Gt(ROSA)26Sor^{tm3(CAG-sfGFP*)Xwy}

The targeting vector is designed by using the Ai9 plasmid and replacing the tdTomato reporter with a Kozak consensus start site (for strong initiation of translation), two Myc-tags, an optimized C)22(-repeat MORF switch, and a farnesylated tamdem superfolder GFP fluorescent reporter with several V5 epitopes (tdsmFP-V5). Superfolder (sfGFP) is a hyperstable GFP variant that accepts large insertions into its loops while retaining folding and fluorescence. To create tdsmFP-V5, sfGFP was altered to embed several V5 epitopes (V5 is a 14 amino acid simian virus 5 epitope) in the GFP scaffold - 3 epitopes at the N terminus, 4 epitopes into the internal 172-173 loop and 3 epitopes at the C terminus. The tandem dimer of smFP-V5 contains a total of 20 V5 epitope tags. The inclusion of the V5 epitopes creates a highly antigenic spaghetti monster fluorescent protein (smGFP) that contains numerous copies of peptide epitopes and simultaneously binds IgG antibodies at each location. The farnesylation sequence (CAAX domain from Ras) targets the reporter to membranous structures including neuronal dendrites and axons. The final vector contains from 5' to 3' a CAG promoter, an FRT site, a loxP-flanked STOP cassette, the tdsmFP-V5 construct (see above), followed by a WPRE, polyA, attB-PGK-FRT-neo-pA-attP inserted in to the Gt(ROSA)26Sor locus. (J:292704)