pHAGE EF1α dCas9-NED
(Plasmid #109369)

• Purpose: (Empty Backbone) Lentiviral vector for expressing effector domains fused to dCas9.
• Depositing Lab: Antal Kiss
• Publication: Goubert et al Methods Mol Biol. 2018;1767:395-415. doi: 10.1007/978-1-4939-7774-1_22.

Backbone

• Vector backbone: pHAGE EF1α dCas9-VP64 (Addgene plasmid #50918)
• Backbone manufacturer: Rene Maehr, Scot Wolfe
• Backbone size (bp): 12547
• Modifications to backbone: The 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 type: Mammalian Expression, Lentiviral, CRISPR
• Promoter: EF-1α
• Selectable markers: Puromycin
• Tag / Fusion Protein:
  • 3xHA (N terminal on backbone)

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): NEB Stable
• Copy number: High Copy

Cloning Information

• Cloning method: Restriction Enzyme
• 3′ sequencing primer: 5'-CAGCGGGGCTGCTAAAGCGCATGC

Resource Information

• A portion of this plasmid was derived from a plasmid made by: Rene Maehr and Scot Wolfe, pHAGE EF1α dCas9-VP64 (Addgene plasmid #50918).
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

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).