Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Katja Leben
Date created: 2016-10-13 11:00:00
Date modified: 2016-10-18 03:48:21
cycLuc_SuMMVs
| Types | DnaRegion |
| Roles | CDS Coding |
| Sequences | BBa_K1965034_sequence (Version 1) |
Description
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 SuMMVs sequence (cycLuc_SuMMVs) contains the circularly permutated firefly luciferase, flanked by intein sequences. The two parts of fLuc are connected with the SuMMVps recognition sequence (EEIHLQ-S).
Characterization
This construct was expressed under CMV promoter and used for testing the orthogonality of different TEVp homologs and studying the activity split SuMMVp measuring the activity of fLuc. The coding sequence for cycLuc_SuMMVs 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.
Notes
-Source
artificial| Sequence Annotation | Location | Component / Role(s) |
| start His-tag intC AU1 cLuc GS-linker nLuc intN stop PEST | 1,3 4,21 22,138 139,156 157,1089 1090,1125 1126,1815 1816,2130 2326,2328 2131,2325 | start_codon feature/start feature/tag tag CDS feature/cds feature/tag tag CDS feature/cds feature/misc sequence_feature CDS feature/cds CDS feature/cds stop_codon feature/stop CDS feature/cds |