Tryptophan 1147 in exon 20, the core indispensable aromatic residue in the ATR activating domain was changed to arginine by introducing a T3439C mutation. In addition an FRT flanked neo cassette was inserted in intron 19. Flp mediated recombination removed the neo cassette leaving a single FRT site in intron 19. Immunoblot analysis confirmed similar levels of protein expressing in heterozygous and wild-type MEFs.