UAS regulatory sequences drive expression of a bicistronic transcript that encodes two open reading frames separated by a 2A linker (this results in separate translation of each protein), which together form an optogenetic tool that can be used to activate the Raf/MEK/ERK signaling pathway. The first ORF encodes a fusion protein composed of CRY2(PHR), mCherry and the Uuuu\Raf1 coding sequence. The second ORF encodes a fusion protein composed of two copies of CIBN(ΔNLS), EGFP and the Tag:CAAX(hKRAS) plasma membrane targeting sequence. Upon illumination with blue light, the CIBN(ΔNLS) domain of the membrane-localized protein encoded by the second ORF binds the CRY2(PHR) domain of the fusion protein encoded by the first ORF, recruiting the CRY2(PHR) domain-containing protein to the membrane, upon which the Uuuu\Raf1 portion should be phosphorylated and activated, activating the Raf/MEK/ERK signaling pathway (PMID:24667437).

UAS regulatory sequences drive expression of a bicistronic transcript that encodes two open reading frames separated by a 2A linker (this results in separate translation of each protein), which together form an optogenetic tool that can be used to activate the Raf/MEK/ERK signaling pathway. The first ORF encodes a fusion protein composed of CRY2(PHR), mCherry and the Uuuu\Raf1 coding sequence. The second ORF encodes a fusion protein composed of two copies of CIBN(ΔNLS), EGFP and the Tag:CAAX(hKRAS) plasma membrane targeting sequence. Upon illumination with blue light, the CIBN(ΔNLS) domain of the membrane-localized protein encoded by the second ORF binds the CRY2(PHR) domain of the fusion protein encoded by the first ORF, recruiting the CRY2(PHR) domain-containing protein to the membrane, upon which the Uuuu\Raf1 portion should be phosphorylated and activated, activating the Raf/MEK/ERK signaling pathway (PMID:24667437).