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This skill provides expert guidance for Golden Gate DNA assembly, focusing on the precise design of primers and the selection of 4-nucleotide overhangs. It streamlines the molecular cloning process by offering structured workflows for BsaI-based assemblies, ensuring proper fragment orientation and enforcing rigorous in silico verification. By simulating PCR products, digestion, and ligation steps before synthesis, the skill helps researchers avoid common pitfalls like internal restriction sites, sub-optimal melting temperatures, and mis-ligation, ensuring the final genetic construct matches the intended design exactly.
