For plate-based small molecule screens, translations were scaled to provide necessary reagents for duplicate reactions in 96-well plates. Each Cfluc-Kinase was translated separately along with Fos-Nfluc using the conditions listed above. After translation and incubation at 30 °C for 90 min, several 24 μL aliquots of lysate were supplemented with 1 μL of buffer A and set aside as negative controls. The remaining lysate was treated with 2 to a final concentration of 125 nM. 24 μL aliquots of lysate treated with 2 were added to each well of a 96-well Lumitrac 200 plate (Grenier Bio-one) containing 1 μL DMSO (for positive controls) or 250 μM inhibitor (final concentration of 10 μM). 76 of the inhibitors used on each plate were from the Tocris Kinase Inhibitor Toolbox (Tocris Bioscience). Four inhibitors, PKC-412 (LC Labs), Sunitinib (LC Labs), Flavopiridol (Alexis), and Roscovitine (LC Labs), were also included. A list of all compounds tested, references, and their location on each plate can be found in the Supplementary Information (Table S2). The final concentrations of 2 and inhibitor were 120 nM and 10 μM respectively. Plates were covered with foil and equilibrated for 1 hour at room temperature. The Fos-Nfluc with Cfluc-Jun control was prepared in parallel under identical conditions, excluding the addition of 2. Using a Centro XS LB 960 plate reader (Berthold Technologies), 80 μL of luciferin assay reagent was injected into each well containing sample and luminescence immediately measured with a 1 sec integration time.