BBa_K1982008 1 BBa_K1982008 tCas9-CIBN (Eukaryotic LACE system) 2016-10-08T11:00:00Z 2016-10-18T10:49:42Z CRY2 and CIB1 are extracted from Arabidopsis thaliana. tCas9 is from GE Share company. VP64 is synthetic construct. The NEU-China iGEM team 2016 designed a fusion protein consisting of tCas9 and CIBN for sequence-specific transactivation of a desired target locus (more information). We used our double truncated tCas9 (BBa_K1982007) impaired in its cleavage activity and fused it to the CIBN (BBa_K1982003). An prokaryotic RBS sequence from the Community collection(BBa_B0034) fused to the beginning of tCas9-CIBN. For detection of expression the fusion protein was tagged with a HA-epitope coding sequence (BBa_K1150016) .Figure 1 illustrates the detailed design of the whole device. Figure 1: Construct design. tCas9 was fused to CIBN. The resulting fusion construct was flanked by RBS sequences and tagged by a HA epitope. The pBad/araC promoter and rrnB T1 terminator were chosen to control gene expression. Optogenetic systems enable precise spatial and temporal control of cell behavior. A light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light.This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive tCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes[1]. we fused the light-inducible heterodimerizering proteins CRY2 and CIB1 from Arabidopsis thaliana to the VP64 transactivation domain and C-terminus of tCas9. false false _2449_ 30176 30176 9 false The CRY2/CIBN interaction is entirely genetically encoded. The binding reverses within minutes in the dark, allowing rapid shutoff of transcription by placing samples in the dark. This fusion protein is for use in LACE(light-activated CRISPR/Cas9 effector) system, and a tCas9 fused to its N terminus. To regulate DNA transcription by blue light, the system is based on CRY2/CIBN interaction in which a light-mediated protein interaction brings together two protein (tCas9 and an activation domain VP64) . If we remove the stimulation of blue light, dark reversion of CRY2 will dissociate the interaction with CIBN and shut off transcription. false Zexu Li annotation2517274 1 Eukaryotic rbs range2517274 1 1 18 annotation2517278 1 TAATAA range2517278 1 4726 4731 annotation2517276 1 ATG range2517276 1 19 21 annotation2517279 1 HA range2517279 1 4669 4725 annotation2517275 1 tCas9 range2517275 1 22 4122 annotation2517277 1 BBa_K1982003 range2517277 1 4123 4668 BBa_K1982008_sequence 1 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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