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pwpi backbone  (Addgene inc)


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    Addgene inc pwpi backbone
    Pwpi Backbone, supplied by Addgene inc, used in various techniques. Bioz Stars score: 95/100, based on 220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pwpi+backbone/pWPI+(Plasmid+%2312254)/bio_rxiv__2025__11__26__690702-58-10-17
    Average 95 stars, based on 220 article reviews
    pwpi backbone - by Bioz Stars, 2026-10
    95/100 stars

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    Related Articles

    Construct:

    Article Title: Systematic identification of post-transcriptional regulatory modules
    Article Snippet: .. In order to construct the cell lines stably expressing BioID2-RBP fusion proteins, we first cloned in an open reading frame of BioID2 enzyme , followed by a linker (YPAFLYKVVYGGGGSGGGGSGGGGS) and attR-flanked ccdB counterselection marker for Gateway cloning, into the pWPI backbone (Addgene #12254). .. The resulting backbone is named pWPI_GW_BioID2_T2A_Blast (Addgene #135448) and is available on Addgene (#214831).

    Article Title: Systematic Identification of Post-Transcriptional Regulatory Modules
    Article Snippet: .. In order to construct the cell lines stably expressing BioID2-RBP fusion proteins, we first cloned in an open reading frame of BioID2 enzyme , followed by a linker (YPAFLYKVVYGGGGSGGGGSGGGGS) and attR-flanked ccdB counterselection marker for Gateway cloning, into the pWPI backbone (Addgene #12254). .. We then used Gateway LR Clonase II Enzyme mix (Thermo Fisher) to clone the open reading frames of the RBPs of interest (from ORFeome entry clone library ( )) into the destination vector.

    Stable Transfection:

    Article Title: Systematic identification of post-transcriptional regulatory modules
    Article Snippet: .. In order to construct the cell lines stably expressing BioID2-RBP fusion proteins, we first cloned in an open reading frame of BioID2 enzyme , followed by a linker (YPAFLYKVVYGGGGSGGGGSGGGGS) and attR-flanked ccdB counterselection marker for Gateway cloning, into the pWPI backbone (Addgene #12254). .. The resulting backbone is named pWPI_GW_BioID2_T2A_Blast (Addgene #135448) and is available on Addgene (#214831).

    Article Title: Systematic Identification of Post-Transcriptional Regulatory Modules
    Article Snippet: .. In order to construct the cell lines stably expressing BioID2-RBP fusion proteins, we first cloned in an open reading frame of BioID2 enzyme , followed by a linker (YPAFLYKVVYGGGGSGGGGSGGGGS) and attR-flanked ccdB counterselection marker for Gateway cloning, into the pWPI backbone (Addgene #12254). .. We then used Gateway LR Clonase II Enzyme mix (Thermo Fisher) to clone the open reading frames of the RBPs of interest (from ORFeome entry clone library ( )) into the destination vector.

    Expressing:

    Article Title: Systematic identification of post-transcriptional regulatory modules
    Article Snippet: .. In order to construct the cell lines stably expressing BioID2-RBP fusion proteins, we first cloned in an open reading frame of BioID2 enzyme , followed by a linker (YPAFLYKVVYGGGGSGGGGSGGGGS) and attR-flanked ccdB counterselection marker for Gateway cloning, into the pWPI backbone (Addgene #12254). .. The resulting backbone is named pWPI_GW_BioID2_T2A_Blast (Addgene #135448) and is available on Addgene (#214831).

    Article Title: Systematic Identification of Post-Transcriptional Regulatory Modules
    Article Snippet: .. In order to construct the cell lines stably expressing BioID2-RBP fusion proteins, we first cloned in an open reading frame of BioID2 enzyme , followed by a linker (YPAFLYKVVYGGGGSGGGGSGGGGS) and attR-flanked ccdB counterselection marker for Gateway cloning, into the pWPI backbone (Addgene #12254). .. We then used Gateway LR Clonase II Enzyme mix (Thermo Fisher) to clone the open reading frames of the RBPs of interest (from ORFeome entry clone library ( )) into the destination vector.

    Clone Assay:

    Article Title: Systematic identification of post-transcriptional regulatory modules
    Article Snippet: .. In order to construct the cell lines stably expressing BioID2-RBP fusion proteins, we first cloned in an open reading frame of BioID2 enzyme , followed by a linker (YPAFLYKVVYGGGGSGGGGSGGGGS) and attR-flanked ccdB counterselection marker for Gateway cloning, into the pWPI backbone (Addgene #12254). .. The resulting backbone is named pWPI_GW_BioID2_T2A_Blast (Addgene #135448) and is available on Addgene (#214831).

