pclipf  (New England Biolabs)


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    Name:
    pCLIPf Vector
    Description:
    pCLIPf Vector 20 ug
    Catalog Number:
    n9215s
    Price:
    166
    Size:
    20 ug
    Category:
    Vectors Plasmids
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    Structured Review

    New England Biolabs pclipf
    pCLIPf Vector
    pCLIPf Vector 20 ug
    https://www.bioz.com/result/pclipf/product/New England Biolabs
    Average 95 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    pclipf - by Bioz Stars, 2020-02
    95/100 stars

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    Related Articles

    Clone Assay:

    Article Title: WDR76 Co-Localizes with Heterochromatin Related Proteins and Rapidly Responds to DNA Damage
    Article Snippet: .. Selected ORFs coding for PARP1, XRCC5, RPA1, CBX1, CBX3, and CBX5 were cloned into a modified pCLIP-tag™-FLAG vector. pCLIP-FLAG was constructed by inserting a DNA fragment encoding a FLAG tag followed by the restriction site Sgf1 into the multiple cloning site of the pCLIP-tag™ vector from New England Biolabs (Ipswich, MA). .. We also constructed CLIP-WDR76 and Halo-RPA1 vectors for use in a reverse tag imaging experiment.

    Article Title: Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia
    Article Snippet: .. Wt and mutant Ank clones were subcloned N-terminal of pSNAPf and pCLIPf (NEB) mammalian expression vectors using forward primer 5′-GGCGCGCCACCATGG ACTACAAA-3′ containing a restriction site for AscI and a Kozak sequence GCCACC and a reverse primer 5′-TCCGGAATTCCTCATTTT CTTCTCTCATC-3′ containing an EcoRI restriction site without stop codon. .. These vectors contained SNAP and CLIP-tags.

    Article Title: Serine Phosphorylation of the Hepatitis C Virus NS5A Protein Controls the Establishment of Replication Complexes
    Article Snippet: Then, mutant NS5A was cloned into either mSGR-luc-JFH-1 or mJFH-1 via flanking BamHI/AfeI sites, and correct insertion was confirmed by sequencing. .. For the insertion of the SNAP and CLIP tags into NS5A, the gene was amplified from the pSNAPf and pCLIPf vectors (NEB) using primers that insert a flexible linker sequence (GSSGSS) on either side of the insertion (primer sequences are available upon request).

    Article Title: The SH3 regulatory domain of the hematopoietic cell kinase Hck binds ELMO via its polyproline motif
    Article Snippet: .. For mammalian expression SH3Hck coding sequence was amplified by PCR (residues 77–141) and cloned in frame with the CLIP tag of the pCLIPf vector (New England Biolabs) The cDNA encoding for the human SH3 domain of DOCK180 (DOCKSH3 : residues 9–69 of DOCK180 from Uniprot Protein Database: Q14185) was optimized for bacterial expression, synthesized (Geneart AG) and subcloned into pGEX 4T3 in frame with a TEV cleavable GST tag for bacterial expression. .. 2.2 Purification of recombinant proteins from bacteria For bacterial production and purification, the recombinant protein constructs were overexpressed in BL21(DE3) E scherichia coli strain using conventional IPTG induction (1 mM) in LB medium for 3 h at 37 °C.

    Article Title: Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia
    Article Snippet: .. Wt and mutant Ank clones were subcloned N-terminal of pSNAPf and pCLIPf (NEB) mammalian expression vectors using forward primer 5′-GGCGCGCCACCATGG ACTACAAA-3′ containing a restriction site for AscI and a Kozak sequence GCCACC and a reverse primer 5′-TCCGGAATTCCTCATTTT CTTCTCTCATC-3′ containing an EcoRI restriction site without stop codon. .. These vectors contained SNAP and CLIP-tags.

    Article Title: A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting
    Article Snippet: .. pSNAP-tag(m) and pCLIP-tag(m) vectors were purchased from NEB. pSNAP-DGCR8: A thrombin site was cloned into the BamHI site of pSNAP-tag(m) to give pSNAP-thrombin. ..

    Article Title: Quantifying Transcription Factor Binding Dynamics at the Single-molecule Level in Live Cells
    Article Snippet: .. This was generated by PCR amplification from HaloTag–GR and sub cloned into the pCLIPf (N9215S, New England Biolabs) backbone with NheI and AgeI sites. .. The mEOS3–GR was generated by purification of the rat GR sequence from a AgeI/XhoI digested fragment of the pEGFP–GR vector [ ], and subsequent sub-cloning into a pre-digested AgeI/XhoI pmEOS3 plasmid, kindly provided by the Lippincott-Schwartz lab.

    Article Title: Newly synthesized claudins but not occludin are added to the basal side of the tight junction
    Article Snippet: .. In some experiments, stable cell lines overexpressing SNAP and CLIP-tagged cldns and mutants were generated by PCR, using pTRE human cldn4 ( ) as a template and cloning into the Bam HI/Xh oI sites in pSNAPf and pCLIPf (New England Biolabs, N9215S). .. SNAP ocln construct was made by PCR from EGFP ocln ( ).

    Amplification:

    Article Title: Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia
    Article Snippet: Wt Ank was amplified from mouse cDNA with primers containing Hind III and EcoRI and cloned in p3xFLAG-CMV-10 mammalian expression vector (Sigma-Aldrich). .. Wt and mutant Ank clones were subcloned N-terminal of pSNAPf and pCLIPf (NEB) mammalian expression vectors using forward primer 5′-GGCGCGCCACCATGG ACTACAAA-3′ containing a restriction site for AscI and a Kozak sequence GCCACC and a reverse primer 5′-TCCGGAATTCCTCATTTT CTTCTCTCATC-3′ containing an EcoRI restriction site without stop codon.

