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pU6-(BbsI)_CBh-Cas9-T2A-mcherry-P2A-Ad4E1B
(Plasmid #64221)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 64221 Standard format: Plasmid sent in bacteria as agar stab 1 $65

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pU6-(BbsI)_CBh-Cas9-T2A-mCherry
  • Backbone manufacturer
    Kuhn lab (Addgene plasmid # 64324)
  • Modifications to backbone
    Mammalian codon–optimized sequence encoding the adenoviral serotype 4 E1B55K protein and mCherry was cloned into the NheI & EcoRI sites of the CRISPR/Cas9-T2A-mCherry plasmid by Gibson assembly.
  • 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-P2A-E1B
  • 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-P2A-E1B (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 see comments and supplemental documents (unknown if destroyed)
  • 3′ cloning site see comments and supplemental documents (unknown if destroyed)
  • 5′ sequencing primer hU6-F (5'-GAGGGCCTATTTCCCATGATT-3')
  • (Common Sequencing Primers)

Resource Information

Depositor Comments

Please note that due to the presence of BbsI sites in the Ad4 coding regions it is NOT possible to directly clone new sgRNA sequences into the BbsI sites downstream of the U6 promoter.

Therefore, precloned U6-sgRNA cassettes, isolated as 1.7 kb PvuI-XbaI fragments from pU6-(BbsI)_CBh-Cas9-T2A-BFP (plasmid# 64323) or pU6-(BbsI)_CBh-Cas9-T2A-mCherry (plasmid# 64324) of this deposit, or any other plasmid derived from pX330 (plasmid# 42230), must be ligated into the PvuI-XbaI digested backbone of this Ad4 containing plasmid.

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-P2A-Ad4E1B was a gift from Ralf Kuehn (Addgene plasmid # 64221 ; http://n2t.net/addgene:64221 ; RRID:Addgene_64221)
  • 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