    Article Title: Systematic Identification of Post-Transcriptional Regulatory Modules
    Article Snippet: .. In order to construct the cell lines stably expressing BioID2-RBP fusion proteins, we first cloned in an open reading frame of BioID2 enzyme , followed by a linker (YPAFLYKVVYGGGGSGGGGSGGGGS) and attR-flanked ccdB counterselection marker for Gateway cloning, into the pWPI backbone (Addgene #12254). .. We then used Gateway LR Clonase II Enzyme mix (Thermo Fisher) to clone the open reading frames of the RBPs of interest (from ORFeome entry clone library ( )) into the destination vector.

    Marker:

    Article Title: Systematic identification of post-transcriptional regulatory modules
    Article Snippet: .. In order to construct the cell lines stably expressing BioID2-RBP fusion proteins, we first cloned in an open reading frame of BioID2 enzyme , followed by a linker (YPAFLYKVVYGGGGSGGGGSGGGGS) and attR-flanked ccdB counterselection marker for Gateway cloning, into the pWPI backbone (Addgene #12254). .. The resulting backbone is named pWPI_GW_BioID2_T2A_Blast (Addgene #135448) and is available on Addgene (#214831).

    Article Title: Systematic Identification of Post-Transcriptional Regulatory Modules
    Article Snippet: .. In order to construct the cell lines stably expressing BioID2-RBP fusion proteins, we first cloned in an open reading frame of BioID2 enzyme , followed by a linker (YPAFLYKVVYGGGGSGGGGSGGGGS) and attR-flanked ccdB counterselection marker for Gateway cloning, into the pWPI backbone (Addgene #12254). .. We then used Gateway LR Clonase II Enzyme mix (Thermo Fisher) to clone the open reading frames of the RBPs of interest (from ORFeome entry clone library ( )) into the destination vector.

    Cloning:

    Article Title: Systematic identification of post-transcriptional regulatory modules
    Article Snippet: .. In order to construct the cell lines stably expressing BioID2-RBP fusion proteins, we first cloned in an open reading frame of BioID2 enzyme , followed by a linker (YPAFLYKVVYGGGGSGGGGSGGGGS) and attR-flanked ccdB counterselection marker for Gateway cloning, into the pWPI backbone (Addgene #12254). .. The resulting backbone is named pWPI_GW_BioID2_T2A_Blast (Addgene #135448) and is available on Addgene (#214831).

    Article Title: Systematic Identification of Post-Transcriptional Regulatory Modules
    Article Snippet: .. In order to construct the cell lines stably expressing BioID2-RBP fusion proteins, we first cloned in an open reading frame of BioID2 enzyme , followed by a linker (YPAFLYKVVYGGGGSGGGGSGGGGS) and attR-flanked ccdB counterselection marker for Gateway cloning, into the pWPI backbone (Addgene #12254). .. We then used Gateway LR Clonase II Enzyme mix (Thermo Fisher) to clone the open reading frames of the RBPs of interest (from ORFeome entry clone library ( )) into the destination vector.

    Amplification:

    Article Title: Homotypic endoplasmic reticulum membrane tethering is critical for flavivirus replication
    Article Snippet: For all ATL2 expression constructs, the NEBuilder HiFi cloning system was used as per the manufacturer’s protocol (New England Biolabs). .. ATL2 mutants were amplified from full-length ATL2-containing plasmids in the pWPI backbone (a gift from Didier Trono, Addgene plasmid #12254); we previously introduced synonymous mutations in the ATL2 gene sequence to remove cryptic bacterial promoters and facilitate plasmid amplification in bacterial strains . .. Sequences were transferred to the pWPI backbone using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) and subsequently transformed into competent DH5α Escherichia coli (Invitrogen).

    Plasmid Preparation:

    Article Title: Homotypic endoplasmic reticulum membrane tethering is critical for flavivirus replication
    Article Snippet: For all ATL2 expression constructs, the NEBuilder HiFi cloning system was used as per the manufacturer’s protocol (New England Biolabs). .. ATL2 mutants were amplified from full-length ATL2-containing plasmids in the pWPI backbone (a gift from Didier Trono, Addgene plasmid #12254); we previously introduced synonymous mutations in the ATL2 gene sequence to remove cryptic bacterial promoters and facilitate plasmid amplification in bacterial strains . .. Sequences were transferred to the pWPI backbone using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) and subsequently transformed into competent DH5α Escherichia coli (Invitrogen).