    Article Title: Serine Phosphorylation of the Hepatitis C Virus NS5A Protein Controls the Establishment of Replication Complexes
    Article Snippet: .. For the insertion of the SNAP and CLIP tags into NS5A, the gene was amplified from the pSNAPf and pCLIPf vectors (NEB) using primers that insert a flexible linker sequence (GSSGSS) on either side of the insertion (primer sequences are available upon request). .. This amplicon was inserted into the aforementioned LITMUS28i subclone by BclI digestion, and correct insertion was verified by sequencing.

    Article Title: The SH3 regulatory domain of the hematopoietic cell kinase Hck binds ELMO via its polyproline motif
    Article Snippet: .. For mammalian expression SH3Hck coding sequence was amplified by PCR (residues 77–141) and cloned in frame with the CLIP tag of the pCLIPf vector (New England Biolabs) The cDNA encoding for the human SH3 domain of DOCK180 (DOCKSH3 : residues 9–69 of DOCK180 from Uniprot Protein Database: Q14185) was optimized for bacterial expression, synthesized (Geneart AG) and subcloned into pGEX 4T3 in frame with a TEV cleavable GST tag for bacterial expression. .. 2.2 Purification of recombinant proteins from bacteria For bacterial production and purification, the recombinant protein constructs were overexpressed in BL21(DE3) E scherichia coli strain using conventional IPTG induction (1 mM) in LB medium for 3 h at 37 °C.

    Article Title: Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia
    Article Snippet: Cloning of p3x-FLAG-wt and mutant Ank in CMV10, pSNAPf, pCLIPf and pRS414 vectors Wt Ank was amplified from mouse cDNA with primers containing Hind III and EcoRI and cloned in p3xFLAG-CMV-10 mammalian expression vector (Sigma-Aldrich). .. Wt and mutant Ank clones were subcloned N-terminal of pSNAPf and pCLIPf (NEB) mammalian expression vectors using forward primer 5′-GGCGCGCCACCATGG ACTACAAA-3′ containing a restriction site for AscI and a Kozak sequence GCCACC and a reverse primer 5′-TCCGGAATTCCTCATTTT CTTCTCTCATC-3′ containing an EcoRI restriction site without stop codon.

    Article Title: Quantifying Transcription Factor Binding Dynamics at the Single-molecule Level in Live Cells
    Article Snippet: .. This was generated by PCR amplification from HaloTag–GR and sub cloned into the pCLIPf (N9215S, New England Biolabs) backbone with NheI and AgeI sites. .. The mEOS3–GR was generated by purification of the rat GR sequence from a AgeI/XhoI digested fragment of the pEGFP–GR vector [ ], and subsequent sub-cloning into a pre-digested AgeI/XhoI pmEOS3 plasmid, kindly provided by the Lippincott-Schwartz lab.

    Stable Transfection:

    Article Title: Newly synthesized claudins but not occludin are added to the basal side of the tight junction
    Article Snippet: .. In some experiments, stable cell lines overexpressing SNAP and CLIP-tagged cldns and mutants were generated by PCR, using pTRE human cldn4 ( ) as a template and cloning into the Bam HI/Xh oI sites in pSNAPf and pCLIPf (New England Biolabs, N9215S). .. SNAP ocln construct was made by PCR from EGFP ocln ( ).

    Synthesized:

    Article Title: The SH3 regulatory domain of the hematopoietic cell kinase Hck binds ELMO via its polyproline motif
    Article Snippet: .. For mammalian expression SH3Hck coding sequence was amplified by PCR (residues 77–141) and cloned in frame with the CLIP tag of the pCLIPf vector (New England Biolabs) The cDNA encoding for the human SH3 domain of DOCK180 (DOCKSH3 : residues 9–69 of DOCK180 from Uniprot Protein Database: Q14185) was optimized for bacterial expression, synthesized (Geneart AG) and subcloned into pGEX 4T3 in frame with a TEV cleavable GST tag for bacterial expression. .. 2.2 Purification of recombinant proteins from bacteria For bacterial production and purification, the recombinant protein constructs were overexpressed in BL21(DE3) E scherichia coli strain using conventional IPTG induction (1 mM) in LB medium for 3 h at 37 °C.

    Construct:

    Article Title: WDR76 Co-Localizes with Heterochromatin Related Proteins and Rapidly Responds to DNA Damage
    Article Snippet: .. Selected ORFs coding for PARP1, XRCC5, RPA1, CBX1, CBX3, and CBX5 were cloned into a modified pCLIP-tag™-FLAG vector. pCLIP-FLAG was constructed by inserting a DNA fragment encoding a FLAG tag followed by the restriction site Sgf1 into the multiple cloning site of the pCLIP-tag™ vector from New England Biolabs (Ipswich, MA). .. We also constructed CLIP-WDR76 and Halo-RPA1 vectors for use in a reverse tag imaging experiment.

    Article Title: Serine Phosphorylation of the Hepatitis C Virus NS5A Protein Controls the Establishment of Replication Complexes
    Article Snippet: Previously, the unique restriction sites flanking NS5A, BamHI and AfeI, were introduced into both the full-length virus and SGR-luc-JFH-1 constructs; these constructs are denoted mJFH-1 and mSGR-luc-JFH-1, respectively, and have shown been to have no effect on virus genome replication or assembly and release ( ). .. For the insertion of the SNAP and CLIP tags into NS5A, the gene was amplified from the pSNAPf and pCLIPf vectors (NEB) using primers that insert a flexible linker sequence (GSSGSS) on either side of the insertion (primer sequences are available upon request).

    Article Title: Quantifying Transcription Factor Binding Dynamics at the Single-molecule Level in Live Cells
    Article Snippet: Paragraph title: 3.1.1. Plasmid constructs ... This was generated by PCR amplification from HaloTag–GR and sub cloned into the pCLIPf (N9215S, New England Biolabs) backbone with NheI and AgeI sites.

