Cloning of pHyPer-pexo and pHyPer-endo vector

To generate the pHyPer-pexo (referred to as Pexo_Hyper), pHyPer-cyto (FP941; Evrogen) was used as a template. The primer Fw_Hyper_BamHI and Rv_Hyper_PTS1_NotI (Table S1) were used to perform PCR. The PCR product was extracted using the clonetech nucleospin gel and PCR cleanup kit (Cat. #740609.5; Takara Clonetech) according to the vendor’s instructions. Both the PCR product and the mEGFP-N1 (Plasmid #54767; Addgene) were digested with BamHI and NotI restriction enzyme and the resulting product was ligated using the DNA Ligation Kit, Mighty Mix (Cat. #6023; Takara Clonetech).

To generate pHyPer-endo (referred to as Endo_Hyper), +HyPer7 (plasmid #136466; Addgene) and the Lamp1-mGFP (plasmid #34831; Addgene) were digested with BamhI and XbaI restriction enzyme. The Hyper7 sequence was then cloned into the Lamp1 backbone by using the DNA Ligation Kit.