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. 2024 Dec 5;8(2):e202402757. doi: 10.26508/lsa.202402757

Figure S3. Schematic illustration of the repeat-associated non-AUG-dependent 45xG4C2 repeat construct with 3xV5-tags in all ORFs.

Figure S3.

As described previously, concatemerized and annealed G4C2/C4G2x15 DNA oligonucleotides blunted with Mung bean nuclease were cloned into the Klenow-filled EcoRI site of pcDNA3.1 to build pcDNA3.1-45xG4C2. A NotI/XbaI cassette encoding 3xV5-tags and three stop codons in all frames was digested from a synthetic custom-synthesised plasmid (Thermo Fisher Scientific) and subcloned into the NotI/XbaI site downstream of 45xG4C2. Note the absence of canonical Kozak-ATG start codons from the transcription start site leading to repeat-associated non-AUG-dependent translation of C9orf72 sense repeat dipeptide repeat proteins. The frame, codon usage and the resulting poly dipeptide are highlighted in the green box. Sense dipeptide repeats are produced in the three reading frames leading to poly(GA), poly(GP), and poly(GR), each with their own V5-tag.