Design and construction of the recombination substrates. p[ACT-LUC] carries an intact transcription unit comprising the firefly (Photinus pyralis) luciferase coding sequence (red) driven from the D. melanogaster actin5C promoter (blue) with transcription terminated by the SV40 small t intron/polyA signal (yellow). The luciferase coding sequence was released from pGEM-luc (Promega) by digestion with Bam HI and Xho I and ligated into the same sites located between the actin5C promoter and SV40 termination sequence in p[ACT-SV] (unpublished data). All relevant restriction enzyme sites are indicated. The left-hand deletion substrate (DL) was generated by Xba I and Bst EII digestion to remove a 561 bp fragment at the 5' end of the luciferase coding sequence. The right-hand deletion substrate (DR) was generated by Eco RV and Xho I digestion to remove a 371 bp fragment at the 3' end of the luciferase coding sequence. The 728 bp region of homology shared by DL and DR is indicated and homologous recombination in this interval has the potential to reconstitute a functional luciferase gene.