BBa_K1965037 1 BBa_K1965037 cycLuc_TEVs 2016-10-13T11:00:00Z 2016-10-18T04:05:20Z artificial 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. false false _2432_ 29974 29974 9 true - false Katja Leben annotation2514438 1 cLuc range2514438 1 157 1089 annotation2514439 1 TEVp cleavage site range2514439 1 1090 1122 annotation2514437 1 AU1 range2514437 1 139 156 annotation2514443 1 stop range2514443 1 2323 2325 annotation2514442 1 PEST degradation tag range2514442 1 2128 2322 annotation2514441 1 IntN range2514441 1 1813 2127 annotation2514440 1 nLuc range2514440 1 1123 1812 annotation2514433 1 start range2514433 1 1 3 annotation2514436 1 IntC range2514436 1 22 138 annotation2514435 1 His range2514435 1 4 21 BBa_K1965037_sequence 1 atgcaccaccaccaccaccacatgatcaaaatagccacacgtaaatatttaggcaaacaaaatgtctatgacattggagttgagcgcgaccataattttgcactcaaaaatggcttcatagcttctaattgtttcaatgatacttatagatatattgacaccgctatcctcagcgtggtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgctggtgcccacactatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggcggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctacggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtggtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctgtgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggacggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacaccccaacatcttcgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggtaaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattctcattaaggccaagaagggatccgaaaatctctatttccagagcggcggtgccaaaaacattaagaagggcccagcgccattctacccactcgaagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatcgcctttaccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaagcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtgttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaacgtgcaaaagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtacaccttcgtgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtaccggattgcccaagggcgtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgccgagtactgtttaagctatgaaacggaaatattgacagtagaatatggattattaccgattggtaaaattgtagaaaagcgcatcgaatgtactgtttatagcgttgataataatggaaatatttatacacaacctgtagcacaatggcacgatcgcggagaacaagaggtgtttgagtattgtttggaagatggttcattgattcgggcaacaaaagaccataagtttatgactgttgatggtcaaatgttgccaattgatgaaatatttgaacgtgaattggatttgatgcgggttgataatttgccgaacggcggcaagatcgccgtcaattctgcttgcaagaactggttcagtagcttaagccactttgtgatccaccttaacagccacggcttccctcccgaggtggaggagcaggccgccggcaccctgcccatgagctgcgcccaggagagcggcatggatagacaccctgctgcttgcgccagcgccaggatcaacgtctaa igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 James Alastair McLaughlin Chris J. Myers 2017-03-06T15:00:00.000Z