Abstract: Malaria parasites utilize effector proteins to remodel the host erythrocyte for development. To reach the host cytosol, these effectors are translocated across the parasite vacuolar membrane by the Plasmodium Translocon of EXported proteins (PTEX). PTEX is powered by the AAA+ chaperone HSP101 which unfolds cargo for passage through a pore formed by EXP2 and PTEX150. While hundreds of proteins are predicted for export, regulation and distinguishing exported cargo remains unclear. HSP101 contains an N-Terminal Domain (NTD) that mediates substrate recognition in related chaperones and may fulfill a role in cargo selection. Additionally, about ~16% of PTEX150 forms an adaptor between HSP101 and EXP2, the function of the rest of this protein is unknown. To evaluate these PTEX features, we developed a DiCre/loxPint-based Near-Isogenic Conditional Excision (NICE) strategy to generate point mutations or in-frame deletions in the endogenous P. falciparum loci. Using this approach, we show that the HSP101 NTD is crucial to the export process. Additionally, serial deletions across the residues N-terminal to the PTEX150 adaptor domain revealed that much of this region is important. Collectively, our work enables detailed dissection of the PTEX mechanism and provides an efficient genetic approach that avoids the time-consuming process of complementing conditional knockdowns.