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pU6-(BbsI)_CBh-Cas9-T2A-mCherry-H1-(BamHI)
(Plasmid #64217)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 64217 Standard format: Plasmid sent in bacteria as agar stab 1 $75

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pU6-(BbsI)_CBh-Cas9-T2A-BFP
  • Backbone manufacturer
    Kuhn lab (Addgene plasmid # 64323)
  • Modifications to backbone
    The human H1 promoter was cloned into the NotI site of the CRISPR/Cas9-T2A-BFP plasmid
  • Vector type
    Mammalian Expression, CRISPR

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin
  • 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-mCherry
  • 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-mCherry (C terminal on insert)

Cloning Information for Gene/Insert 1

Gene/Insert 2

  • Gene/Insert name
    hU6 promoter; BbsI sites for sgRNA
  • 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)

Gene/Insert 3

  • Gene/Insert name
    H1 promoter; BamHI site for shRNA
  • Promoter H1

Cloning Information for Gene/Insert 3

  • Cloning method Restriction Enzyme
  • 5′ cloning site BamHI (unknown if destroyed)
  • 3′ cloning site AflII (unknown if destroyed)
  • 5′ sequencing primer H1 (5'-tcgctatgtgttctgggaaa-3')
  • (Common Sequencing Primers)

Resource Information

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-mCherry-H1-(BamHI) was a gift from Ralf Kuehn (Addgene plasmid # 64217 ; http://n2t.net/addgene:64217 ; RRID:Addgene_64217)
  • 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