N-Terminal Split Cas9 D10A Nickase with GyrA intein
(Plasmid #58695)

• Purpose: Expresses N-terminus of D10A SpCas9 nickase domain fused to a GyrA intein, flanked by ITRs for AAV packaging. Combine with C-Terminal Split Cas9 Gyra Intein for full length SpCas9 nickase production
• Depositing Lab: Gang Bao
• Publication: Fine et al Sci Rep. 2015 Jul 1;5:10777. doi: 10.1038/srep10777.

Backbone

• Vector backbone: Plasmid 42235: pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A)
• Backbone size w/o insert (bp): 6412
• Total vector size (bp): 6769
• Modifications to backbone: Inserted 5' ITR using Afl3 site in pX335 backbone as well as split the hSpCas9 D10A nickase using EcoRI/EcoRV and replaced the c-terminus with the GyrA Nsplit intein for protein trans-splicing with the C-Terminal Split Cas9 with GyrA intein plasmid
• Vector type: Mammalian Expression, AAV, CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): Top10
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: D10A Nickase humanized S. pyogenes Cas9 with Gyra Nsplit Intein
• Alt name: GyraN_hSpCas9n (D10A)
• Alt name: tsCas9-Nick
• Species: H. sapiens (human)
• Insert Size (bp): 357
• Mutation: D10A nickase converting mutation to SpCas9
• Promoter: CBh
• Tags / Fusion Proteins:
  • GyrA Nsplit Intein (C terminal on insert)
  • NLS (N terminal on insert)
  • HA Tag (N terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: EcoRV (not destroyed)
• 3′ cloning site: EcoRI (not destroyed)
• 5′ sequencing primer: cctccgggctgtaattagc
• 3′ sequencing primer: AGGGTCAAGGAAGGCACG

Resource Information

• A portion of this plasmid was derived from a plasmid made by: The Bao Lab modified the plasmids pX330 and pX335, which were received from AddGene (#42230 and #42335)and originally deposited by Feng Zhang

Terms and Licenses

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