C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system

Jaïs, Philippe H; Decroly, Etienne; Jacquet, Eric; Le Boulch, Marine; Jaïs, Aurélien; Jean-Jean, Olivier; Eaton, Heather; Ponien, Prishila; Verdier, Fréderique; Canard, Bruno; Goncalves, Sergio; Chiron, Stéphane; Le Gall, Maude; Mayeux, Patrick; Shmulevitz, Maya

Plasmids containing the Firefly Luciferase gene were used to optimize C3P3-G1 enzymes and DNA template sequences. They consist of a phage RNA polymerase promoter, variable 5 -UTR, Kozak consensus sequence followed by the ORF of the Luciferase gene from Photinus pyralis and stop codon, variable 3 -UTR, poly [A] track that was routinely of 40 adenosine residues, followed by a selfcleavage RNA sequence that was generally the genomic ribozyme sequence from the hepatitis D virus, and terminated by the bacteriophage T7 10 transcription stop. Restriction enzymatic sites were inserted between each motif of the luciferase plasmids to allow easy swapping of each motif by subcloning. The plasmids are identified by the corresponding ORF (e.g. Luciferase) preceded by the phage promoter (e.g. pT710-Luciferase).