Sequence overlap between two genes is common across all genomes, with viruses having particularly high proportions of these gene overlaps. The natural biological function and effects on fitness of gene overlaps are not fully understood and their effects on gene cluster and genome-level refactoring are unknown.The model bacteriophage φX174 genome displays complex sequence architecture in which ~26% of nucleotides are involved in encoding more than one gene. In this study we use an engineered φX174 phage containing a genome with all gene overlaps removed. Here we have temporally measured the proteome of a synthetically engineered and wild-type φX174 during infection. We find that almost half of all phage proteins (5/11) have abnormal expression profiles after genome modularisation.