Igs7^{tm143.1(tetO-RCaMP1.07)Hze}

The replacement vector is designed with (from 5' to 3') an FRT3 site, two copies of chicken beta-globin HS4 insulator element, a modified Tet response element/promoter, a floxed-STOP cassette, the R-CaMP1.07 sequence (details below), a WPRE, a BGH polyA, two copies of chicken beta-globin HS4 insulator element, an AttB site, a PGK-5'hygro cassette, an RNA splice donor and a FRT5 site. Embryonic stem cells previously targeted with FRT3::AttB::PGK-neoR-polyA::FRT5::splice acceptor::3'hygro cassette::SV40 polyA:AttP in TIGRE, were re-transfected with the pTRE-LSL-R-CaMP1.07 replacement vector and Flp recombinase vector for recombinase-mediated cassette exchange (RCME). 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). R-CaMP1.07 is a red calcium-sensing molecule that functions at cellular calcium levels and is a modified version of R-CaMP1.01 (which was created by insertion of random mutations into R-GECO1). R-CaMP1.07 is composed (from N-term to C-term) of a 35 aa polyHis plasmid leader sequence, a 13 aa chicken smooth muscle myosin light chain kinase, a circularly permutated red fluorescent protein (mApple; aa 146-231 followed by aa 1-145), a rat calmodulin DNA fragment, a 15 aa linker and a self-cleaving peptide (F2A). (J:241918)