pMB63
(Plasmid #47945)

• Purpose: Expresses C. elegans optimized Cas9 from the eef-1A.1 (eft-3) 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): 8380
• Vector type: Worm Expression, CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): BL21
• Growth instructions: Plasmid isolation from standard DH5alpha results in low yield. We isolate this plasmid from BL21 cells, which results in normal yields.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: Cas9
• Species: Synthetic
• Insert Size (bp): 4254
• Promoter: eef-1A.1 (eft-3)
• 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:
  • Snapgene file
  • 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.

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

Please note: eft-3 has officially been changed to eef-1A.1 Please see the eef-1A.1 WormBase entry: http://www.wormbase.org/species/c_elegans/gene/WBGene00001168#05-9g-3