px330-mcherry
(Plasmid #98750)

• Purpose: (Empty Backbone) Cas9 from S.pyogenes with CMV-mcherry cassette, and cloning backbone for sgRNA
• Depositing Lab: Jinsong Li
• Publication: Wu et al Cell Stem Cell. 2013 Dec 5;13(6):659-62. doi: 10.1016/j.stem.2013.10.016.

Backbone

• Vector backbone: pX330
• Backbone manufacturer: Zhang lab (Addgene plasmid #42230)
• Modifications to backbone: CMV- mcherry-pA amplified from mCherry-C1 (clontech) and inserted in to pX330 in NotI site
• Vector type: Mammalian Expression, CRISPR
• Promoter: U6
• Selectable markers: mCherry

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): Stbl3
• Copy number: High Copy

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ sequencing primer: CATCGTGGAACAGTACGAA
• 3′ sequencing primer: CGTTCACGGAGCCCTCCA

Resource Information

• Supplemental Documents:
  • px330-mcherry.gb
• Articles Citing this Plasmid:
  • 26 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Because there exists a BbsI cleavage site on CMV-mcherry-pA cassette as well, it is suggested that the BbsI digested product need to be recovered directly instead of agarose gel purification.

User Instruction:
Digest the px330-mcherry plasmid with BbsⅠ enzyme. After that, run a small amount of the digest on an agarose gel electrophoresis to check the digestion efficiency. If the px330-mcherry plasmid has been well digested, recover the remaining digestion products using a DNA purification kit (such as Tiangen, DP214-03) for restriction enzyme cleanup. In our opinion, any commonly used DNA or PCR purification kit procedure would work well, but not agarose gel purification. We do not suggest combined digestion and ligation. For all other steps (including ligation of annealed oligos into this plasmid), refer to the Zhang lab protocol for pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene plasmid #42230).