pcDNA3.1-Syn-GFP-Neogenin plasmid construction

For the construction of the pcDNA3.1-Syn-GFP-NEO1 vector, the NEO1 coding sequence without signal peptide (aa 46-1492) was PCR-amplified from wild type mouse NEO1 (pCMVXL-6-NEO1; a kind gift of Denise Davis). This fragment was cloned into the blunt-made MluI/NotI sites of the PCI-Syn-GlyS267Q plasmid, containing the rat synapsin I promoter (a kind gift of Manfred Kiliman (Hoesche et al., 1993) Syn-NEO1 fragment was released from the PCI vector backbone by ClaI restriction and ligated into the EcoRV site of pcDNA3.1 (pcDNA3.1(-)/myc-his; Invitrogen). A signal peptide, GFP and 3xFLAG tag were PCR-amplified from the pRK5-DR/GABA(A)a1 vector (a kind gift of Guus Smit) and ligated N-terminal of the NEO1 coding sequence into the newly generated restriction sites AgeI and PshAI. For construction of the pcDNA3.1-CMV-GFP-NEO1 vector, the GFP-NEO1 fragment was isolated from the pcDNA3.1-Syn-GFP-NEO1 vector using ClaI and PshA restriction. The restriction sites were made blunt-ended and the GFP-NEO1 fragment was ligated into the EcoRV site of pcDNA3.1 (pcDNA3.1(-)/myc-his; Invitrogen).