Strict on-demand control of protein synthesis is a crucial aspect of synthetic biology. The 5′-terminal untranslated region (5′-UTR) is an essential bacterial genetic element that can be designed for the regulation of translation initiation. However, there is insufficient systematical data on the consistency of 5′-UTR function among various bacterial cells andin vitroprotein synthesis systems, which is crucial for the standardization and modularization of genetic elements in synthetic biology. Here, more than 400 expression cassettes comprising the GFP gene under the regulation of various 5′-UTRs were systematically characterized to evaluate the protein translation consistency in the two popularEscherichia colistrains of JM109 and BL21, as well as anin vitroprotein expression system based on cell lysate. In contrast to the very strong correlation between the two cellular systems, the consistency betweenin vivoandin vitroprotein translation was lost, whereby bothin vivoandin vitrotranslation evidently deviated from the estimation of the standard statistical thermodynamic model. Finally, we found that the absence of nucleotide C and complex secondary structure in the 5′-UTR significantly improve the efficiency of protein translation, bothin vitroandin vivo.