Abstract Redesign or modification of the cellular physiology requires a quantitatively well-controlled expression system known as the “tunable expression.” Although the modification of promoters demonstrates the great impact on the translation efficiency, it is difficult to detect the proper variants required for tunable expression. The 5′-untranslated region (UTR), however, can be an important target for tunable expressions because the ribosome binding affinity is directly modulated by the sequence variants of the Shine–Dalgarno (SD) sequence and the AU-rich sequence, which are the ribosome binding sites and a SD-sequence-independent translation enhancer, respectively. This study developed a simple method to obtain numerous 5′-UTR variants and analyze their translation efficiency based on the PCR-based site-directed mutagenesis and the expressional PCR using coupled in vitro transcription/translation system derived from Escherichia coli and eGFP gene as a template. SD sequence variants (18) and AU-rich sequence variants (36), which have a wide range of relative expression levels ranging from 0.1 to 2.0, were obtained. The translation efficiency was affected by the ribosome binding affinity and its accessibility that is dependent on the secondary structure around the 5′-UTR.