BBa_K1750001 1 BBa_K1750001 YFP fused to CIB1 N-terminal domain 2015-09-17T11:00:00Z 2015-09-18T01:22:38Z CIB1 - genomic sequence of A. thaliana, YFP - genomic sequence of A. thaliana A. victoria Yellow fluorescent protein, which produces yellow light (excitation 400nm, emission 530nm), fused to the carboxy-terminus of cryptochrome-interacting basic-helix-loop-helix1 (CIB1), a transcription factor derived from A. thaliana in which it initiates flowering upon binding the photoreceptor cryptochrome 2 (CRY2). We used the N-terminal domain of CIB1 (a.a. 1-170), that lacks the conserved basic helix-loop-helix domain which mediates dimerisation and DNA binding. This device can be used as a reporter for binding to CRY2 fused to a second fluorescent protein, e.g. cyan fluorescent protein as detected by FRET assay. This part is expressed strongly in the pETMCSII vector. Expression in E. coli showed strong YFP fluorescence but SDS-PAGE analysis suggested that the CIB1 segment may be proteolysed in the cell, which would hinder its ability to bind CRY2. false false _2172_ 25444 25444 9 false Initially designed CRY2-CFP-CIB1-YFP, CRY2-YFP-CIB1-YFP, CIB1-CFP and CIB1-YFP constructs with ribosomal binding sites at the start of each fusion protein (if contained both CRY2 and CIB1 fusion proteins), HIS/MYC tags and a flexible linker between the fused proteins, and a Terminator at the end. We redesigned the construct in the order RBS-CIB1-Linker-YFP-H6-Terminator to allow purification and fluorescence measurements of individual reporter proteins. The N-terminal HIS tag also facilitated purification of the protein. false Janelle San Juan annotation2469864 1 YFP range2469864 1 517 1230 annotation2469865 1 linker range2469865 1 1231 1233 annotation2469863 1 CIB1 (N-terminal domain) range2469863 1 1 510 annotation2469862 1 linker range2469862 1 511 516 annotation2469866 1 HIS tag (H6) range2469866 1 1234 1251 annotation2469868 1 STOP codon range2469868 1 1252 1254 BBa_K1750001_sequence 1 atgaatggcgctatcggcggtgatctgctgctgaactttccagacatgtccgttctggaacgccagcgtgcgcatctgaaatacctgaacccgaccttcgactctcctctggctggtttcttcgcagactcttctatgatcaccggcggcgaaatggactcttacctgtctaccgctggtctgaacctgccaatgatgtacggcgaaaccactgtagaaggcgactctcgtctgagtatttccccagaaactaccctgggcaccggtaacttcaaaaaacgtaagttcgacactgaaactaaagactgcaacgaaaaaaaaaaaaagatgaccatgaaccgtgatgatctggtagaagaaggtgaagaagaaaaatctaaaatcaccgagcagaacaacggttctaccaaatccatcaaaaaaatgaaacacaaagcaaagaaagaagaaaacaacttctctaacgactcctctaaagtgactaaagaactggaaaagaccgactacatcggcagtgtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagctgatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgggctacggcctgcagtgcttcgcccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcaccgccgacaagcagaagaacggcatcaaggccaacttcaagatccgccacaacatcgaggacggcggcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagggccaccatcaccatcaccattaa igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 Chris J. Myers James Alastair McLaughlin 2017-03-06T15:00:00.000Z