Igs2^{tm1(CAG-cas9*)Mmw}

The targeting construct contains a full-length attB site, the CMV enhancer/chicken beta-actin core promoter (CAGGS), a loxP-STOP(3xSV40 polyA)- loxP cassette (LSL), a 3xFLAG sequence, a nuclear localization signal (NLS), a human codon-optimized S. pyogenes cas9 gene (hSpCas9), a second NLS and the SV40 polyA signal. The embryonic stem cells used were previously targeted with the H11 allele ( Igs2tm3.1Luo) or H11P3; a unique sequence from the promoter of the yeast his3 gene followed by three shortened tandem attP sites and one FRT5 site. One founder mouse carried the correctly targeted insertion of pattB-LSL-Cas9 into the first and second attP sites of the H11 locus. The resulting H11 knock-in allele is therefore attL-CAGGS-LSL-3xFLAG-NLS-hSpCas9-NLS-polyA-attR-attP-FRT5. (J:101977, J:222466)