PurposedCas9VP48 on Gateway donor vector pCR8/GW/TOPO. Note: This is not for expression. It has to be transferred to a gateway destination vector for expression
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||48214||Standard format: Plasmid sent in bacteria as agar stab||1||$85|
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 2799
- Total vector size (bp) 7148
Vector typeCRISPR ; Gateway Donor
Growth in Bacteria
Bacterial Resistance(s)Spectinomycin, 50 μg/mL
Gene/Insert namedCas9(D10A;H840A) fusion with VP48 activation domain
Insert Size (bp)4349
/ Fusion Proteins
- VP48 (C terminal on insert)
- HA Tag (N terminal on insert)
- 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)
A portion of this plasmid was derived from a plasmid made byA 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
- Not Available to Industry
- Zeocin® is an InvivoGen trademark.
For more information including protocols and
updates, please go to http://www.crispr-on.org
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 ; http://n2t.net/addgene:48214 ; RRID:Addgene_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