FIG. 1.

Overview of Gal4, Split Gal4, KalTA4, and Split KalTA4 systems. (A) Gal4/UAS system: Gal4 driver lines contain a transgene encoding Gal4 (DBD and AD in a single open-reading frame) under a specific promoter (P). In a cell where P is active, Gal4 protein is expressed and can bind UAS in effector lines, activating transcription of the downstream gene-of-interest (gray arrow). (B) Split Gal4 system: Gal4 DBD hemi-driver is encoded under one promoter (P1) and Gal4 AD hemi-driver in a separate open-reading frame under a different promoter (P2). In a cell where both promoters are active, the two domains assemble and reconstitute Gal4 function, leading to expression of genes-of-interest only in the intersection of two expression domains. (C) Structure of Gal4, KalTA4, split Gal4, and split KalTA4 coding sequences. Compared to Gal4, KalTA4 includes a stronger Kozak sequence (K), zebrafish-optimized codon usage (zf), the core repeats of the VP16 AD (TA4), and a β-globin intron; these features were used to generate the split KalTA4 hemi-drivers. ATG: start codon, TAG/TAA: stop codon, Gly: decaglycine linker, Zip: heterodimerizing leucine zippers, nls: nuclear localization signal. Numbers indicate length of feature in amino acid residues. NheI and SpeI restriction sites from the original split Gal4 constructs were maintained in split KalTA4. AD, activation domain; DBD, DNA-binding domain; UAS, upstream activating sequence.