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

The C3P3-G1 enzyme sequences were subcloned into the pCMVScript plasmid backbone (Stratagene, La Jolla, CA, USA), following the removal of the T7 10 promoter sequence (i.e. the promoter commonly used in E. coli exogenous expression vectors, such as the pET series). All pCMV-C3P3-G1 plasmids and derivatives had the same design: IE1 promoter/enhancer from the human cytomegalovirus (CMV), 5 -untranslated region (5 -UTR), Kozak consensus sequence followed by the ORFs of the C3P3-G1 enzymes, 3 -untranslated region , and SV40 polyadenylation signal. Substitution of the coding sequence components from the C3P3-G1 enzymes was enabled by digestion at endonuclease restriction enzyme sites of six nucleotides located between each domain of the coding sequences of the C3P3-G1 enzyme (i.e. NH 2 terminus, upstream and downstream to the linker) and eight nucleotides immediately downstream to stop codon. The C3P3-G1 enzyme plasmids are identified by the different modules in the ORF of the C3P3-G1 enzyme downstream to the IE1 human CMV promoter/enhancer. For instance, NP868R-(G 4 S) 4 -K1ERNAP(R551S) designates the fusion of the NP868R capping enzyme with the mutant R551S K1E RNA polymerase through a (G 4 S) 4