The VP16(AD)::PIF6 hemidriver contains the transcriptional activation domain from the Herpes simplex virus Type 1 VP16 gene (UniProtKB:P06492) fused to an N-terminal fragment of the Arabidopsis thaliana PIF6 gene (TAIR:39734); the PIF6 fragment contains only the activated phytochrome binding domain. In the 'PhotoGal4' split-GAL4 system, VP16(AD)::PIF6 is combined with GAL4(DBD)::PHYB. Upon incorporation of its chromophore phycocyanobilin (PCB) and when stimulated by red light of approximately 630nm, the PHYB portion of the GAL4(DBD)::PHYB hemidriver undergoes a conformational change and is activated, allowing it to heterodimerize with VP16(AD)::PIF6 via the PIF6 portion. This reconstitutes a functional transcriptional activator that can drive transcription of a downstream gene of interest and in this case, the reconstituted transcriptional activator binds specifically to the UAS regulatory sequence due to the presence of the GAL4 DNA-binding domain in GAL4(DBD)::PHYB. Since transcription is only activated at the intersection of the expression patterns of the two hemidrivers, the PhotoGal4 system can be used to drive expression in a restricted pattern. In addition, the light-dependent activation of GAL4(DBD)::PHYB is reversible; upon illumination of far-red light (>730nm) the PHYB portion reverts to the inactive state and dissociates from the PIF6-containing hemidriver, providing fine spatiotemporal control. The PhotoGal4 system is designed for use in ex vivo culture of cells and tissues. Alternatively, VP16(AD)::PIF6 can be combined with GAL4(DBD)::PHYB(Y276H), a DNA-binding domain-containing hemidriver in which the PHYB sequence has been mutated such that heterodimerization is no longer light-dependent but requires only the presence of the phycocyanobilin chromophore. The PHYB phycocyanobilin chromophore is not present in animal cells and must be supplied exogenously in both cases (FBrf0246250). VP16(AD)::PIF6 can potentially be combined in vivo with any hemidriver containing a DNA-binding domain fused to the Arabidopsis thaliana PHYB gene (TAIR:26548) to generate a functional transcriptional activator; in each case the condition(s) required to stimulate heterodimerization of the two hemidrivers will depend on the nature of the PHYB sequence in the DNA-binding domain PHYB-based hemidriver and the DNA-binding specificity of the reconstituted transcriptional activator will depend upon the DNA-binding domain present in the DNA-binding domain PHYB-based hemidriver.