Aavs1 puro pgk1 3xflag twin strep

Manufactured by Addgene

The AAVS1_Puro_PGK1_3xFLAG_Twin_Strep is a lab equipment product that serves as a plasmid vector. It contains the AAVS1 targeting sequence, a puromycin resistance gene, and a 3xFLAG and Twin-Strep tag sequence under the control of the PGK1 promoter.

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Lab products found in correlation

Pvax1 vector, by Thermo Fisher Scientific (1 mentions) Hcas9, by Addgene (1 mentions) Pspcas9 bb 2a puro px459 v2, by Addgene (1 mentions) Pfuultra 2 fusion hotstart dna polymerase, by Agilent Technologies (1 mentions) Memerald sec61β, by Addgene (1 mentions) Pmscarlet i c1, by Addgene (1 mentions) Gblock, by Integrated DNA Technologies (1 mentions)

3 protocols using aavs1 puro pgk1 3xflag twin strep

CRISPR Cas9 Inhibitor Protein Expression

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SpCas9 and associated sgRNAs were expressed from pX458^{5 (link)} (Addgene plasmid #48138, a gift from Feng Zhang). Human expression vectors for S. thermophilus St1Cas9 and associated sgRNA were a gift from Keith Joung (Addgene plasmids #65775 and #65778)^{59 (link)}. Spacer (guide) sequences for SpCas9 and St1Cas9 have been described previously^{59 (link),60 (link)} and are provided in Supplementary Table 3. Untagged AcrIIA proteins were expressed transiently from a modified pVAX1 vector (Thermo Fisher Scientific) containing a beta-globin intron. The AcrIIA ORFs were codon-optimized for expression in human cells and synthesized as gBlocks gene fragments (IDT) (Supplementary Table 4). To establish cell lines stably expressing the various AcrIIAs, gBlock gene fragments were cloned into AAVS1_Puro_PGK1_3xFLAG_Twin_Strep (Addgene plasmid #68375)^{58 (link)}.

Hynes A.P., Rousseau G.M., Agudelo D., Goulet A., Amigues B., Loehr J., Romero D.A., Fremaux C., Horvath P., Doyon Y., Cambillau C, & Moineau S. (2018). Widespread anti-CRISPR proteins in virulent bacteriophages inhibit a range of Cas9 proteins. Nature Communications, 9, 2919.

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LDAF1-FLAG-Seipin(1-310) Complex Expression

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The following plasmids were kind gifts: ERoxBFP (Addgene plasmid #68126) from Erik Snapp, mEmerald-Sec61β (Addgene plasmid #54249) from Michael Davidson, hCas9 (Addgene plasmid #41815) and gRNA-AAVS1-T2 (Addgene plasmid #41818) from George Church, AAVS1_Puro_PGK1_3xFLAG_Twin_Strep (Addgene plasmid #68375) from Yannick Doyon, pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene plasmid #62988) from Feng Zhang, and pmScarlet-i_C1 (Addgene plasmids #85044) from Dorus Gadella. pEGFP-N1 and pEGFP-C1 plasmids were purchased from Clontech Laboratories, pSMART-HC-Amp plasmid was purchased from Lucigen. pCAG-LNK vector was modified from pCAGEN (Addgene plasmid #11160) as described (Scheich et al., 2007).
For plasmid construction, all PCRs were performed using PfuUltra II Fusion HotStart DNA Polymerase (#600672, Agilent Technologies) and restriction enzymes were from New England Biolabs. The synthetic DNAs (gBlock, Integrated DNA Technologies) that were used in this study and cloning strategies of the other plasmids (including primer information) were summarized in Table S3 and S4, respectively.
For expression of the LDAF1-FLAG-seipin(1-310) complex, pCAG-LDAF1-FLAG-Seipin(1-310) plasmid was generated by pCAG-LNK-LDAF1-FLAG and pCAG-LNK-Seipin(1-310) using approach described in Scheich et al.(Scheich et al., 2007). The detailed cloning strategies were summarized in Table S3 and S4.

Chung J., Wu X., Lambert T.J., Lai Z.W., Walther T.C, & Farese RV J.r. (2019). Lipid droplet assembly factor-1 and seipin form a lipid droplet assembly complex. Developmental cell, 51(5), 551-563.e7.

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AAVS1-targeted MADR expression system

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AAVS1 targeting MADR vector was derived from AAVS1-targeting vector AAVS1_Puro_PGK1_3xFLAG_Twin_Strep (Addgene 68375). TagBFP2-V5-nls-P2A-puroR-Cag-LoxP-TdTomato-FRT was inserted into this AAVS1 vector, and a human cell line was transfected with it and selected in puromycin. MADR-smFP-myc (bright) and MADR-TagBFP2–3flag WPRE was transfected into the resulting stable cell line with Cag-Flpo-2A-Cre to induce the MADR reaction.

Kim G.B., Pacheco D.R., Saxon D., Yang A., Sabet S., Dutra-Clarke M., Levy R., Watkins A., Park H., Akhtar A.A., Linesch P., Kobritz N., Chandra S.S., Grausam K., Ayala-Sarmiento A., Molina J., Sedivakova K., Hoang K., Tsyporin J., Gareau D.S., Filbin M.G., Bannykh S., Santiskulvong C., Wang Y., Tang J., Suva M.L., Chen B., Danielpour M, & Breunig J.J. (2019). Rapid generation of somatic mouse mosaics with locus-specific, stably-integrated transgenic elements. Cell, 179(1), 251-267.e24.

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