pHAGE EF1α dCas9-NED
Purpose(Empty Backbone) Lentiviral vector for expressing effector domains fused to dCas9.
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||109369||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
Vector backbonepHAGE EF1α dCas9-VP64 (Addgene plasmid #50918)
Backbone manufacturerRene Maehr, Scot Wolfe
- Backbone size (bp) 12547
Modifications to backboneThe ORF encoding VP64 was excised from pHAGE EF1α dCas9-VP64 by BamHI-NotI double digestion and replaced with an oligonucleotide containing MluI and AsiSI restriction sites.
Vector typeMammalian Expression, Lentiviral, CRISPR
- Promoter EF-1α
/ Fusion Protein
- 3xHA (N terminal on backbone)
Growth in Bacteria
Growth Strain(s)NEB Stable
Copy numberHigh Copy
- Cloning method Restriction Enzyme
- 3′ sequencing primer 5'-CAGCGGGGCTGCTAAAGCGCATGC (Common Sequencing Primers)
The DNA fragment encoding the effector domain is synthesized by PCR and cloned between the MluI and AsiSI sites of pHAGE EF1α dCas9-NED to create in-frame fusion between dCas9 (D10A, H840A) and the effector domain. If the experimental strategy requires testing the effector domain in a transient transfection system before establishing stably expressing cell lines, it is recommended to construct the dCas9-effector domain fusion first in pdCas9-NED (Addgene plasmid #109358), then excise the coding sequence of the effector domain by AscI-PacI double digestion, and clone the fragment between the MluI and AsiSI sites of pHAGE EF1α dCas9-NED. Ligation of the MluI end to the compatible AscI end preserves the reading frame (Goubert et al. 2018).
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:pHAGE EF1α dCas9-NED was a gift from Antal Kiss (Addgene plasmid # 109369 ; http://n2t.net/addgene:109369 ; RRID:Addgene_109369)
For your References section:Establishment of Cell Lines Stably Expressing dCas9-Fusions to Address Kinetics of Epigenetic Editing. Goubert D, Koncz M, Kiss A, Rots MG. Methods Mol Biol. 2018;1767:395-415. doi: 10.1007/978-1-4939-7774-1_22. 10.1007/978-1-4939-7774-1_22 PubMed 29524148