One loxP sequence was added 260 bp upstream from the translational initiation codon of Slc18a3 (within the first intron of Chat), and a second loxP was added approximately 1.5 kb downstream from the stop codon. This flanking region included the second exon of Chat. The TK-Neo cassette was added immediately after the second loxP and was followed by a third loxP (all in the same direction). Cre-mediated recombination removed the neo cassette.