Igs7^{tm170.1(tetO-GFP*/TPTE2*,CAG-tTA2)Hze}

The vector is designed with (from 5' to 3') an FRT3 site, 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 three reading frames linked to synthetic pA-hGHpA-PGKpA), a green fluorescent voltage indicator protein ASAP2s sequence with soma and proximal dendrite targeting sequences (ASAP2s-Kv; details below), a 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 lox2272-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-TKpA), a synthetic modified tetracycline-regulated transactivator gene (tTA2S), a WPRE, a BGH polyA, an AttB site, a PGK-5'hygro cassette, an RNA splice donor and a FRT5 site. 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. GFP*/TPTE2* has VSD (voltage sensor domwin) sequences (GgVSD) from the Gallus gallus voltage-sensitive phosphatase TPTE2, including the R154Q mutation for desired voltage response. A circularly permuted superfolder GFP 1-10 OPT variant (cpsfGFP-OPT) is inserted into the GgVSD extracellular loop between the third and fourth transmembrane segments (S3-S4). The GgVSD includes the R154Q mutation for desired voltage response (equivalent to R153Q in original descriptions). The GFP 1-10 OPT includes the substitutions from folding reporter GFP (F99S, M153T, V163A, F64L, S65T) and superfolder GFP (S30R, Y145F, I171V, A206V), and also has seven additional substitutions (N39I, T105K, E111V, I128T, K166T, I167V, S205T). Together, these mutations result in improved brightness and dynamic range, while maintaining efficient expression at the membrane. In addition, this voltage sensor is tagged with a 65-amino-acid cytosolic segment of a rat Kv2.1 to restrict protein localization to the soma and proximal dendrites. PhiC31-mediated recombination removed the AttB/AttP-flanked sequence and replaced it with the recombined AttB/AttP site (AttL). (J:260362)