pdCas9-DNMT3A-PuroR_v2 | Addgene 74407 product information

This webpage contains legacy information. The product is either no longer available from the supplier or has been delisted at Labome.

product summary

company name :

Addgene

product type :

cDNA

product name :

pdCas9-DNMT3A-PuroR_v2

catalog :

74407

citations: 3

Reference
Rodden L, Gilliam K, Lam C, Rojsajjakul T, Mesaros C, Dionisi C, et al. DNA methylation in Friedreich ataxia silences expression of frataxin isoform E. Sci Rep. 2022;12:5031 pubmed publisher
Cali C, Park D, Lee E. Targeted DNA methylation of neurodegenerative disease genes via homology directed repair. Nucleic Acids Res. 2019;47:11609-11622 pubmed publisher
Vojta A, DobriniÄ‡ P, TadiÄ‡ V, BoÄkor L, KoraÄ‡ P, Julg B, et al. Repurposing the CRISPR-Cas9 system for targeted DNA methylation. Nucleic Acids Res. 2016;44:5615-28 pubmed publisher

product information

Catalog Number :

74407

Product Name :

pdCas9-DNMT3A-PuroR_v2

article :

doi	10.1093/nar/gkw159
id	18013
pubmed_id	26969735

bacterial resistance :

Ampicillin

cloning :

backbone

PX462

backbone_mutation

backbone_origin

backbone_size

4246

promoter

sequencing_primer_3

sequencing_primer_5

vector_types

Mammalian Expression

CRISPR

growth notes :

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.

growth strain :

Expression of human codon-optimized dCas9-DNMT3A fusion with T2A-PuroR (v2) for targeted DNA methylation in mammalian cells; cloning backbone for sgRNA

origin :

pi :

alt_names

dCas9-DNMT3A-PuroR

SpdCas9-DNMT3A-T2A-PuroR

dCas9-DNMT3A

cloning

clone_method	Restriction Enzyme
cloning_site_3	FseI
cloning_site_5	BamHI
promoter	CBh
sequencing_primer_3	BGH Rev
sequencing_primer_5	GS-linker For 5' GAAGAGGTACACCAGCACCAAAG 3'
site_3_destroyed
site_5_destroyed

entrez_gene

aliases	DNMT3A2, HESJAS, M.HsaIIIA, TBRS
gene	DNMT3A
id	1788

genbank_ids

AKA60242.1

NP_072046.2

mutation

D10A and H840A in S.pyogenes Cas9

name

S. pyogenes dCas9 fused with the catalytic domain of human DNMT3A (amino acids P602-V912) and T2A-PuroR_v2

shRNA_sequence

size

5906

species

9606

Homo sapiens

100

Synthetic

32644

Other

S. pyogenes