TgTn(pb-Gt(ROSA)26Sor/CAG-EYFP*,-mCherry*,-Notch1*,-EGFP*,-Rbpj*,-mCerulean*)#Ben

This allele has three chromatin/nuclear-targeted fluorescent protein sequences uniquely flanked with pairs of incompatible lox sites (in the same direction), all 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 N-PhiMut gene (non-cytotoxic/non-fluorescent Phialidium yellow fluorescent protein variant PhiYFP with nuclear localization signal), bovine GH polyA signal sequence, a second loxN site, the HA-H2B-Cherry gene (monomeric DsRed fluorescent protein variant mCherry with N-terminal hemagglutinin (HA) tag and 125 amino acid human histone 2B chromatin/nucleus tag sequence), in-frame Thosea asigna virus 2A cleavage site (T2A; mediates ribosomal skipping), in-frame mouse Notch1 intracellular domain (NICD) and PEST domain sequence (NICD-PEST), bovine GH polyA signal sequence, a second lox2272 site, the H2B-EGFP-V5 gene (enhanced green fluorescent protein with 127 amino acid and human histone 2B chromatin/nucleus tag plus C-terminal paramyxovirus V5 tag sequence), in-frame T2A sequence, bovine GH polyA signal sequence, a second loxP site, the His-H2B-Cerulean gene (monomeric enhanced cyan fluorescent protein variant mCerulean with N-terminal 6xHis affinity tag and 125 amino acid human histone 2B chromatin/nucleus tag sequence), in-frame T2A sequence, in-frame sequence for a dominant-negative form of mouse Rbpj (DN-Rbpj, with a p.R218H mutation), bovine GH polyA signal sequence, an FRT site flanked neomycin resistance gene cassette, and a second 2xINS sequence. The neo cassette was removed through subsequent flp-mediated recombination. (J:252386)