The 176 kb bacterial artificial chromosome, RP24-256F2, containing the entire Chat and Slc18a3 (VAChT) genes was modified to insert an optimized FLP recombinase (FLPo) followed by a roxP-flanked PGK-prokaryotic gb2 promoter-neoR/kanR cassette under the direction of the Chat promoter. Induction of Dre recombinase removes the kanamycin cassette in recombineering competent bacteria. Next, a 3X Stop is inserted into the Slc18a3 (or VAChT) blocking transcription. (J:101977)