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pAC1-pCR8-dCas9VP48
(Plasmid #48214)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 48214 Plasmid sent as bacteria in agar stab 1 $65

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pCR8/GW/TOPO
  • Backbone manufacturer
    Invitrogen
  • Backbone size w/o insert (bp) 2799
  • Total vector size (bp) 7148
  • Vector type
    CRISPR ; Gateway Donor

Growth in Bacteria

  • Bacterial Resistance(s)
    Spectinomycin
  • Growth Temperature
    37°C
  • Growth Strain(s)
    DH5alpha
  • Copy number
    Unknown

Gene/Insert

  • Gene/Insert name
    dCas9(D10A;H840A) fusion with VP48 activation domain
  • Alt name
    dCas9VP48
  • Species
    Synthetic
  • Insert Size (bp)
    4349
  • Mutation
    D10A;H840A
  • Tags / Fusion Proteins
    • VP48 (C terminal on insert)
    • HA Tag (N terminal on insert)

Cloning Information

  • Cloning method Restriction Enzyme
  • 5′ cloning site EcoRI (not destroyed)
  • 3′ cloning site EcoRI (not destroyed)
  • 5′ sequencing primer M13F
  • 3′ sequencing primer M13R
  • (Common Sequencing Primers)

Resource Information

  • Supplemental Documents
  • A portion of this plasmid was derived from a plasmid made by
    A two-step fusion PCR was performed to amplify Cas9 Nickase ORF without stop codon from the pX335 vector (Addgene: 42335), incorporate H840A mutation, EcoRI-AgeI restriction site on the 5′ end as well as an FseI site on the 3′ end (EcoRI-AgeIdCas9- FseI fragment). The 3× minimal VP16 activation domain coding fragment (VP48) was excised from a vector (Addgene: 20342) containing NLSM2rtTA coding sequence by FseI and EcoRI digestion (FseI-TA-EcoRI fragment). The two fragments were ligated into pCR8/GW/TOPO (Invitrogen) vector digested by EcoRI to generate a gateway compatible dCas9VP48 coding plasmid.
  • Terms and Licenses

Depositor Comments

For more information including protocols and
updates, please go to http://www.crispr-on.org

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:

    pAC1-pCR8-dCas9VP48 was a gift from Rudolf Jaenisch (Addgene plasmid # 48214)
  • For your References section:

    Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, Rangarajan S, Shivalila CS, Dadon DB, Jaenisch R. Cell Res. 2013 Aug 27. doi: 10.1038/cr.2013.122. 10.1038/cr.2013.122 PubMed 23979020