Cloning and Transformation Protocol

All constructs for imaging were cloned into the standard peGFP-N2 vector (BD Biosciences Clontech, Heidelberg, Germany). Constructs used for planar lipid bilayer experiments were cloned into pET24-dellac (Merck, Darmstadt, Germany). Fragments were amplified with relevant overhangs using either Phusion polymerase (Thermo Fisher, Langenselbold, Germany) or Q5 polymerase (NEB, Frankfurt am Main, Germany) according to manufacturer’s specifications. The fragments were subsequently fused by either overlap-extension PCR [23 (link)] or Gibson Assembly [24 (link)]. The correct construct size was checked by standard agarose-gelelectrophoresis. Bands showing the expected size were purified with the Zymoclean Gel DNA Recovery Kit (Zymo Research, Freiburg, Germany) and the purified DNA was taken for heat shock transformation in competent Escherichia coli (DH5α). The amplified plasmid DNA was extracted with the ZR Plasmid Miniprep Kit Classic (Zymo Research, Freiburg, Germany) and sent sequenced (Microsynth Seqlab, Göttingen, Germany).