Gt(ROSA)26Sor^{tm4.1(CAG-sbGLuc/COP3/EYFP)Ute}

The targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an FRT site, a loxP-flanked STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), the excitatory luminopsin 3 sequence, a woodchuck hepatitis virus post-transcriptional regulatory element, a polyA signal, an attB site, a PGK-frt-Neo-polyA cassette and an attP site. This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus. PhiC31-mediated recombination removed the AttB/AttP-flanked sequence (PGK-Neo-polyA::frt5::RNA splice acceptor::3'hygro-polyA) and replaced it with the recombined AttB/AttP site (AttL). The excitatory luminopsin 3 (LMO3) is designed with the sbGLuc (slow-burn luciferase variant with improved bioluminescence from Gaussia princeps) sequence fused in-frame to the extracellular amino terminus of the VChR1 (red-shifted channelrhodopsin-1 variant from Volvox carteri) sequence fused in-frame to the amino terminus of an EYFP (enhanced yellow fluorescent protein) sequence. sbGLuc is a slow-burn luciferase variant that harbors two point mutations (M43L and M110L) that increase the half-life of light emission (10-fold greater than wildtype GLuc) while preserving the intensity of the luminescence signal. (J:101977)