Synthetic Gene Construction for DPR Proteins

Synthetic genes for DPR sequences with ATG start codon, reduced GC content and very few remaining GGGGCC repeats were made to order with C-terminal epitope tags (Life technologies, Geneart, Regensburg, Germany). For details and design rational see Fig. S1a. The full sequence information is available in the supplemental methods. Synthetic genes and the original GGGCCG-based poly-GP construct with an ATG start codon were subcloned into pEF6/V5-His vector (Life technologies) or a lentiviral vector driven by human synapsin promoter (FhSynW2). To replace the ATG start codon in the GA₁₄₉-myc construct with a TAG stop codon we cloned annealed oligonucleotides between an SgrAI site at the 5′ end of the open reading frame and the EcoRI site in the vector. As a negative control GFP from pEGFP-N1 (Clontech) was subcloned into pEF6/V5-His and FhSynW2. The GGGGCC repeat constructs without ATG start codon had been described previously [36 ]. Rat and human Unc119 cDNA was expressed from a lentiviral vector driven by human ubiquitin promoter containing an N-terminal HA-tag (FUW2-HA). We used shRNA targeting rat Unc119 (GAGAGGCACTACTTTCGAA) or a control targeting firefly luciferase (CGTACGCGGAATACTTCGA) driven by the H1 promoter in the vector FUW coexpressing TagRFP both for transfection and transduction. Lentivirus was produced in HEK293FT cells (Life Technologies) as described previously [17 (link)]. The Q₁₀₂-GFP construct in pCS2 vector was a gift from B. Schmid [44 (link)].