Part:BBa_K1699005

gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm under U6 promoter

gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm under U6 (RNA polymerase III) promoter. gRNA sequence is complementary to 3 different loci in the synthetic promoter pMLPm (1), and gRNA-dCas9-VP64 complex can promote transcription downstream of synthetic promoter.

Usage and Biology

Guide RNA is a hundred base-long molecule with a unique two dimensional structure which binds dCas9-VP64 and guides it to a dsDNA sequence complementary to 21-22 base pairs on the 5' end of the molecule. Upon binding to promoter, dCas9-VP64-gRNA complex will promote transcription of genes downstream of binding site. gRNA scaffold sequence for SpdCas9-VP64 was used (2).

Characterization

This part was designed as a control construct for two cancer-specific promoter-based design of CRISPR-mediated transcriptional activation. In the design, gRNA is ribozyme-flanked (to enable synthesis under RNA polymerase II promoter) and is under control of human survivin promoter.

U6-gMLP: AAV vector for the synthesis of gRNA for dCas9-VP64 targeting synthetic activation promoter pMLPm (abbreviated as gMLP) under the control of human U6 promoter (Fig. 1).

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Fig. 1. Plasmid map of AAV vector carrying gMLP under the control of human U6 promoter.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 250
  
• 1000
  COMPATIBLE WITH RFC[1000]