Table 2. Reaction conditions were adapted to CrPV IGR IRES-mediated translation in eukaryotic cell-free systems.

Coupled cell-free systems	Sf21 cell extract	CHO cell extract	K562 cell extract
Expression vector	60 nM/60 nM	60 nM/20 nM	60 nM/20 nM
KOAc	75 mM/120 mM	75 mM/150 mM	75 mM/120 mM
Mg(OAc)2	2.9 mM/3.4 mM	2.9 mM/3.9 mM	2.9 mM/3.4 mM
Incubation temperature	27°C/27°C	30°C/33°C	30°C/33°C

Reaction parameters used for cap-dependent translation in coupled reactions (left) were optimized for CrPV IGR IRES-mediated translation (right). Optimization was performed by cell-free expression of active LUC using the pIX3.0-CrPV IGR IRES (GCT) vector as a template. Reactions were incubated for 3 h in a thermomixer.