LentiCRISPRv2-mCherry
(Plasmid #99154)

• Purpose: Lentiviral vector encoding sgRNA cloning site + hSpCAS9-P2A-mCherry.
• Depositing Lab: Agata Smogorzewska
• Publication: LentiCRISPRv2-mCherry (unpublished)

Backbone

• Vector backbone: LentiCRISPRv2
• Backbone manufacturer: Feng Zhang
• Backbone size w/o insert (bp): 10000
• Total vector size (bp): 14984
• Modifications to backbone: mCherry P2A-linked to hSpCAS9 by InFusion cloning.
• Vector type: Mammalian Expression, Lentiviral, CRISPR
• Selectable markers: mCherry

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): NEB Stable
• Growth instructions: Use SapI digest to check for unwanted recombination of lentiviral plasmid. Only amplify in RecA- bacteria (eg. Stbl3).
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: Cas9
• Alt name: S. pyogenes CRISPR-Cas9
• Species: Synthetic
• Insert Size (bp): 4200
• Promoter: EFS-NS
• Tag / Fusion Protein:
  • FLAG (C terminal on insert)

Cloning Information for Gene/Insert 1

• Cloning method: Restriction Enzyme
• 5′ cloning site: AgeI, XbaI, AfeI (not destroyed)
• 3′ cloning site: BamHI (not destroyed)
• 5′ sequencing primer: hEF1aprom-F
• 3′ sequencing primer: WPRE-R

Gene/Insert 2

• Gene/Insert name: mCherry
• Species: Discoma sp.
• Insert Size (bp): 708
• Promoter: EFS-NS

Cloning Information for Gene/Insert 2

• Cloning method: Ligation Independent Cloning
• 5′ sequencing primer: ACC AAA GAG GTG CTG GAC G
• 3′ sequencing primer: WPRE-R

Resource Information

• Supplemental Documents:
  • LentiCRISPRv2_mCherry.gb
  • Cutting validation data post-FACS sort
• A portion of this plasmid was derived from a plasmid made by: LentiCRISPRv2 was produced by Feng Zhang, MIT. mCherry is the copyright of Clontech.
• Articles Citing this Plasmid:
  • 29 References

Terms and Licenses

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

Depositor Comments

This LentiCRISPRv2 variant was produced by restriction digest and multi-fragment InFusion (Clontech) assembly. It has been validated for cutting efficiency following transduction and FACS-sorting for mCherry. Suitable for single- and multi-colour guided CRISPR editing in stem cells and cell lines.

sgRNA of choice can be cloned in using BsmBI digest. Follow original LentiCRISPRv2 cloning protocol.