416 restricted with Rsr II; lane 11, icMHV-A59#1 amplicon from nucleotides 22060 to 25416, uncut; lane 12, icMHV-A59#1 amplicon from nucleotides 22060 to 25416 restricted with Rsr II; lane 13, 1-kb ladder.

    Article Snippet: Each of the plasmids was grown to a high concentration, isolated, and digested, or double digested, with Esp 3I, Bgl I, or Not I according to the manufacturer's directions (New England Biolabs) (Fig. ).

    Techniques: Clone Assay, Infection, Isolation, Reverse Transcription Polymerase Chain Reaction, Marker, Purification, End-sequence Profiling, Amplification

    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

    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

    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