Significance The ability to design and build nonnative DNA sequences conferring specific and useful engineered functions on cells defines the field of synthetic biology. Here, we describe a synthetic biology device built for Saccharomyces cerevisiae. The telomerator inducibly and precisely linearizes circular DNA molecules in vivo, yielding derivatives that encode functional telomeres at each end. The telomerator provides the means to engineer artificial chromosomes, typically constructed as circular molecules, because telomerator-mediated linearization permits specific positioning of genes to modulate their expression. The telomerator also provides a tool to probe spatial relationships between functional elements on chromosomes such as telomeres, centromeres, and replication origins. In the future, the telomerator may be applied to engineer artificial chromosomes in other organisms.