    Article Title: Cbl-mediated K63-linked ubiquitination of JAK2 enhances JAK2 phosphorylation and signal transduction
    Article Snippet: .. Plasmids pcDNA3-GMRα and pGMRβ-clip are pcDNA3 vector-based expression systems encoding GMRα and GMRβ-clip proteins, respectively, as previously described . pGMRβ-clip was constructed by inserting the PCR-amplified clip coding region from pCLIP vector (New England BioLabs,Ipswich, MA, USA) into the C terminus of the GMRβ gene in pCDNA3-GMRβ. .. The Cbl cDNA was kindly provided by Dr. Wallace Y. Langdon (School of Pathology and Laboratory Medicine, University of Western Australia, Australia), and sub-cloned into the pcDNA6 vector.

    Article Title: Newly synthesized claudins but not occludin are added to the basal side of the tight junction
    Article Snippet: In some experiments, stable cell lines overexpressing SNAP and CLIP-tagged cldns and mutants were generated by PCR, using pTRE human cldn4 ( ) as a template and cloning into the Bam HI/Xh oI sites in pSNAPf and pCLIPf (New England Biolabs, N9215S). .. SNAP ocln construct was made by PCR from EGFP ocln ( ).

    Luciferase:

    Article Title: Serine Phosphorylation of the Hepatitis C Virus NS5A Protein Controls the Establishment of Replication Complexes
    Article Snippet: DNA constructs of and the full-length pJFH-1 virus ( ) and the luciferase subgenomic replicon (SGR-luc-JFH-1) ( ) were used throughout the study. .. For the insertion of the SNAP and CLIP tags into NS5A, the gene was amplified from the pSNAPf and pCLIPf vectors (NEB) using primers that insert a flexible linker sequence (GSSGSS) on either side of the insertion (primer sequences are available upon request).

    Expressing:

    Article Title: WDR76 Co-Localizes with Heterochromatin Related Proteins and Rapidly Responds to DNA Damage
    Article Snippet: Paragraph title: Construction of vectors for protein expression in HEK293 cells ... Selected ORFs coding for PARP1, XRCC5, RPA1, CBX1, CBX3, and CBX5 were cloned into a modified pCLIP-tag™-FLAG vector. pCLIP-FLAG was constructed by inserting a DNA fragment encoding a FLAG tag followed by the restriction site Sgf1 into the multiple cloning site of the pCLIP-tag™ vector from New England Biolabs (Ipswich, MA).

    Article Title: Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia
    Article Snippet: .. Wt and mutant Ank clones were subcloned N-terminal of pSNAPf and pCLIPf (NEB) mammalian expression vectors using forward primer 5′-GGCGCGCCACCATGG ACTACAAA-3′ containing a restriction site for AscI and a Kozak sequence GCCACC and a reverse primer 5′-TCCGGAATTCCTCATTTT CTTCTCTCATC-3′ containing an EcoRI restriction site without stop codon. .. These vectors contained SNAP and CLIP-tags.

    Article Title: Serine Phosphorylation of the Hepatitis C Virus NS5A Protein Controls the Establishment of Replication Complexes
    Article Snippet: For the insertion of the SNAP and CLIP tags into NS5A, the gene was amplified from the pSNAPf and pCLIPf vectors (NEB) using primers that insert a flexible linker sequence (GSSGSS) on either side of the insertion (primer sequences are available upon request). .. For plasmid expression, mutated NS5A-coding sequences were subcloned into the pCMV(NS3-5B) vector as described previously ( ).

    Article Title: The SH3 regulatory domain of the hematopoietic cell kinase Hck binds ELMO via its polyproline motif
    Article Snippet: .. For mammalian expression SH3Hck coding sequence was amplified by PCR (residues 77–141) and cloned in frame with the CLIP tag of the pCLIPf vector (New England Biolabs) The cDNA encoding for the human SH3 domain of DOCK180 (DOCKSH3 : residues 9–69 of DOCK180 from Uniprot Protein Database: Q14185) was optimized for bacterial expression, synthesized (Geneart AG) and subcloned into pGEX 4T3 in frame with a TEV cleavable GST tag for bacterial expression. .. 2.2 Purification of recombinant proteins from bacteria For bacterial production and purification, the recombinant protein constructs were overexpressed in BL21(DE3) E scherichia coli strain using conventional IPTG induction (1 mM) in LB medium for 3 h at 37 °C.

    Article Title: Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia
    Article Snippet: .. Wt and mutant Ank clones were subcloned N-terminal of pSNAPf and pCLIPf (NEB) mammalian expression vectors using forward primer 5′-GGCGCGCCACCATGG ACTACAAA-3′ containing a restriction site for AscI and a Kozak sequence GCCACC and a reverse primer 5′-TCCGGAATTCCTCATTTT CTTCTCTCATC-3′ containing an EcoRI restriction site without stop codon. .. These vectors contained SNAP and CLIP-tags.

    Article Title: Cbl-mediated K63-linked ubiquitination of JAK2 enhances JAK2 phosphorylation and signal transduction
    Article Snippet: .. Plasmids pcDNA3-GMRα and pGMRβ-clip are pcDNA3 vector-based expression systems encoding GMRα and GMRβ-clip proteins, respectively, as previously described . pGMRβ-clip was constructed by inserting the PCR-amplified clip coding region from pCLIP vector (New England BioLabs,Ipswich, MA, USA) into the C terminus of the GMRβ gene in pCDNA3-GMRβ. .. The Cbl cDNA was kindly provided by Dr. Wallace Y. Langdon (School of Pathology and Laboratory Medicine, University of Western Australia, Australia), and sub-cloned into the pcDNA6 vector.

