pdCas9-NED
(Plasmid #109358)

• Purpose: (Empty Backbone) Mammalian 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: pMLM3705 (Addgene plasmid #47754).
• Backbone manufacturer: Joung lab
• Backbone size (bp): 7636
• Modifications to backbone: The ORF encoding VP64 was excised from pMLM3705 (Addgene plasmid #47754) by AscI-AgeI double-digestion and replaced with an oligonucleotide containing a PacI site.
• Vector type: Mammalian Expression, CRISPR
• Promoter: CMV
• Tag / Fusion Protein:
  • dCas9, SV40 NLS, 3xFLAG (N terminal on backbone)

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Cloning Information

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

Resource Information

• A portion of this plasmid was derived from a plasmid made by: Keith Joung (Addgene plasmid #47754).
• Articles Citing this Plasmid:
  • 3 References

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 AscI and PacI sites of pdCas9-NED to create in-frame fusion between dCas9 (D10A, H840A) and the effector domain. For experiments involving cell lines stably expressing the dCas9-effector domain fusion protein, the coding sequence of the effector domain can be excised from the pdCas9-NED-based recombinant plasmid and transferred into pHAGE EF1α dCas9-NED (Addgene plasmid #109369). Cloning the AscI-PacI fragment between the MluI and AsiSI sites of pHAGE EF1α dCas9-NED preserves the reading frame of the fusion protein (Goubert et al., 2018).