One of the key factors influencing expression efficiency is the design of the expression vector. Therefore, we started with the design of a vector based on pSB1C3 that would allow successful cell-free expression. Additionally, we received an expression vector containing a GFP coding sequence, an HA and a double His-tag kindly provided by AG Roth, as an external standard that had already performed well in a cell-free expression system. The third vector we used for our experiments was the pBESTlucTM encoding a luciferase for performing the luciferase assay.