pdCas9-DNMT3A-PuroR (ANV)_v2 | Addgene 74408 product information

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product summary

company name :

Addgene

product type :

cDNA

product name :

pdCas9-DNMT3A-PuroR (ANV)_v2

catalog :

74408

citations: 1

Reference
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 :

74408

Product Name :

pdCas9-DNMT3A-PuroR (ANV)_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. The DNMT3A active site motif ENV was mutated to ANV (E756A). 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 mutant human codon-optimized dCas9-DNMT3A fusion (inactive catalytic domain of DNMT3A) with T2A-PuroR (v2); cloning backbone for sgRNA

origin :

pi :

alt_names

dCas9-DNMT3A-PuroR (ANV)

SpdCas9-DNMT3A-T2A-PuroR (ANV)

dCas9-DNMT3A (ANV)

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; E756A inactivating mutation in H. sapiens DNMT3A

name

S. pyogenes dCas9 fused with the inactivated (E756A) 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