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lenti-sgRNA puro
(Plasmid #104990)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 104990 Standard format: Plasmid sent in bacteria as agar stab 1 $75

This material is available to academics and nonprofits only.

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
  • 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

Terms and Licenses

Trademarks:
  • Zeocin® is an InvivoGen trademark.

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.

How to cite this plasmid ( Back to top)

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.

  • For your Materials & Methods section:

    lenti-sgRNA puro was a gift from Brett Stringer (Addgene plasmid # 104990 ; http://n2t.net/addgene:104990 ; RRID:Addgene_104990)
  • For your References section:

    A reference collection of patient-derived cell line and xenograft models of proneural, classical and mesenchymal glioblastoma. Stringer BW, Day BW, D'Souza RCJ, Jamieson PR, Ensbey KS, Bruce ZC, Lim YC, Goasdoue K, Offenhauser C, Akgul S, Allan S, Robertson T, Lucas P, Tollesson G, Campbell S, Winter C, Do H, Dobrovic A, Inglis PL, Jeffree RL, Johns TG, Boyd AW. Sci Rep. 2019 Mar 20;9(1):4902. doi: 10.1038/s41598-019-41277-z. 10.1038/s41598-019-41277-z PubMed 30894629