pdCas9-DNMT3A-PuroR (ANV)_v2
(Plasmid #74408)

• Purpose: Expression of mutant human codon-optimized dCas9-DNMT3A fusion (inactive catalytic domain of DNMT3A) with T2A-PuroR (v2); cloning backbone for sgRNA
• Depositing Lab: Vlatka Zoldoš
• Publication: Vojta et al Nucleic Acids Res. 2016 Mar 11. pii: gkw159.

Backbone

• Vector backbone: PX462
• Backbone size w/o insert (bp): 4246
• Total vector size (bp): 10152
• Vector type: Mammalian Expression, CRISPR
• Selectable markers: Puromycin

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: S. pyogenes dCas9 fused with the inactivated (E756A) catalytic domain of human DNMT3A (amino acids P602-V912) and T2A-PuroR (v2)
• Alt name: dCas9-DNMT3A-PuroR (ANV)
• Alt name: SpdCas9-DNMT3A-T2A-PuroR (ANV)
• Alt name: dCas9-DNMT3A (ANV)
• Species: H. sapiens (human), Synthetic; S. pyogenes
• Insert Size (bp): 5906
• Mutation: D10A and H840A in S.pyogenes Cas9; E756A inactivating mutation in H. sapiens DNMT3A
• GenBank ID: AKA60242.1 NP_072046.2
• Entrez Gene: DNMT3A (a.k.a. DNMT3A2, HESJAS, M.HsaIIIA, TBRS)
• Promoter: CBh
• Tags / Fusion Proteins:
  • 3xFLAG (N terminal on insert)
  • SV40 NLS (N terminal on insert)
  • T2A-PuroR (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: BamHI (not destroyed)
• 3′ cloning site: FseI (not destroyed)
• 5′ sequencing primer: GS-linker For 5' GAAGAGGTACACCAGCACCAAAG 3'
• 3′ sequencing primer: BGH Rev

Resource Information

• Supplemental Documents:
  • pdCas9-DNMT3A-PuroR (ANV)_v2 annotated GenBank file
• A portion of this plasmid was derived from a plasmid made by: The plasmid is derived from Addgene plasmids #35521 and #48141.

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor 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. 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.