Part:BBa_K779400

U6-TetO-FF1 MammoBlock

The U6-TetO-FF1 part encodes conditional expression of the short RNA sequence FF1. The mammalian RNA Pol III U6 promoter incorporates tetracycline-responsive elements that allow for tightly regulated RNA expression under specific conditions. In the presence of the Tet repressor (TetR), two Tet operator (TetO) sites become bound by the TetR and prevent RNA Pol III from binding, subsequently inhibiting transcription of the downstream sequence. In the absence of TetR, RNA transcription proceeds normally. The short FF1 RNA sequence can be used as an RNA interference mechanism through its binding to a complementary region present in the transcript of another gene, thus inhibiting translation of that sequence and silencing expression of that gene.

100,000 HEK293 Cells were transfected with varying molar ratios of U6-tetO:mirFF1 to Hef1a:eYFP 4xFF1, and 1:1 molar ratio of Hef1a:eYFP 4xFF1 and Hef1a:TagBFP (a transfection marker), standardized to a total of 500ng plasmid DNA using 1.65 uL of lipofectamine 2000. As the ratio of mirFF1 increases from 0.25X to 8X, there is a corresponding decrease in the yellow fluorescent signal, indicating gene knockdown. The histograms show the population of cells shifting from yellow towards the non-fluorescent region, by 10². This region was determined by analyzing a no-transfection control.

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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 248