C-Terminal Split Cas9 with GyrA intein
(Plasmid #58694)

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

Backbone

• Vector backbone: Plasmid 42230: pX330-U6-Chimeric_BB-CBh-hSpCas9
• Backbone size w/o insert (bp): 6580
• Total vector size (bp): 6812
• Modifications to backbone: Inserted 5' ITR using Afl3 cloning site of pX330 vector and split hSpCas9 using AgeI/PmlI to replace 5' portion of hSpCas9 with Gyra Csplit intein that when combined with the GyraNsplit intein backbone splices the two components together forming full length hSpCas9
• Vector type: Mammalian Expression, AAV, CRISPR

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: Humanized C-Terminal S. pyogenes Cas9 with GyrA Csplit Intein
• Alt name: GyraC_hSpCas9
• Alt name: tsCas9-C
• Species: H. sapiens (human)
• Insert Size (bp): 237
• Promoter: CBh
• Tags / Fusion Proteins:
  • GyrA Csplit Intein (N terminal on insert)
  • NLS (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: AgeI (not destroyed)
• 3′ cloning site: PmlI (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 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