    Modification:

    Article Title: WDR76 Co-Localizes with Heterochromatin Related Proteins and Rapidly Responds to DNA Damage
    Article Snippet: .. Selected ORFs coding for PARP1, XRCC5, RPA1, CBX1, CBX3, and CBX5 were cloned into a modified pCLIP-tag™-FLAG vector. pCLIP-FLAG was constructed by inserting a DNA fragment encoding a FLAG tag followed by the restriction site Sgf1 into the multiple cloning site of the pCLIP-tag™ vector from New England Biolabs (Ipswich, MA). .. We also constructed CLIP-WDR76 and Halo-RPA1 vectors for use in a reverse tag imaging experiment.

    Article Title: The SH3 regulatory domain of the hematopoietic cell kinase Hck binds ELMO via its polyproline motif
    Article Snippet: Full length and truncated forms of ELMO1 were generated by PCR (ELMO1–727 , ELMO532–727 , ELMO532–707 , ) and subcloned either using ligation-independent cloning in frame with a Tobacco Etch Virus (TEV) protease cleavable 6-Histine tag for bacterial expression (pPROEXHtb) or by conventional ligation into a modified pEGFP-N1 vector for mammalian expression, in which the EGFP ORF is replaced by a TEV cleavable SNAP tag (New England Biolabs). .. For mammalian expression SH3Hck coding sequence was amplified by PCR (residues 77–141) and cloned in frame with the CLIP tag of the pCLIPf vector (New England Biolabs) The cDNA encoding for the human SH3 domain of DOCK180 (DOCKSH3 : residues 9–69 of DOCK180 from Uniprot Protein Database: Q14185) was optimized for bacterial expression, synthesized (Geneart AG) and subcloned into pGEX 4T3 in frame with a TEV cleavable GST tag for bacterial expression.

    Western Blot:

    Article Title: Cbl-mediated K63-linked ubiquitination of JAK2 enhances JAK2 phosphorylation and signal transduction
    Article Snippet: Plasmids pcDNA3-GMRα and pGMRβ-clip are pcDNA3 vector-based expression systems encoding GMRα and GMRβ-clip proteins, respectively, as previously described . pGMRβ-clip was constructed by inserting the PCR-amplified clip coding region from pCLIP vector (New England BioLabs,Ipswich, MA, USA) into the C terminus of the GMRβ gene in pCDNA3-GMRβ. .. The Cbl cDNA was kindly provided by Dr. Wallace Y. Langdon (School of Pathology and Laboratory Medicine, University of Western Australia, Australia), and sub-cloned into the pcDNA6 vector.

    Transfection:

    Article Title: Newly synthesized claudins but not occludin are added to the basal side of the tight junction
    Article Snippet: Paragraph title: Cell culture and transfection ... In some experiments, stable cell lines overexpressing SNAP and CLIP-tagged cldns and mutants were generated by PCR, using pTRE human cldn4 ( ) as a template and cloning into the Bam HI/Xh oI sites in pSNAPf and pCLIPf (New England Biolabs, N9215S).

    Ligation:

    Article Title: The SH3 regulatory domain of the hematopoietic cell kinase Hck binds ELMO via its polyproline motif
    Article Snippet: Full length and truncated forms of ELMO1 were generated by PCR (ELMO1–727 , ELMO532–727 , ELMO532–707 , ) and subcloned either using ligation-independent cloning in frame with a Tobacco Etch Virus (TEV) protease cleavable 6-Histine tag for bacterial expression (pPROEXHtb) or by conventional ligation into a modified pEGFP-N1 vector for mammalian expression, in which the EGFP ORF is replaced by a TEV cleavable SNAP tag (New England Biolabs). .. For mammalian expression SH3Hck coding sequence was amplified by PCR (residues 77–141) and cloned in frame with the CLIP tag of the pCLIPf vector (New England Biolabs) The cDNA encoding for the human SH3 domain of DOCK180 (DOCKSH3 : residues 9–69 of DOCK180 from Uniprot Protein Database: Q14185) was optimized for bacterial expression, synthesized (Geneart AG) and subcloned into pGEX 4T3 in frame with a TEV cleavable GST tag for bacterial expression.

    Cell Culture:

    Article Title: Newly synthesized claudins but not occludin are added to the basal side of the tight junction
    Article Snippet: Paragraph title: Cell culture and transfection ... In some experiments, stable cell lines overexpressing SNAP and CLIP-tagged cldns and mutants were generated by PCR, using pTRE human cldn4 ( ) as a template and cloning into the Bam HI/Xh oI sites in pSNAPf and pCLIPf (New England Biolabs, N9215S).

    Generated:

    Article Title: The SH3 regulatory domain of the hematopoietic cell kinase Hck binds ELMO via its polyproline motif
    Article Snippet: Full length and truncated forms of ELMO1 were generated by PCR (ELMO1–727 , ELMO532–727 , ELMO532–707 , ) and subcloned either using ligation-independent cloning in frame with a Tobacco Etch Virus (TEV) protease cleavable 6-Histine tag for bacterial expression (pPROEXHtb) or by conventional ligation into a modified pEGFP-N1 vector for mammalian expression, in which the EGFP ORF is replaced by a TEV cleavable SNAP tag (New England Biolabs). .. For mammalian expression SH3Hck coding sequence was amplified by PCR (residues 77–141) and cloned in frame with the CLIP tag of the pCLIPf vector (New England Biolabs) The cDNA encoding for the human SH3 domain of DOCK180 (DOCKSH3 : residues 9–69 of DOCK180 from Uniprot Protein Database: Q14185) was optimized for bacterial expression, synthesized (Geneart AG) and subcloned into pGEX 4T3 in frame with a TEV cleavable GST tag for bacterial expression.

    Article Title: Quantifying Transcription Factor Binding Dynamics at the Single-molecule Level in Live Cells
    Article Snippet: .. This was generated by PCR amplification from HaloTag–GR and sub cloned into the pCLIPf (N9215S, New England Biolabs) backbone with NheI and AgeI sites. .. The mEOS3–GR was generated by purification of the rat GR sequence from a AgeI/XhoI digested fragment of the pEGFP–GR vector [ ], and subsequent sub-cloning into a pre-digested AgeI/XhoI pmEOS3 plasmid, kindly provided by the Lippincott-Schwartz lab.

