Igs7^{tm210(tetO-GCaMP7f,CAG-tTA2)Tasic}

The targeting vector contains (from 5' to 3') a Bxb1 attL site, a partial green fluorescent protein sequence (non-functional 500bp fragment to increase the space between the Bxb1 attL 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 (tetO), a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), a GCaMP7 sensitive and fast variant calcium indicator sequence (jGCaMP7f; details below) followed by a 6xHis tag, T7 tag and XPress tag sequence, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a BGH polyA, two copies of chicken beta-globin HS4 insulator element, a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG; from pCAGGS), a 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 mRNA splice donor, a Bxb1 attR site, SA, 3'hygro, SV40pA, and PhiC31 attP. The "Janelia GCaMP7" sensitive and fast variant calcium indicator is an ultrasensitive detector of neuronal activity with faster decay and response kinetics. jThe allele carries 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, this allele has the H78K mutation in GFP and the A317L, R381T, T383S, and G392R mutations in CaM. (J:101977)