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The major ampullate silk of orb-weaving spiders is renowned for its exceptional mechanical properties, including high tensile strength and extensibility. The development of artificial spider silk presents a promising alternative to traditional fibers with significant environmental impacts. This study aims to elucidate the relationship between sequence motifs of natural spider silk and the mechanical properties of artificial spider silk. Using the Spider Silkome Database, we identified motifs correlated with specific physical properties and substituted them into MaSp2-based mini-spidroin BP1. We then measured the mechanical properties of the resulting recombinant artificial spider silk through tensile tests, observed structural properties via birefringence measurement and wide-angle X-ray scattering, and evaluated the water response through boiled water shrinkage tests. Introducing a positively correlated motif increased the tensile strength by 9.3%, while a negatively correlated motif decreased it by 5.1%, confirming the sequence–property relationship. These findings demonstrate that targeted motif substitution can effectively control the physical properties of artificial spider silk, facilitating the development of sustainable biomaterials with tailored mechanical properties for diverse industrial applications.