    Article Title: Cbl-mediated K63-linked ubiquitination of JAK2 enhances JAK2 phosphorylation and signal transduction
    Article Snippet: Plasmids pcDNA3-GMRα and pGMRβ-clip are pcDNA3 vector-based expression systems encoding GMRα and GMRβ-clip proteins, respectively, as previously described . pGMRβ-clip was constructed by inserting the PCR-amplified clip coding region from pCLIP vector (New England BioLabs,Ipswich, MA, USA) into the C terminus of the GMRβ gene in pCDNA3-GMRβ. .. The Cbl C381A mutant was generated using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA).

    Article Title: Newly synthesized claudins but not occludin are added to the basal side of the tight junction
    Article Snippet: .. In some experiments, stable cell lines overexpressing SNAP and CLIP-tagged cldns and mutants were generated by PCR, using pTRE human cldn4 ( ) as a template and cloning into the Bam HI/Xh oI sites in pSNAPf and pCLIPf (New England Biolabs, N9215S). .. SNAP ocln construct was made by PCR from EGFP ocln ( ).

    Imaging:

    Article Title: WDR76 Co-Localizes with Heterochromatin Related Proteins and Rapidly Responds to DNA Damage
    Article Snippet: Selected ORFs coding for PARP1, XRCC5, RPA1, CBX1, CBX3, and CBX5 were cloned into a modified pCLIP-tag™-FLAG vector. pCLIP-FLAG was constructed by inserting a DNA fragment encoding a FLAG tag followed by the restriction site Sgf1 into the multiple cloning site of the pCLIP-tag™ vector from New England Biolabs (Ipswich, MA). .. We also constructed CLIP-WDR76 and Halo-RPA1 vectors for use in a reverse tag imaging experiment.

    Polymerase Chain Reaction:

    Article Title: The SH3 regulatory domain of the hematopoietic cell kinase Hck binds ELMO via its polyproline motif
    Article Snippet: .. For mammalian expression SH3Hck coding sequence was amplified by PCR (residues 77–141) and cloned in frame with the CLIP tag of the pCLIPf vector (New England Biolabs) The cDNA encoding for the human SH3 domain of DOCK180 (DOCKSH3 : residues 9–69 of DOCK180 from Uniprot Protein Database: Q14185) was optimized for bacterial expression, synthesized (Geneart AG) and subcloned into pGEX 4T3 in frame with a TEV cleavable GST tag for bacterial expression. .. 2.2 Purification of recombinant proteins from bacteria For bacterial production and purification, the recombinant protein constructs were overexpressed in BL21(DE3) E scherichia coli strain using conventional IPTG induction (1 mM) in LB medium for 3 h at 37 °C.

    Article Title: Quantifying Transcription Factor Binding Dynamics at the Single-molecule Level in Live Cells
    Article Snippet: .. This was generated by PCR amplification from HaloTag–GR and sub cloned into the pCLIPf (N9215S, New England Biolabs) backbone with NheI and AgeI sites. .. The mEOS3–GR was generated by purification of the rat GR sequence from a AgeI/XhoI digested fragment of the pEGFP–GR vector [ ], and subsequent sub-cloning into a pre-digested AgeI/XhoI pmEOS3 plasmid, kindly provided by the Lippincott-Schwartz lab.

    Article Title: Cbl-mediated K63-linked ubiquitination of JAK2 enhances JAK2 phosphorylation and signal transduction
    Article Snippet: .. Plasmids pcDNA3-GMRα and pGMRβ-clip are pcDNA3 vector-based expression systems encoding GMRα and GMRβ-clip proteins, respectively, as previously described . pGMRβ-clip was constructed by inserting the PCR-amplified clip coding region from pCLIP vector (New England BioLabs,Ipswich, MA, USA) into the C terminus of the GMRβ gene in pCDNA3-GMRβ. .. The Cbl cDNA was kindly provided by Dr. Wallace Y. Langdon (School of Pathology and Laboratory Medicine, University of Western Australia, Australia), and sub-cloned into the pcDNA6 vector.

    Article Title: Newly synthesized claudins but not occludin are added to the basal side of the tight junction
    Article Snippet: .. In some experiments, stable cell lines overexpressing SNAP and CLIP-tagged cldns and mutants were generated by PCR, using pTRE human cldn4 ( ) as a template and cloning into the Bam HI/Xh oI sites in pSNAPf and pCLIPf (New England Biolabs, N9215S). .. SNAP ocln construct was made by PCR from EGFP ocln ( ).

    Mutagenesis:

    Article Title: Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia
    Article Snippet: .. Wt and mutant Ank clones were subcloned N-terminal of pSNAPf and pCLIPf (NEB) mammalian expression vectors using forward primer 5′-GGCGCGCCACCATGG ACTACAAA-3′ containing a restriction site for AscI and a Kozak sequence GCCACC and a reverse primer 5′-TCCGGAATTCCTCATTTT CTTCTCTCATC-3′ containing an EcoRI restriction site without stop codon. .. These vectors contained SNAP and CLIP-tags.

    Article Title: Serine Phosphorylation of the Hepatitis C Virus NS5A Protein Controls the Establishment of Replication Complexes
    Article Snippet: Then, mutant NS5A was cloned into either mSGR-luc-JFH-1 or mJFH-1 via flanking BamHI/AfeI sites, and correct insertion was confirmed by sequencing. .. For the insertion of the SNAP and CLIP tags into NS5A, the gene was amplified from the pSNAPf and pCLIPf vectors (NEB) using primers that insert a flexible linker sequence (GSSGSS) on either side of the insertion (primer sequences are available upon request).

