The vector contains (from 5' to 3') a Bxb1 attB site, a partial green fluorescent protein sequence (non-functional 500bp fragment to increase the space between the Bxb1 attB 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 CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter, a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), a synthetic enhanced green fluorescent protein sequence (EGFP), a woodchuck hepatitis virus post-transcriptional regulatory element, a BGH polyA, two copies of chicken beta-globin HS4 insulator element, the CAG promoter, an FRT-flanked STOP cassette, a monomeric orange fluorescent protein sequence (mOrange2; details below) followed by 2xHA tag sequence, a WPRE, a BGH polyA, a PhiC31 attB site, a WPRE, a PGK polyA, two copies of chicken beta-globin HS4 insulator element, the CAG promoter, a nox-flanked STOP cassette, a sequence encoding the monomeric far-red fluorescent protein mKate2 (details below), a viral 2A oligopeptide that mediates ribosomal skipping, a WPRE, a BGH polyA, a PhiC31 attB site, a PGK-5'hygro cassette, an RNA splice donor, Bxb1 attR-SA-3'hygro-SV40pA-PhiC31 attP. mOrange2 is a mammalian-codon optimized, photostable, fast-bleaching, monomeric orange fluorescent protein. Compared to its predecessor, mOrange2 has substantially enhanced photostability (Q64H, F99Y, E160K and G196D mutations) and also exhibits slightly shifted excitation and emission peaks (549nm and 565nm, respectively). Kate2 is a monomeric far-red fluorescent protein optimized for high-brightness, photostability, pH resistance and low-toxicity. Compared with its predecessor, the fluorescence of mKate2 is far more pH-stable, thus increasing its brightness at physiological pH and making mKate2 a more reliable reporter for imaging in acidic organelles. (J:101977)
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