1：Experimental Design and Method Selection:

Employed a strategy to split Flpo recombinase into two pieces that reassemble upon blue light stimulation using the Magnet system for heterodimerization. Screened nine split-site variants and optimized configurations with nuclear localization signals (NLS).

2：Sample Selection and Data Sources:

Used HEK293T cells for in vitro screening, embryonic mouse brains (E15) for in utero electroporation, and adult C57BL/6 mice, RCE:FRT reporter mice, and ROSA26RCE:FRT/Ai14 mice for in vivo validation. Data included fluorescence imaging and behavioral assays.

3：List of Experimental Equipment and Materials:

AAV vectors (serotype DJ/8), lentiviral constructs, plasmids (e.g., pQC-mCherry-IX, pAAV vectors), optic fibers (? 200 μm, 50-60 μm), blue diode laser (473 nm), LEDs (white or 470 nm), stereotaxic equipment, confocal microscope (Nikon A1), automated epifluorescence microscope (ImageXpress Micro XLS), power meter (PM120D), and various antibodies (anti-HA, anti-Cav3.1).

4：1). Experimental Procedures and Operational Workflow:

4. Experimental Procedures and Operational Workflow: Constructed PA-Flp variants via Gibson assembly, transfected HEK293T cells, performed in utero electroporation in embryonic mice, stereotaxically injected AAVs into specific brain regions (e.g., hippocampus, medial septum), delivered light stimulation via optic fibers or noninvasive LEDs, and conducted behavioral tests (object-exploration task). Mice were sacrificed, brains sectioned, and imaged for fluorescence analysis.

5：Data Analysis Methods:

Used CellProfiler, MetaXpress, Nikon imaging software, and MetaMorph for image analysis. Statistical analysis included Student's t-test, one-way ANOVA, and two-way repeated measures ANOVA with Bonferroni post-hoc tests.