P{lox(Trojan-dVP16AD)x3} is a transgenic donor construct containing three tandem "Trojan dVP16AD" cassettes, one in each intron phase. The Trojan dVP16AD sequence in each case consists of a splice acceptor site followed by the T2A peptide, a VP16(AD)::Zip+ hemidriver and an Hsp70 transcription termination signal. Each cassette is flanked by attB sites nested within a pair of uniquely compatible lox sites (lox2272 for the phase 0 cassette, loxN for the phase 1 cassette, and loxP for the phase 2 cassette). Expression of P1cre recombinase will excise and circularise all three cassettes, which can then be integrated into a target in the genome that contains inverted attP sites, such as the Mi{MIC} element, using phiC31:int integrase. Integration of the cassette with the appropriate phase in the correct orientation into a coding intron of a native Drosophila gene of interest will result in the cassette behaving as a "Trojan" exon: the splice acceptor site ensures that the T2A-VP16(AD)::Zip+ open reading frame is incorporated into the mRNA of the native Drosophila gene, while the T2A sequence truncates the native gene product and promotes the separate translation of the VP16(AD)::Zip+ open reading frame. Thus VP16(AD)::Zip+ should be expressed under the control of the regulatory sequences of the native Drosophila gene of interest in the resulting fly line.