pTW<Syt-CLIPm>, pTW<Syt-SNAPm>, pTW<CLIPf-Syb> and pTW<SNAPf-Syb> constructs were made using the Multisite Gateway technology platform (Invitrogen). CLIPf and SNAPf are engineered versions of the original versions CLIP26m (CLIPm) and SNAP26m (SNAPm), respectively, that display faster labeling kinetics [Sun:2011dq, Pellett:2011oq]. pTW is a pUASt vector containing a Gateway cassette. All CLIPm, CLIPf, SNAPm and SNAPf coding sequences were amplified from pCLIPm, pCLIPf, pSNAPm or pSNAPf plamids (NEB), respectively, to generate Gateway pDONR entry clones. Drosophila synaptotagmin 1 (syt1, GenBank M55048) was amplified from P{UAS-syt.eGFP}1 flies (Bloomington, [Zhang:2002uq]) using syt forward and EGFP reverse primers to avoid amplification of endogenous syt1. The resulting fragment was subcloned into the pJET vector (Fermentas) to generate pJET<Syt>. PCR on pJET<Syt> was performed with primers containing Gateway B1 and B5r sites. Drosophila n-synaptobrevin (n-syb, GenBank S66686) was amplified from P{UAS-n-syb.eGFP}2 flies using syb forward and EGFP reverse primers to avoid amplification of endogenous n-syb. The resulting fragment was subcloned into pJET to generate pJET<Syb>. PCR on pJET<Syb> with primers containing B5 and B2 Gateway sites was performed. Because the C-terminus of Synaptotagmin is cytoplasmatic (whereas the N-terminus is luminal), all Synaptotagmin constructs described in this study are C-terminal, i.e. cytoplasmic fusions. In contrast, all Synaptobrevin constructs described here are N-terminal fusions, i.e. cytoplasmic.