pMSCV-Cas9-2A-GFP-sgRNA
(Plasmid #124889)

• Purpose: Contains spCas9-2A-GFP and guide RNA scaffold in single retroviral vector
• Depositing Lab: Harvey Lodish
• Publication: Li et al Haematologica. 2016 Jun;101(6):e216-9. doi: 10.3324/haematol.2015.135723. Epub 2016 Mar 11.

Backbone

• Vector backbone: pMCSV
• Backbone size w/o insert (bp): 3000
• Total vector size (bp): 10500
• Vector type: Retroviral

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: Cas9
• Alt name: S. pyogenes CRISPR-Cas9
• Species: Synthetic; Streptococcus Pyogenes
• Insert Size (bp): 4200
• Promoter: MSCV
• Tag / Fusion Protein:
  • 2A-GFP (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: XhoI (not destroyed)
• 3′ cloning site: EcoRI (not destroyed)
• 5′ sequencing primer: Gag

Resource Information

• Supplemental Documents:
  • pXZ201-Cas9-2A-GFP-BbsI-gRNA.gcc
  • guid RNA cloning protocol
• A portion of this plasmid was derived from a plasmid made by: spCas9 kind gift from Feng Zhang lab
• Articles Citing this Plasmid:
  • 2 References

Terms and Licenses

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

Depositor Comments

Guide RNA to be cloned into BbsI site. Supplied modified golden-gate cloning method is required given that extra BbsI site is located within the 2A sequence.

To design guide RNAs:
-add ACCG to 5' end of positive strand (positive strand defined as strand which will contact PAM sequence at 3' end)
-add AAAC to 5' end of negative strand