In this study, we present the first methodology (to the best of our knowledge) enabling reconstruction of the eukaryotic 40S ribosome subunit with completely customized r-proteins, referred to as “Ribo-swap” (Figure 1). In Ribo-swap, a designer platform yeast was developed through relocating the 56 genes encoding the whole set of 32 40S r-proteins from native genome loci to a synthetic gene cluster, which could be further swapped by another one encoding customer-designed 40S r-proteins to reconstruct customer 40S ribosome subunits in yeast.