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pU6-(BbsI)_CBh-Cas9-T2A-BFP
(Plasmid #64323)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 64323 Standard format: Plasmid sent in bacteria as agar stab 1 $85

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pX330
  • Backbone manufacturer
    Zhang lab (Addgene plasmid # 42230)
  • Modifications to backbone
    The T2A-BFP fragment was cloned into FseI & EcoRI sites of pX330 (Addgene plasmid # 42230). The BFP template was derived from MSCV-IRES-BFP, a kind gift of Frank Rosenbauer and Martin Janz (Charite, Berlin)
  • Vector type
    Mammalian Expression, CRISPR

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin, 100 μg/mL
  • Growth Temperature
    37°C
  • Growth Strain(s)
    DH5alpha
  • Copy number
    High Copy

Gene/Insert 1

  • Gene/Insert name
    Cas9
  • Alt name
    3xFLAG-NLS-Cas9-NLS-T2A-EBFP2
  • Species
    Streptococcus pyogenes
  • Insert Size (bp)
    4500
  • GenBank ID
    NC_002737.1
  • Promoter CBh
  • Tags / Fusion Proteins
    • 3xFLAG (N terminal on insert)
    • NLS (N terminal on insert)
    • NLS (C terminal on insert)
    • T2A-EBFP2 (C terminal on insert)

Cloning Information for Gene/Insert 1

Gene/Insert 2

  • Gene/Insert name
    sgRNA cassette
  • Promoter U6

Cloning Information for Gene/Insert 2

  • Cloning method Restriction Enzyme
  • 5′ cloning site BbsI (unknown if destroyed)
  • 3′ cloning site BbsI (unknown if destroyed)
  • 5′ sequencing primer hU6-F (5'-GAGGGCCTATTTCCCATGATT-3')
  • (Common Sequencing Primers)

Resource Information

Terms and Licenses

  • Academic/Nonprofit Terms
  • Industry Terms
    • Not Available to Industry
Trademarks:
  • Zeocin® is an InvivoGen trademark.

Depositor Comments

Please see the supplemental documents "Plasmid gb file" and "Cloning protocol" for more information on using this plasmid.

To confirm a sgRNA sequence cloned into this plasmid using the BbsI sites, use the hU6-F primer (5'-GAGGGCCTATTTCCCATGATT-3').

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:

    pU6-(BbsI)_CBh-Cas9-T2A-BFP was a gift from Ralf Kuehn (Addgene plasmid # 64323 ; http://n2t.net/addgene:64323 ; RRID:Addgene_64323)
  • For your References section:

    Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kuhn R. Nat Biotechnol. 2015 Mar 24. doi: 10.1038/nbt.3198. 10.1038/nbt.3198 PubMed 25803306