pTMt_NES_TCS(Q’G)_HA-dCas9(N)_P2A-Puro-WPRE
(Plasmid #101101)

• Purpose: Encodes dCas9(N) fused to transmembrane tether
• Depositing Lab: Tudor Fulga
• Publication: Baeumler et al Cell Rep. 2017 Sep 12;20(11):2639-2653. doi: 10.1016/j.celrep.2017.08.044.

Backbone

• Vector backbone: pX855
• Modifications to backbone: The pX855 vector (gift from Feng Zhang, Addgene plasmid #62887) was modified as follows: the U6 promoter/sgRNA scaffold/U6 terminator cassette was removed; the dCas9(N) N-terminal NES and the C-terminal FRB+NES were also removed; a transmembrane tether (TMt; modified from the pDisplay Vector (Invitrogen)) containing the Igκ signal sequence, (GGGS)2 linker, myc epitope tag, PDGF receptor transmembrane domain and the XTEN linker, was synthesized as a gBlock (IDT). This transmembrane tether was then fused to the N-terminus of HA-dCas9(N) via a NES sequence and a TEV cleavage site (ENLYFQG); the puromycin resistance gene and the WPRE stabilising element from pCW-Cas9 (gift from Eric Lander and David Sabatini, Addgene plasmid #50661) were inserted downstream of dCas9(N).
• Vector type: Mammalian Expression, CRISPR, Synthetic Biology
• Selectable markers: Puromycin

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: TMt-dCas9(N)
• Tags / Fusion Proteins:
  • Myc (N terminal on insert)
  • HA (N terminal on insert)

Resource Information

• Supplemental Documents:
  • pTMt_NES_TCS(Q’G)_HA-dCas9(N)_P2A-Puro-WPRE.gb
• A portion of this plasmid was derived from a plasmid made by: pX855 vector (gift from Feng Zhang, Addgene plasmid #62887); pCW-Cas9 (gift from Eric Lander and David Sabatini, Addgene plasmid #50661)

Terms and Licenses

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