pIA33
(Plasmid #120803)

• Purpose: E. coli-C. difficile shuttle vector for CRISPR interference (CRISPRi) in C. difficile; sgRNA targets rfp; Pxyl::dCas9-opt Pgdh::sgRNA-rfp
• Depositing Labs: Craig Ellermeier, David S. Weiss
• Publication: Muh et al J Bacteriol. 2019 Feb 11. pii: JB.00711-18. doi: 10.1128/JB.00711-18.

Backbone

• Vector backbone: pAP114
• Backbone size w/o insert (bp): 6400
• Total vector size (bp): 12940
• Vector type: Bacterial Expression, CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Chloramphenicol, 25 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: Low copy in C. difficile following conjugation and selected with thiamphenicol
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: deactivated nuclease dCas9, codon-optimized for C. difficile
• Alt name: dCas9-opt
• Species: S. pyogenes
• Insert Size (bp): 4107
• Mutation: Codon optimized for C. difficile; H840A and D10A to deactivate nuclease
• Promoter: Pxyl

Cloning Information for Gene/Insert 1

• Cloning method: Gibson Cloning
• 5′ sequencing primer: unknown
• 3′ sequencing primer: unknown

Gene/Insert 2

• Gene/Insert name: single guide RNA targeting red fluorescent protein
• Alt name: sgRNA-rfp
• Species: Synthetic; S. pyogenes
• Insert Size (bp): 140
• Mutation: Base-pairing region targets red fluorescent protein (rfp)
• Promoter: Pgdh

Cloning Information for Gene/Insert 2

• Cloning method: Gibson Cloning
• 5′ sequencing primer: unknown
• 3′ sequencing primer: unknown

Resource Information

• Supplemental Documents:
  • pIA33.gb

Terms and Licenses

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