pTMt_TCS(Q’G)_NLS-HA-dCas9(C)-VP64_T2A_MCP-P65-HSF1
(Plasmid #101102)

• Purpose: Encodes dCas9(C)-VP64 with SAM components 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: pX856
• Modifications to backbone: The pX856 vector (gift from Feng Zhang, Addgene plasmid #62888) was modified as follows: the U6 promoter/sgRNA scaffold/U6 terminator cassette was removed; the dCas9(C)VP64 N-terminal NLS+FKBP were also removed; a transmembrane tether (TMt) 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(C)VP64 via a TEV cleavage site (ENLYFQG) and NLS. The MCP-P65-HSF1 from plasmid MS2-P65-HSF1_GFP (gift from Feng Zhang, Addgene plasmid #61423) was fused to the C-terminus of dCas9(C)VP64 via at T2A site.
• Vector type: Mammalian Expression, CRISPR, Synthetic Biology

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(C)-VP64
• Tags / Fusion Proteins:
  • Myc (N terminal on insert)
  • HA (N terminal on insert)

Resource Information

• Supplemental Documents:
  • pTMt_TCS(Q’G)_NLS-HA-dCas9(C)-VP64_T2A_MCP-P65-HSF1.gb
• A portion of this plasmid was derived from a plasmid made by: pX856 vector (gift from Feng Zhang, Addgene plasmid #62888); plasmid MS2-P65-HSF1_GFP (gift from Feng Zhang, Addgene plasmid #61423)

Terms and Licenses

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