In bacteria, high A and low G content of the 5′ end of the coding sequence (CDS) promotes low RNA stability, facilitating ribosomal initiation and subsequently a high protein to transcript ratio. Additionally, 5′ NGG codons are suppressive owing to peptidyl-tRNA drop off. It was, therefore, surprising that the first large-scale transgene experiment to interrogate the 5′ effect by codon randomization found the NGG, G-rich codon AGG to be the most associated with high transgene output. 

In this study we show that this is not replicated in other large transgene datasets, where AGG and NGG are associated with low efficiency. More generally, there is limited agreement between the first experiment and others. This we find to be a consequence of non-random construct design. The results of this research have implications for both transgene and experimental design.