Fig. 6

Assembly of the Level 0.5 and Level 1 vectors. a The reporter gene (Gene 1) and its associated terminator (“T1”) were cloned into the Level 0 vector EC10161, using the Type IIS restriction enzyme BsaI. The resulting Level 0.5 vector contains loxP sites flanking the inserted reporter gene and terminator. b The desired promoter sequence, loxP-flanked reporter gene, gene of interest (Gene 2), and corresponding terminator (T2) are cloned into a Level 1, position 3 vector (e.g. pAGM8031) in a reaction involving both BsaI, for the canonical Level 0 and Level 1 parts, and Esp3I, for the loxP-containing Level 0.5 vector (see a). In both cases, additional components can be included during the cloning step, such as targeting sequences