This allele has three membrane-targeted fluorescent protein sequences - each uniquely flanked with pairs of incompatible lox sites (in the same direction) inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus on BAC RP23-401D9. PiggyBAC transposon inverted repeats were added to the BAC to facilitate single-copy and complete insertion into the genome with the PiggyBAC system. The targeting vector is designed with (from 5' to 3') two copies of chicken beta-globin HS4 insulator element (2xINS; increases downstream gene expression and minimizes regulatory interference), a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a loxN site, a lox2272 site, a loxP site, the non-cytotoxic/non-fluorescent Phialidium yellow fluorescent protein variant PhiYFP with nuclear localization signal (N-PhiMut), bovine GH polyA signal sequence, a second loxN site, the MbTomato gene (non-oligomerizing tandem-dimer DsRed fluorescent protein variant tdTomato with N-terminal membrane-targeting tag), in-frame Thosea asigna virus 2A cleavage site (T2A; mediates ribosomal skipping) followed by in-frame mutated human KDR cDNA for the active form (where sequence coding for the extra-cellular domain was deleted), bovine GH polyA signal sequence, a second lox2272 site, the MbYFP gene (monomeric enhanced yellow fluorescent protein [mYFPA206K] with N-terminal membrane-targeting tag), in-frame T2A sequence followed by in-frame mutated mouse Kdr cDNA (with a p.Y1173F mutation that renders the residue non-phosphorylatable and abolishes the peptide's downstream signalling upon binding Vegf), bovine GH polyA signal sequence, a second loxP site, the MbKate2 gene (monomeric far-red fluorescent protein mKate2 with N-terminal membrane-targeting tag), bovine GH polyA signal sequence, and a second 2xINS sequence. (J:252386)
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