lenti-sgRNA hygro
(Plasmid #104991)

• Purpose: (Empty Backbone) This 3rd generation lentiviral plasmid expresses a S. pyogenes CRISPR chimeric RNA element with customizable sgRNA from a U6 promoter and hygromycin 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 hygro (Addgene #98291)
• Backbone manufacturer: Brett Stringer from Feng Zhang plasmid lentiCRISPR v2 (Addgene plasmid #52961)
• Backbone size (bp): 11022
• 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-hygro cassette of lentiCRISPRv2 hygro (Addgene plasmid #98291) was removed by XbaI/MluI digestion and the hygromycin 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 puro, Addgene plasmid #104990; lentiGuide neo, Addgene plasmid #104992; and lentiGuide blast, Addgene plasmid #104993).
• Vector type: Mammalian Expression, Lentiviral, CRISPR
• Selectable markers: Hygromycin

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: The hygromycin resistance gene sequence was amplified by PCR from pF_GVP16_hygro (a gift from John Silke, Cell Death and Differentiation (2008) 15, 213–219). The BsmBI restriction site it contains was removed by site-directed mutagenesis.
• Articles Citing this Plasmid:
  • 6 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 hygromycin resistance gene from an EF-1a core promoter.
This plasmid does NOT contain Cas9. It should be used in conjunction with lentiCas9 puro, 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