AV44_pCAG.Cas9-D10A.gRNA.S1
(Plasmid #199258)

• Purpose: Expression construct encoding SpCas9-D10A nickase and an AAVS1-targeting guide RNA
• Depositing Lab: Manuel Goncalves
• Publication: Wang et al Nucleic Acids Res. 2021 Jan 25;49(2):1173-1198. doi: 10.1093/nar/gkaa1236.

Backbone

• Vector backbone: pBR322
• Backbone size w/o insert (bp): 3196
• Total vector size (bp): 9451
• Vector type: Mammalian Expression, CRISPR

Growth in Bacteria

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

Gene/Insert 1

• Gene/Insert name: SpCas9-D10A nickase
• Species: Streptococcus pyogenes
• Insert Size (bp): 4137
• Mutation: D10A
• Promoter: CAG promoter
• Tag / Fusion Protein:
  • SV40 NLS (C terminal on insert)

Cloning Information for Gene/Insert 1

• Cloning method: Unknown
• 5′ sequencing primer: M13 rev
• 3′ sequencing primer: M13 fwd

Gene/Insert 2

• Gene/Insert name: AAVS1-targeting gRNA
• Alt name: AAVS1-T2
• Species: Synthetic
• Promoter: RNA polymerase III promoter for human U6 snRNA

Cloning Information for Gene/Insert 2

• Cloning method: Restriction Enzyme
• 5′ cloning site: BveI (destroyed during cloning)
• 3′ cloning site: BveI (destroyed during cloning)
• 5′ sequencing primer: M13 rev
• 3′ sequencing primer: M13 fwd

Resource Information

• Supplemental Documents:
  • AV44_pCAG.Cas9-D10A.gRNA.S1.gbk
• A portion of this plasmid was derived from a plasmid made by: The SpCas9 insert, derived from Addgene plasmid #41815, and gRNA AAVS1-T2 are described in: Mali et al. Science 2013 339:823-826.

Terms and Licenses

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

Depositor Comments

The SpCas9 insert was modified via the introduction of the RuvC-disabling mutation D10A and placed in a CAG-based expression construct together with an AAVS1-targeting gRNA.