TAP110–PTP was created by amplification of the TAP110 open reading frame (ORF) (Tb927.11.7590) positions 2242 to 2922 from genomic NYsm DNA and was cloned between the ApaI and EagI (NEB) sites of the pLEW100 based PTP tagging vector (Schimanski et al., 2005 (link)). We linearized the resulting plasmid with XcmI (NEB) prior to transfection. TAP110 RNAi targeting the ORF (positions 2081 to 2629) was cloned into a tet-inducible RNAi vector (Bochud-Allemann and Schneider, 2002 (link)) in two steps by cloning with the restriction enzymes BamHI HF, HindIII HF, XbaI and XhoI (NEB) to generate the later hairpin loop double-stranded RNA (dsRNA) for RNAi. The final plasmid was linearized with NotI HF (NEB) prior to transfection. The ORF of TAP110 was amplified and inserted without the stop codon by cloning with the restriction enzymes HindIII HF and XhoI (NEB) into a modified pLew100 vector for overexpression (Wenger et al., 2017 (link); Wirtz et al., 1999 (link)).
For the Tb927.11.6660-PTP construct, the ORF positions 2397 to 2805 were amplified as described above and cloned between the ApaI and EagI sites. We used SnaBI (NEB) to linearize the plasmid prior to transfection.
For the Tb927.11.6660-PTP construct, the ORF positions 2397 to 2805 were amplified as described above and cloned between the ApaI and EagI sites. We used SnaBI (NEB) to linearize the plasmid prior to transfection.
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