Igs7^{tm195(tetO-GCaMP7s,CAG-tTA2)Tasic}

The targeting vector contains (from 5' to 3') a Bxb1 attB site, a partial green fluorescent protein sequence (non-functional 500bp fragment to increase the space between the Bxb1 attB site and the insulator sequence to improve stability of the targeting vector), two copies of chicken beta-globin HS4 insulator element (to reduce reporter gene expression in absence of transactivators), a Tet response element/promoter (TRE2; details below), a loxP-flanked STOP cassette (stop codons in all thr ee reading frames linked to synthetic pA-hGHpA-PGKpA), a GCaMP7 sensitive and slow variant calcium indicator sequence (jGCaMP7s; details below) followed by a 6xHis tag, T7 tag and XPress tag sequence, a woodchuck hepatitis virus post-transcriptional regulatory element, a BGH polyA, two copies of chicken beta-globin HS4 insulator element, a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter , an FRT-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-TKpA), an internal ribosome entry site (IRES) sequence, a synthetic modified tetracycline-regulated transactivator gene (tTA2(S)), a WPRE, a BGH polyA, a PhiC31 attB site, a PGK-5'hygro cassette, an RNA splice donor,a Bxb1 attR-SA-3'hygro-SV40pA-PhiC31 attP. The TRE2 promoter used here is Tet-responsive P)hCMV*-1(; containing the Tet response element (seven copies of the 19 bp tet operator sequence [tetO]) just upstream of a minimal cytomegalovirus promoter (P)min CMV(), which lacks the enhancer that is part of the complete CMV promoter. Consequently, P)hCMV*-1( is silent in the absence of tTA or rtTA binding to tetO. The "Janelia GCaMP7" sensitive and slow variant calcium indicator (jGCaMP7s) is an ultrasensitive detector of neuronal activity with slower decay and response kinetics. The jGCaMP7s fusion gene is an improved version of GCaMP6s. Like GCaMP6s, jGCaMP7s has a chicken smooth muscle M13 fragment of myosin light chain kinase (also called calmodulin-binding peptide [CBP]), a circularly permutated EGFP (cpEGFP; aa 149-238 followed by aa 1-144 [with mutations to increase dynamic range/baseline fluorescence, as well as increased sensitivity/slower kinetics]), and a rat calmodulin DNA fragment (CaM; aa 2-148 [with mutations to increase the fluorescence change for small calcium transients]). Amino acid substitutions in the cpEGFP-to-CaM linker improve sensor function. In addition, jGCaMP7s has the A52V mutation in CBP and the A317L mutation in CaM. (J:101977)