Part:BBa_K1965037
cycLuc_TEVs
The cyclic luciferase system enhances the circularly permuted luciferase (cpLuc) (Fan2008, Wigdal2008) by fusing two fragments of an intein to the ends of cpLuc. Inteins are protein fragments that allow protein splicing and cyclization by formation of a new peptide bond between the N- and C-termini of the protein. We expected this reporter to result in a higher signal due to the stabilization of the protein by cyclization (Figure 1). To further optimize the dynamic range of the system, a PEST sequence for fast digestion of the protein was included at the C-terminus of the protein. This sequence targets any of the unspliced protein to degradation, while the spliced cyclic protein remains stable, since the PEST sequence is excised along with the intein fragments during the splicing reaction (Kanno2007, Kanno2009).
Cyclic luciferase with the TEVs sequence (cycLuc_TEVs) contains the circularly permutated firefly luciferase, flanked by intein sequences. The two parts of fLuc are connected with the TEVps recognition sequence (ENLYFQ-S).
Characterization This construct was expressed under CMV promoter and used for testing the orthogonality of different TEVp homologs and studying the activity split TEVp measuring the activity of fLuc. The coding sequence for cycLuc_TEVs was deposited in pSB1C3.
For testing the orthogonality, HEK293T cells were cotransfected by the plasmids with the whole protease and cycLuc reporters as shown on Figure 2.
For testing the activity of split proteases, HEK293T cells were cotransfected by the plasmids with rapamycin inducible split protease and corresponding cycLuc reporter as shown on Figure 3.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1090
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 865
Illegal NgoMIV site found at 886
Illegal NgoMIV site found at 1177
Illegal NgoMIV site found at 2239
Illegal AgeI site found at 589 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 771
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