    Article Title: Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia
    Article Snippet: .. Wt and mutant Ank clones were subcloned N-terminal of pSNAPf and pCLIPf (NEB) mammalian expression vectors using forward primer 5′-GGCGCGCCACCATGG ACTACAAA-3′ containing a restriction site for AscI and a Kozak sequence GCCACC and a reverse primer 5′-TCCGGAATTCCTCATTTT CTTCTCTCATC-3′ containing an EcoRI restriction site without stop codon. .. These vectors contained SNAP and CLIP-tags.

    Article Title: Cbl-mediated K63-linked ubiquitination of JAK2 enhances JAK2 phosphorylation and signal transduction
    Article Snippet: Plasmids pcDNA3-GMRα and pGMRβ-clip are pcDNA3 vector-based expression systems encoding GMRα and GMRβ-clip proteins, respectively, as previously described . pGMRβ-clip was constructed by inserting the PCR-amplified clip coding region from pCLIP vector (New England BioLabs,Ipswich, MA, USA) into the C terminus of the GMRβ gene in pCDNA3-GMRβ. .. The Cbl C381A mutant was generated using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA).

    Subcloning:

    Article Title: Quantifying Transcription Factor Binding Dynamics at the Single-molecule Level in Live Cells
    Article Snippet: This was generated by PCR amplification from HaloTag–GR and sub cloned into the pCLIPf (N9215S, New England Biolabs) backbone with NheI and AgeI sites. .. The mEOS3–GR was generated by purification of the rat GR sequence from a AgeI/XhoI digested fragment of the pEGFP–GR vector [ ], and subsequent sub-cloning into a pre-digested AgeI/XhoI pmEOS3 plasmid, kindly provided by the Lippincott-Schwartz lab.

    Purification:

    Article Title: Quantifying Transcription Factor Binding Dynamics at the Single-molecule Level in Live Cells
    Article Snippet: This was generated by PCR amplification from HaloTag–GR and sub cloned into the pCLIPf (N9215S, New England Biolabs) backbone with NheI and AgeI sites. .. The mEOS3–GR was generated by purification of the rat GR sequence from a AgeI/XhoI digested fragment of the pEGFP–GR vector [ ], and subsequent sub-cloning into a pre-digested AgeI/XhoI pmEOS3 plasmid, kindly provided by the Lippincott-Schwartz lab.

    Sequencing:

    Article Title: Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia
    Article Snippet: .. Wt and mutant Ank clones were subcloned N-terminal of pSNAPf and pCLIPf (NEB) mammalian expression vectors using forward primer 5′-GGCGCGCCACCATGG ACTACAAA-3′ containing a restriction site for AscI and a Kozak sequence GCCACC and a reverse primer 5′-TCCGGAATTCCTCATTTT CTTCTCTCATC-3′ containing an EcoRI restriction site without stop codon. .. These vectors contained SNAP and CLIP-tags.

    Article Title: Serine Phosphorylation of the Hepatitis C Virus NS5A Protein Controls the Establishment of Replication Complexes
    Article Snippet: .. For the insertion of the SNAP and CLIP tags into NS5A, the gene was amplified from the pSNAPf and pCLIPf vectors (NEB) using primers that insert a flexible linker sequence (GSSGSS) on either side of the insertion (primer sequences are available upon request). .. This amplicon was inserted into the aforementioned LITMUS28i subclone by BclI digestion, and correct insertion was verified by sequencing.

    Article Title: The SH3 regulatory domain of the hematopoietic cell kinase Hck binds ELMO via its polyproline motif
    Article Snippet: .. For mammalian expression SH3Hck coding sequence was amplified by PCR (residues 77–141) and cloned in frame with the CLIP tag of the pCLIPf vector (New England Biolabs) The cDNA encoding for the human SH3 domain of DOCK180 (DOCKSH3 : residues 9–69 of DOCK180 from Uniprot Protein Database: Q14185) was optimized for bacterial expression, synthesized (Geneart AG) and subcloned into pGEX 4T3 in frame with a TEV cleavable GST tag for bacterial expression. .. 2.2 Purification of recombinant proteins from bacteria For bacterial production and purification, the recombinant protein constructs were overexpressed in BL21(DE3) E scherichia coli strain using conventional IPTG induction (1 mM) in LB medium for 3 h at 37 °C.

    Article Title: Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia
    Article Snippet: .. Wt and mutant Ank clones were subcloned N-terminal of pSNAPf and pCLIPf (NEB) mammalian expression vectors using forward primer 5′-GGCGCGCCACCATGG ACTACAAA-3′ containing a restriction site for AscI and a Kozak sequence GCCACC and a reverse primer 5′-TCCGGAATTCCTCATTTT CTTCTCTCATC-3′ containing an EcoRI restriction site without stop codon. .. These vectors contained SNAP and CLIP-tags.

    Article Title: Quantifying Transcription Factor Binding Dynamics at the Single-molecule Level in Live Cells
    Article Snippet: This was generated by PCR amplification from HaloTag–GR and sub cloned into the pCLIPf (N9215S, New England Biolabs) backbone with NheI and AgeI sites. .. The mEOS3–GR was generated by purification of the rat GR sequence from a AgeI/XhoI digested fragment of the pEGFP–GR vector [ ], and subsequent sub-cloning into a pre-digested AgeI/XhoI pmEOS3 plasmid, kindly provided by the Lippincott-Schwartz lab.

    CRISPR:

    Article Title: Newly synthesized claudins but not occludin are added to the basal side of the tight junction
    Article Snippet: In some experiments, stable cell lines overexpressing SNAP and CLIP-tagged cldns and mutants were generated by PCR, using pTRE human cldn4 ( ) as a template and cloning into the Bam HI/Xh oI sites in pSNAPf and pCLIPf (New England Biolabs, N9215S). .. SNAP- and CLIP-tagged cldns and ocln, SNAP tag alone, and CRISPR/Cas9 constructs were transfected into MDCK II Tet-off cells using Lipofectamine 2000.

