Part:BBa_K4830027

mCherry_C-terminus stop codon_Clover

The mCherry_C-terminus stop codon_Clover is protein coding sequence containing the mCherry fluorescent protein with a premature stop codon installed in the C-terminus before the Clover fluorescent protein. The presence of the premature stop codon prevents the expression of coding regions found downstream. This is particularly useful in assays such as the Traffic Light Reporter (TLR) assay.

Usage and Biology

The mCherry_C-terminus stop codon is utilized in the Traffic Light Reporter (TLR) assay, where a Clover fluorescent protein (commonly known as GFP) is found downstream. In this TLR assay, the Clover is not expressed due to the presence of the stop codon before the GFP. As such, the TLR assay is used to screen for DNA editing activity that swaps the stop codon into a functional amino acid, restoring the expression of Clover.

The entire coding sequence can be cloned into suitable plasmids and subsequently transfected into mammalian cells for in vitro DNA editing activity assay.

Characterization

The pegRNA in combination with ngRNA were used to test the efficiency of the Prime Editor containing the alternative Reverse Trancriptases. The pegRNA served as template to install the edit. 3 plasmids containing PE, pegRNA and ngRNA were trasfected into HEK293T cells, and editing efficiency was evaluated 72hr after transfection using flow cytometry by evaluating either the mean fluorescence intensity of the green fluorescent protein (GFP), or the percentage of cells containing the GFP.

TLR pegRNA 1 (BBa_K4830019) was co-transfected with TLR ngRNA 1 (BBa_K4830020); while TLR pegRNA 2 (BBa_K4830021) was co-transfected with TLR ngRNA 2 (BBa_K4830021). The targeting sequence was the premature stop codon found on the mCherry protein (BBa_K4830027).

Fig. 1 Bar graph shows the mean fluorescence intensity (MFI) for GFP, and scatter points denote the %GFP cells in one of the replicates of the initial TLR assay.

Sequence and Features

The sequence shows the mCherry fluorescent protein with a stop codon on the C-terminus. This is followed with a Clover fluorescent protein downstream.

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 352
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 352
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 352
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 352
  
• 1000
  COMPATIBLE WITH RFC[1000]