pMB67
(Plasmid #47947)

• Purpose: Expresses C. elegans optimized Cas9 from the hsp-16.48 promoter
• Depositing Lab: Mike Boxem
• Publication: Waaijers et al Genetics. 2013 Aug 26.

Backbone

• Vector backbone: pBluescript SK+
• Backbone manufacturer: Stratagene
• Backbone size w/o insert (bp): 2958
• Total vector size (bp): 8127
• Vector type: Worm Expression, CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): EPI400
• Growth instructions: Plasmid isolation from standard DH5alpha results in low yield. The plasmid may need to first be retransformed into BL21 cells to obtain normal DNA yields. Requires extra growth time.
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: Cas9
• Species: Synthetic
• Insert Size (bp): 4254
• Promoter: hsp-16.48
• Tags / Fusion Proteins:
  • 3xFLAG (N terminal on insert)
  • SV40 NLS (N terminal on insert)
  • egl-13 NLS (C terminal on insert)

Cloning Information

• Cloning method: Unknown
• 5′ sequencing primer: M13F
• 3′ sequencing primer: M13R

Resource Information

• Supplemental Documents:
  • genbank file
  • features

Terms and Licenses

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

Depositor Comments

To generate the unique SpeI site, a short, 37 bp, linker was inserted into an existing NdeI site. This linker seems to have been inserted 2 or more times, creating a short repeat region that we have been unable to sequence across. Sequencing with the M13 reverse primer will fail.

This plasmid is provided in EPI400 bacteria which may need to be induced to obtain a high copy number. Please refer to this protocol: http://www.epibio.com/docs/default-source/protocols/copycutter-epi400-electrocompetent-e-coli-copycutter-epi400-chemically-competent-e-coli-copycutter-induction-solution.pdf?sfvrsn=10 for more information.

For more information on Boxem Lab CRISPR Plasmids please refer to: http://www.addgene.org/crispr/boxem/