Significance The mechancial and transport properties of every material are intimately coupled to the generation and formation of defects. In many systems, the nucleation process of defects is highly unpredictable due to the multiple coexisting nucleation mechanisms. A strategy to design and manipulate defect nucleation and formation can improve our understanding and in principle lead to the ability to control performance. Here we present a simple approach of designing 2D defects—named antiphase boundaries—using an atomic-controlled high-quality interface. These defects have a well-defined origin, location, and nucleation mechanism. Via advanced synthesis technology, atomically resolved electron microscopy, and theory, we reveal that these defects display distinctive properties such as physical merging and antipolar structural phases to potentially achieve memory devices.