lenti-sgRNA puro
(Plasmid #104990)

• Purpose: (Empty Backbone) This 3rd generation lentiviral plasmid expresses a S. pyogenes CRISPR chimeric RNA element with customizable sgRNA from a U6 promoter and puromycin resistance from an EF-1a promoter.
• Depositing Lab: Brett Stringer
• Publication: Stringer et al Sci Rep. 2019 Mar 20;9(1):4902. doi: 10.1038/s41598-019-41277-z.

Backbone

• Vector backbone: lentiCRISPRv2 puro (Addgene #98290)
• Backbone manufacturer: Brett Stringer from Feng Zhang plasmid lentiCRISPR v2 (Addgene plasmid #52961)
• Backbone size (bp): 10638
• Modifications to backbone: The first of the two MluI restriction sites of lentiCRISPR v2 (Addgene plasmid #52961) was mutated (to create lentiCRISPRv2 puro, Addgene plasmid #98290) to allow the subcloning of alternative P2A-antibiotic resistance cassettes between the BamHI and remaining MluI restriction site (see lentiCRISPRv2 hygro, Addgene plasmid #98291; lentiCRISPRv2 neo, Addgene plasmid #98292; and lentiCRISPRv2 blast, Addgene plasmid #98293). The Cas9-P2A-puro cassette of lentiCRISPRv2 puro (Addgene plasmid #98290) was removed by XbaI/MluI digestion and the puromycin resistance gene sequence was cloned in its place between the XbaI and MluI restriction sites downstream of the EF-1alpha core promoter (see also lentiGuide hygro, Addgene plasmid #104991; lentiGuide neo, Addgene plasmid #104992; and lentiGuide blast, Addgene plasmid #104993).
• Vector type: Mammalian Expression, Lentiviral, CRISPR
• Selectable markers: Puromycin

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): Stbl4
• Growth instructions: Single colonies of transformed STBL3 or STBL4 cells cultured in 5 ml of LB broth containing 100 micrograms/ml ampicillin for 20 hours at 37C, 200 rpm give good yields for plasmid minipreps.
• Copy number: High Copy

Resource Information

• Supplemental Documents:
  • Cloning protocol
• Addgene Notes:
  • Addgene Diagnostic Digest
• A portion of this plasmid was derived from a plasmid made by: Feng Zhang (Addgene plasmid # 52961)
• Articles Citing this Plasmid:
  • 8 References

Terms and Licenses

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

Depositor Comments

This 3rd generation lentiviral plasmid expresses a S. pyogenes CRISPR chimeric RNA element with a customizable sgRNA from a U6 promoter and puromycin resistance from an EF-1a core promoter.
This plasmid does NOT contain Cas9. It should be used in conjunction with lentiCas9 hygro, lentiCas9 neo or lentiCas9 blast or with cell lines expressing Cas9.
After cloning a double-stranded oligonucleotide specifying a gRNA between the two BsmBI restriction sites, use hU6-F (5'-GAGGGCCTATTTCCCATGATT-3') or LKO.1 5’(5'- GACTATCATATGCTTACCGT-3') to sequence the chimeric RNA element.
Instruction for gRNA double-stranded oligonucleotide design and cloning can be found in the Zhang lab protocol, https://media.addgene.org/data/plasmids/52/52961/52961-attachment_B3xTwla0bkYD.pdf
lentiGuide puro is essentially the same as the Feng Zhang lab plasmid lentiGuide-Puro (Plasmid #52963) but lacks one of the two MluI restriction sites, permitting subcloning of alternative selectable markers (e.g. hygro, neo, blast) between the XbaI and remaining MluI restriction site.