pKM197
(Plasmid #132665)

• Purpose: pyrE-targeted CRISPR-Cas9 control plasmid. Cas9 expression is controlled by the xylR promoter and results in improved conjugation efficiency. Induction with xylose results in a pyrE mutant.
• Depositing Lab: Joseph Sorg
• Publication: Bhattacharjee et al mSphere. 2020 Mar 4;5(2):e00941-19. doi: 10.1128/mSphere.00941-19.

Backbone

• Vector backbone: pJS116
• Backbone size w/o insert (bp): 6324
• Modifications to backbone: Addition of Cas9 (codon optimized for C. difficile expression), tetR, region of homology for pyrE deletion, gRNA, gdh promoter
• Vector type: E. coli - C. difficile shuttle vector

Growth in Bacteria

• Bacterial Resistance(s): Chloramphenicol, 25 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: Upstream & Downstream pyrE deletion region
• Species: C. difficile
• Insert Size (bp): 2000

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: tttataaaatagttttatctacaatttttttatcaggaaacagcta
• 3′ sequencing primer: CTGCAAATGCAGGCTTCTTATTTTTA

Resource Information

• Supplemental Documents:
  • pkm197.gb

Terms and Licenses

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