    Plasmid Preparation:

    Article Title: WDR76 Co-Localizes with Heterochromatin Related Proteins and Rapidly Responds to DNA Damage
    Article Snippet: .. Selected ORFs coding for PARP1, XRCC5, RPA1, CBX1, CBX3, and CBX5 were cloned into a modified pCLIP-tag™-FLAG vector. pCLIP-FLAG was constructed by inserting a DNA fragment encoding a FLAG tag followed by the restriction site Sgf1 into the multiple cloning site of the pCLIP-tag™ vector from New England Biolabs (Ipswich, MA). .. We also constructed CLIP-WDR76 and Halo-RPA1 vectors for use in a reverse tag imaging experiment.

    Article Title: Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia
    Article Snippet: Wt Ank was amplified from mouse cDNA with primers containing Hind III and EcoRI and cloned in p3xFLAG-CMV-10 mammalian expression vector (Sigma-Aldrich). .. Wt and mutant Ank clones were subcloned N-terminal of pSNAPf and pCLIPf (NEB) mammalian expression vectors using forward primer 5′-GGCGCGCCACCATGG ACTACAAA-3′ containing a restriction site for AscI and a Kozak sequence GCCACC and a reverse primer 5′-TCCGGAATTCCTCATTTT CTTCTCTCATC-3′ containing an EcoRI restriction site without stop codon.

    Article Title: Serine Phosphorylation of the Hepatitis C Virus NS5A Protein Controls the Establishment of Replication Complexes
    Article Snippet: For the insertion of the SNAP and CLIP tags into NS5A, the gene was amplified from the pSNAPf and pCLIPf vectors (NEB) using primers that insert a flexible linker sequence (GSSGSS) on either side of the insertion (primer sequences are available upon request). .. For plasmid expression, mutated NS5A-coding sequences were subcloned into the pCMV(NS3-5B) vector as described previously ( ).

    Article Title: The SH3 regulatory domain of the hematopoietic cell kinase Hck binds ELMO via its polyproline motif
    Article Snippet: .. For mammalian expression SH3Hck coding sequence was amplified by PCR (residues 77–141) and cloned in frame with the CLIP tag of the pCLIPf vector (New England Biolabs) The cDNA encoding for the human SH3 domain of DOCK180 (DOCKSH3 : residues 9–69 of DOCK180 from Uniprot Protein Database: Q14185) was optimized for bacterial expression, synthesized (Geneart AG) and subcloned into pGEX 4T3 in frame with a TEV cleavable GST tag for bacterial expression. .. 2.2 Purification of recombinant proteins from bacteria For bacterial production and purification, the recombinant protein constructs were overexpressed in BL21(DE3) E scherichia coli strain using conventional IPTG induction (1 mM) in LB medium for 3 h at 37 °C.

    Article Title: Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia
    Article Snippet: Cloning of p3x-FLAG-wt and mutant Ank in CMV10, pSNAPf, pCLIPf and pRS414 vectors Wt Ank was amplified from mouse cDNA with primers containing Hind III and EcoRI and cloned in p3xFLAG-CMV-10 mammalian expression vector (Sigma-Aldrich). .. Wt and mutant Ank clones were subcloned N-terminal of pSNAPf and pCLIPf (NEB) mammalian expression vectors using forward primer 5′-GGCGCGCCACCATGG ACTACAAA-3′ containing a restriction site for AscI and a Kozak sequence GCCACC and a reverse primer 5′-TCCGGAATTCCTCATTTT CTTCTCTCATC-3′ containing an EcoRI restriction site without stop codon.

    Article Title: A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting
    Article Snippet: pSNAP-tag(m) and pCLIP-tag(m) vectors were purchased from NEB. pSNAP-DGCR8: A thrombin site was cloned into the BamHI site of pSNAP-tag(m) to give pSNAP-thrombin. .. This product was inserted into the XhoI and NotI sites of the pSNAP-thrombin vector to give pSNAP-DGCR8. pCS3–6Myc-FLAG-TEV-Myc-Drosha: Oligonucleotides with the FLAG and TEV sequences were purchased in order to have HindIII and KpnI compatible overhangs after annealing.

    Article Title: Quantifying Transcription Factor Binding Dynamics at the Single-molecule Level in Live Cells
    Article Snippet: Paragraph title: 3.1.1. Plasmid constructs ... This was generated by PCR amplification from HaloTag–GR and sub cloned into the pCLIPf (N9215S, New England Biolabs) backbone with NheI and AgeI sites.

    Article Title: Cbl-mediated K63-linked ubiquitination of JAK2 enhances JAK2 phosphorylation and signal transduction
    Article Snippet: .. Plasmids pcDNA3-GMRα and pGMRβ-clip are pcDNA3 vector-based expression systems encoding GMRα and GMRβ-clip proteins, respectively, as previously described . pGMRβ-clip was constructed by inserting the PCR-amplified clip coding region from pCLIP vector (New England BioLabs,Ipswich, MA, USA) into the C terminus of the GMRβ gene in pCDNA3-GMRβ. .. The Cbl cDNA was kindly provided by Dr. Wallace Y. Langdon (School of Pathology and Laboratory Medicine, University of Western Australia, Australia), and sub-cloned into the pcDNA6 vector.

    Article Title: Newly synthesized claudins but not occludin are added to the basal side of the tight junction
    Article Snippet: In some experiments, stable cell lines overexpressing SNAP and CLIP-tagged cldns and mutants were generated by PCR, using pTRE human cldn4 ( ) as a template and cloning into the Bam HI/Xh oI sites in pSNAPf and pCLIPf (New England Biolabs, N9215S). .. Cldn2 KOs were made using the pSpCas9(BB)-2A-Puro vector (62988; Addgene; Ran et al., 2013; gRNA sequences in Supplemental Table 1).

