URL: http://www.addgene.org/74407
Proper Citation: RRID:Addgene_74407
Insert Name: S. pyogenes dCas9 fused with the catalytic domain of human DNMT3A (amino acids P602-V912) and T2A-PuroR_v2
Organism: Homo sapiens
Bacterial Resistance: Ampicillin
Defining Citation: PMID:26969735
Vector Backbone Description: Backbone Size:4246; Vector Backbone:PX462; Vector Types:Mammalian Expression, CRISPR; Bacterial Resistance:Ampicillin
Comments: The catalytic domain of human DNMT3A (amino acids P602-V912) was derived from the plasmid pcDNA3/Myc-DNMT3A (Addgene, plasmid #35521) (Chen et al., 2005, J Cell Biochem 95: 902-917). Undesired BbsI restriction site was removed by site-directed mutagenesis, without affecting the amino acid sequence. Plasmid pSpCas9n(BB)-2A-Puro (PX462) (Addgene, plasmid #48141) (Ran et al., 2013, Nat Protoc 8: 2281-2308) was used as a backbone. Additional H840A mutation was introduced into Cas9n D10A nickase. Puromycin gene was mutated by site-directed mutagenesis (H166R) to improve its efficacy.
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Source: Addgene