The ability to synthesize large DNA assemblies has led to milestone achievements in prokaryotic systems and budding yeast chromosome biology motivating efforts to develop synthetic chromosomes in mammalian systems. Chromosomes in mammals and many other eukaryotes control their inheritance through an epigenetic locus: the centromere. Harnessing centromere epigenetics permits human artificial chromosome (HAC) formation but is not sufficient to avoid rampant multimerization of the initial DNA molecule that is introduced. Here, we describe an overhauled approach that efficiently forms stable, single-copy HACs. Their single-copy nature is achieved by starting with a construct that is sufficiently large to house the distinct chromatin types present at the inner and outer centromere. These HACs harbor full centromere function without the need to initially multimerize, thus permitting faithful chromosome engineering in the context of metazoan cells.