Kcnma1^em1Geh

CRISPR/Cas9 genome editing is used to replace 195 bp of exon 1 (including the first start codon) with an 855 bp insertion containing Igkappa signal sequence, an N-terminal HA epitope and an improved fluorogen activating protein sequence (L5** FAP) - such that the insertion is fused immediately 5' of the Kcnma1 downstream start codon. The L5** FAP sequence, originally derived from the human IGLV7-46 germline gamma allele (which appears to have undergone immune somatic hypermutation), was further engineered to have mutations conferring improved spectral and thermodynamic properties (L89S for increased quantum yield and E50D for greater MG affinity), as well as to function as a dimer. (J:273623)