UASt sequences regulate expression of a fusion protein composed of the Tag:TM(hPDGFRB) transmembrane domain, the CIBN(ΔNLS)::N-TEV split protease fragment (with a nuclear export signal in the linker between the CIBN(ΔNLS) and N-TEV portions), Tag:AsLOV2(BLITz-1) (TEVp protease target site that is inaccessible in the dark), GFP and the intracellular domain of N (amino acids 1766-2703). Upon illumination with blue light, Tag:AsLOV2(BLITz-1) undergoes a conformational change, resulting in the TEVp protease target site becoming accessible. In addition, if the transgene is co-expressed with a source of the CRY2(PHR)::C-TEV split protease fragment, light exposure also allows the CIBN(ΔNLS)::N-TEV and CRY2(PHR)::C-TEV peptides to associate and reconstitute a functional TEVp protease, which can cleave the now accessible TEVp protease target site, releasing a GFP-tagged N intracellular domain.