    Sequencing:

    Article Title: Homotypic endoplasmic reticulum membrane tethering is critical for flavivirus replication
    Article Snippet: For all ATL2 expression constructs, the NEBuilder HiFi cloning system was used as per the manufacturer’s protocol (New England Biolabs). .. ATL2 mutants were amplified from full-length ATL2-containing plasmids in the pWPI backbone (a gift from Didier Trono, Addgene plasmid #12254); we previously introduced synonymous mutations in the ATL2 gene sequence to remove cryptic bacterial promoters and facilitate plasmid amplification in bacterial strains . .. Sequences were transferred to the pWPI backbone using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) and subsequently transformed into competent DH5α Escherichia coli (Invitrogen).

    other:

    Article Title: Control of Gene Expression by RNA Binding Proteins and RNA Secondary Structure Elements
    Article Snippet: Guide RNA sequences for CRISPRi-mediated gene knockdown were cloned into pCRISPRia-v2 (Addgene #84832) via BstXI-BlpI sites.



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    Image Search Results


    The USB1 de novo variant is catalytically active and correctly processes U6 snRNA. (A) Total RNA extracted from the indicated BEBV cell lines were treated with T4 PNK or with buffer only (PNKBuff) in mild acidic conditions. RNA was subsequently treated with poly(A) polymerase (PAP). Nontreated RNA was loaded as a control, ( n = 2). (B) 3′ RACE analysis of U6 oligo(U) tails in the indicated cell lines. At least 24 clones per sample in each experiment ( n = 2) were sequenced. Bars and error bars are averages of the number of U's within U6 oligo(U) tails and SEM from two independent experiments. (C and D) Indicated cell lines were treated with actinomycin D for 0, 4, and 8 h. RNA samples were processed by northern blotting for detection of U6 and 5S ( n = 2). L: marker of known length (67 nucleotides). U6 signals were normalized through the corresponding 5S signals and successively expressed as fold decrease over U6 signal at time 0. Error bars are averages of SEM from two independent experiments. (E and F) U6 relative abundance quantification by qPCR analysis on patients and control cell lines (ctr1 n = 2, ctr2 n = 3, ctr3 n = 1, USB1 −/− n = 3, P1 n = 3) (E), and USB1 −/− cells transduced with the indicated lentiviral constructs ( n = 2) (F). U6 signals were normalized through the corresponding 5S signals and successively expressed as fold decrease over U6 signal at time 0. Error bars are averages of SEM from two independent experiments. EV, empty vector. Source data are available for this figure: .

    Journal: Journal of Human Immunity

    Article Title: A heterozygous USB1 variant linked to immunodeficiency

    doi: 10.70962/jhi.20250110

    Figure Lengend Snippet: The USB1 de novo variant is catalytically active and correctly processes U6 snRNA. (A) Total RNA extracted from the indicated BEBV cell lines were treated with T4 PNK or with buffer only (PNKBuff) in mild acidic conditions. RNA was subsequently treated with poly(A) polymerase (PAP). Nontreated RNA was loaded as a control, ( n = 2). (B) 3′ RACE analysis of U6 oligo(U) tails in the indicated cell lines. At least 24 clones per sample in each experiment ( n = 2) were sequenced. Bars and error bars are averages of the number of U's within U6 oligo(U) tails and SEM from two independent experiments. (C and D) Indicated cell lines were treated with actinomycin D for 0, 4, and 8 h. RNA samples were processed by northern blotting for detection of U6 and 5S ( n = 2). L: marker of known length (67 nucleotides). U6 signals were normalized through the corresponding 5S signals and successively expressed as fold decrease over U6 signal at time 0. Error bars are averages of SEM from two independent experiments. (E and F) U6 relative abundance quantification by qPCR analysis on patients and control cell lines (ctr1 n = 2, ctr2 n = 3, ctr3 n = 1, USB1 −/− n = 3, P1 n = 3) (E), and USB1 −/− cells transduced with the indicated lentiviral constructs ( n = 2) (F). U6 signals were normalized through the corresponding 5S signals and successively expressed as fold decrease over U6 signal at time 0. Error bars are averages of SEM from two independent experiments. EV, empty vector. Source data are available for this figure: .

    Article Snippet: Those inserts (with the addition of an HA tag at the C-terminal when indicated) were subcloned into the lentiviral pWPI backbone (RRID:Addgene_12254) by GenScript.

    Techniques: Variant Assay, Control, Clone Assay, Northern Blot, Marker, Transduction, Construct, Plasmid Preparation