β-actin YFP-Sec61β encodes full-length human ER marker Sec61β, fused to EYFP and was a gift from C. Hoogenraad (Utrecht University, Utrecht, The Netherlands). pB80-KIF1A(1-365)-VVDfast-FLAG-SSPB(micro), encoding opto-kinesin, was derived from pB80-KIF1A(1-365)-VVDfast-GFP-SSPB(micro) (18 (link)) by replacing GFP with a 2xFLAG-tag (DYKDHDGDYKDHD). pB80-LAMP1-mCherry-iLID was cloned in the mammalian expression vector pβactin-16-pl (chicken β-actin promoter, referred to hereafter as pB80) (42 (link)), in which the multiple cloning site was replaced by (Asc I–Xba I–Hind III–Nhe I–Sna BI–Mlu I–Age I). The full-length human lysosome marker LAMP1, without stop codon, was tagged C-terminally with the red fluorescent protein mCherry and the photosensitive heterodimerization module iLID, interspaced by two synthetic 29–amino acid GGGS linkers. The iLID module was derived from pLL7.0-Venus-iLID-Mito (43 (link)), a gift from B. Kuhlman (University of North Carolina at Chapel Hill, NC; Addgene plasmid #60413). Full-length wild-type human LAMP1 was derived from LAMP1-mGFP (44 (link)), a gift from E. Dell’Angelica (University of California, Los Angeles, CA; Addgene plasmid #34831). All constructs were validated by sequencing of the full open reading frame.