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.

UAS-TLN-CLIPm and UAS-TLN-SNAPm constructs were generated as follows: First, fusion PCR was performed on a pUAST-TLN-cherry plasmid to remove the mCherry coding sequence and flanking linker sequences (pUAST-TLNΔcherry). A 1.5 kb fragment from the XhoI restriction site to the sequence corresponding to amino acids TVRVA of mouse Telencephalin/ICAM-5 and a 1.0 kb fragment ranging from amino acids GPWLW of Telencephalin/ICAM-5 to the MfeI restriction site were amplified. Using primers that introduced flanking XhoI and BglII sites, using flanking primers containing XhoI and BglII, respectively (and removing two Stop codons) the entire 2.7 kb fragment was amplified and cloned into pUAST-TLNΔcherry (pUAST-TLN), yielding pUAST-TLN-BglII. CLIPm and SNAPm sequences were amplified using BglII-containing primers pairs and inserted into pUAST-TLN, yielding pUAST-TLN-CLIP and pUAST-TLN-SNAP.

pTW<PAT3SP-CD4-CLIPf> and pTW<PAT3SP-CD4-SNAPf> constructs were made using the Multisite Gateway technology platform. SNAPf was amplified from pSNAPf with primers containing B3fw and B2 Gateway sites, respectively to generate pDONR<B3-SNAPf-B2>. PAT3 signal peptide (SP) and CD4 sequences were amplified from UAS-CD4::spGFP1-10 flies [Gordon:2009qf].

UAS-myr-SNAPf was generated as follows: the GFP coding sequence from a pJFRC-MUH-myr-GFP construct [Pfeiffer:2010fu] was removed using BamHI/KpnI restriction sites and the SNAPf coding sequence (amplified from pSNAPf, NEB) was inserted.

UAS-myr-TMP was generated as follows: the GFP coding sequence was excised from a pJFRC81-L21 construct [Pfeiffer:2012ye] using BamHI/KpnI restriction sites and replaced with the eDHFR coding sequence (codon-optimized for Drosophila) and an N-terminal myristoylation signal (pJFRC81-L21_myrTMP).

UAS-myr-Halo was generated as follows: the TMP coding sequence was excised from UAS-myr-TMP (see above) using BamHI/KpnI restriction sites and replaced with a Halo-tag sequence amplified from the pHT2 Halo-tag plasmid (Promega) and subcloned into pJET. The insert was then excised with KpnI/BamHI and ligated into pJFRC81-L21_myrTMP (see above), from which the TMP (eDHFR) sequence had been excised.