Definitive demonstration by synthesis of genome annotation completeness

While the possibilities of living systems are theoretically near-infinite, the reality of biology at any instant is bounded. Here, we demonstrate how the discovery of functional information encoded in natural DNA sequences can be formally completed. Using bacteriophage øX174 as a model system, we identify and disrupt all conserved but unknown open reading frames, demonstrating by experiment that no cryptic functions remain to be discovered. Our proof by synthesis definitively affirms that the øX174 protein-coding annotations are complete and showcases how genomics and synthetic biology can be combined to operationally complete the genetics of natural living systems.