    FLAG-tag:

    Article Title: WDR76 Co-Localizes with Heterochromatin Related Proteins and Rapidly Responds to DNA Damage
    Article Snippet: .. Selected ORFs coding for PARP1, XRCC5, RPA1, CBX1, CBX3, and CBX5 were cloned into a modified pCLIP-tag™-FLAG vector. pCLIP-FLAG was constructed by inserting a DNA fragment encoding a FLAG tag followed by the restriction site Sgf1 into the multiple cloning site of the pCLIP-tag™ vector from New England Biolabs (Ipswich, MA). .. We also constructed CLIP-WDR76 and Halo-RPA1 vectors for use in a reverse tag imaging experiment.

    Marker:

    Article Title: Quantifying Transcription Factor Binding Dynamics at the Single-molecule Level in Live Cells
    Article Snippet: This was generated by PCR amplification from HaloTag–GR and sub cloned into the pCLIPf (N9215S, New England Biolabs) backbone with NheI and AgeI sites. .. Finally, pEGFP-NF1[ ] was used as an homogenous nuclear marker in .

    Cross-linking Immunoprecipitation:

    Article Title: WDR76 Co-Localizes with Heterochromatin Related Proteins and Rapidly Responds to DNA Damage
    Article Snippet: Selected ORFs coding for PARP1, XRCC5, RPA1, CBX1, CBX3, and CBX5 were cloned into a modified pCLIP-tag™-FLAG vector. pCLIP-FLAG was constructed by inserting a DNA fragment encoding a FLAG tag followed by the restriction site Sgf1 into the multiple cloning site of the pCLIP-tag™ vector from New England Biolabs (Ipswich, MA). .. We also constructed CLIP-WDR76 and Halo-RPA1 vectors for use in a reverse tag imaging experiment.

    Article Title: Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia
    Article Snippet: Wt and mutant Ank clones were subcloned N-terminal of pSNAPf and pCLIPf (NEB) mammalian expression vectors using forward primer 5′-GGCGCGCCACCATGG ACTACAAA-3′ containing a restriction site for AscI and a Kozak sequence GCCACC and a reverse primer 5′-TCCGGAATTCCTCATTTT CTTCTCTCATC-3′ containing an EcoRI restriction site without stop codon. .. These vectors contained SNAP and CLIP-tags.

    Article Title: Serine Phosphorylation of the Hepatitis C Virus NS5A Protein Controls the Establishment of Replication Complexes
    Article Snippet: .. For the insertion of the SNAP and CLIP tags into NS5A, the gene was amplified from the pSNAPf and pCLIPf vectors (NEB) using primers that insert a flexible linker sequence (GSSGSS) on either side of the insertion (primer sequences are available upon request). .. This amplicon was inserted into the aforementioned LITMUS28i subclone by BclI digestion, and correct insertion was verified by sequencing.

    Article Title: The SH3 regulatory domain of the hematopoietic cell kinase Hck binds ELMO via its polyproline motif
    Article Snippet: .. For mammalian expression SH3Hck coding sequence was amplified by PCR (residues 77–141) and cloned in frame with the CLIP tag of the pCLIPf vector (New England Biolabs) The cDNA encoding for the human SH3 domain of DOCK180 (DOCKSH3 : residues 9–69 of DOCK180 from Uniprot Protein Database: Q14185) was optimized for bacterial expression, synthesized (Geneart AG) and subcloned into pGEX 4T3 in frame with a TEV cleavable GST tag for bacterial expression. .. 2.2 Purification of recombinant proteins from bacteria For bacterial production and purification, the recombinant protein constructs were overexpressed in BL21(DE3) E scherichia coli strain using conventional IPTG induction (1 mM) in LB medium for 3 h at 37 °C.

    Article Title: Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia
    Article Snippet: Wt and mutant Ank clones were subcloned N-terminal of pSNAPf and pCLIPf (NEB) mammalian expression vectors using forward primer 5′-GGCGCGCCACCATGG ACTACAAA-3′ containing a restriction site for AscI and a Kozak sequence GCCACC and a reverse primer 5′-TCCGGAATTCCTCATTTT CTTCTCTCATC-3′ containing an EcoRI restriction site without stop codon. .. These vectors contained SNAP and CLIP-tags.

    Article Title: Quantifying Transcription Factor Binding Dynamics at the Single-molecule Level in Live Cells
    Article Snippet: The CLIP-tag–GR expresses the rat GR with CLIP-tag fused in the C-terminal domain under the CMV promoter. .. This was generated by PCR amplification from HaloTag–GR and sub cloned into the pCLIPf (N9215S, New England Biolabs) backbone with NheI and AgeI sites.

    Article Title: Cbl-mediated K63-linked ubiquitination of JAK2 enhances JAK2 phosphorylation and signal transduction
    Article Snippet: .. Plasmids pcDNA3-GMRα and pGMRβ-clip are pcDNA3 vector-based expression systems encoding GMRα and GMRβ-clip proteins, respectively, as previously described . pGMRβ-clip was constructed by inserting the PCR-amplified clip coding region from pCLIP vector (New England BioLabs,Ipswich, MA, USA) into the C terminus of the GMRβ gene in pCDNA3-GMRβ. .. The Cbl cDNA was kindly provided by Dr. Wallace Y. Langdon (School of Pathology and Laboratory Medicine, University of Western Australia, Australia), and sub-cloned into the pcDNA6 vector.

    Article Title: Newly synthesized claudins but not occludin are added to the basal side of the tight junction
    Article Snippet: .. In some experiments, stable cell lines overexpressing SNAP and CLIP-tagged cldns and mutants were generated by PCR, using pTRE human cldn4 ( ) as a template and cloning into the Bam HI/Xh oI sites in pSNAPf and pCLIPf (New England Biolabs, N9215S). .. SNAP ocln construct was made by PCR from EGFP ocln ( ).

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    New England Biolabs pclipf vectors
    Pclipf Vectors, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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