8xlexAop sequences regulate expression of a transcript that encodes two open reading frames separated by a T2A linker (this results in separate translation of each protein). The two open reading frames together encode an an optogenetic tool, "Photo-induced Gα Modulator-Inhibitor of Gαq" (PiGM-Iq), that can be used to inhibit Gαq signaling. The first open reading frame encodes a fusion protein in which a minimal regulator of G protein signaling (RGS) domain is fused to CRY2(PHR). The minimal RGS domain consists of the Rnor\Rgs2 gene from which the first 53 amino acids have been deleted; this removes an adenylate cyclase inhibiting peptide, plus G protein-coupled receptor (GPCR) targeting and membrane association sequences, and thus in the absence of light induction, the CRY2(PHR) tagged RGS domain is located in the cytosol and does not bind GTP-bound Gαq. The second open reading frame encodes EGFP tagged at the N-terminal end with CIBN(ΔNLS) and at the C-terminal end with a Tag:CAAX(Unk) motif that targets the protein to the membrane. Upon illumination with blue light, the CRY2(PHR) and CIBN(ΔNLS) motifs heterodimerize, recruiting the minimal RGS domain to its membrane substrate, GTP-bound Gαq, where it can catalyze the hydrolysis of GTP to GDP to inhibit Gαq signaling.

8xlexAop sequences regulate expression of a transcript that encodes two open reading frames separated by a T2A linker (this results in separate translation of each protein). The two open reading frames together encode an an optogenetic tool, "Photo-induced Gα Modulator-Inhibitor of Gαq" (PiGM-Iq), that can be used to inhibit Gαq signaling. The first open reading frame encodes a fusion protein in which a minimal regulator of G protein signaling (RGS) domain is fused to CRY2(PHR). The minimal RGS domain consists of the Rnor\Rgs2 gene from which the first 53 amino acids have been deleted; this removes an adenylate cyclase inhibiting peptide, plus G protein-coupled receptor (GPCR) targeting and membrane association sequences, and thus in the absence of light induction, the CRY2(PHR) tagged RGS domain is located in the cytosol and does not bind GTP-bound Gαq. The second open reading frame encodes EGFP tagged at the N-terminal end with CIBN(ΔNLS) and at the C-terminal end with a Tag:CAAX(Unk) motif that targets the protein to the membrane. Upon illumination with blue light, the CRY2(PHR) and CIBN(ΔNLS) motifs heterodimerize, recruiting the minimal RGS domain to its membrane substrate, GTP-bound Gαq, where it can catalyze the hydrolysis of GTP to GDP to inhibit Gαq signaling.