DNA Assembly

Overview

This skill provides procedural knowledge for Golden Gate assembly primer design, a molecular cloning technique that uses Type IIS restriction enzymes (like BsaI) to create scarless multi-fragment assemblies. The skill emphasizes rigorous verification and simulation to ensure designed primers produce the expected assembled product.

Workflow

Phase 1: Understand the Assembly Requirements

Before designing primers, thoroughly analyze:

1. Input sequences: Read and parse all input FASTA files to understand the fragments to be assembled
2. Output sequence: Understand the expected final assembled product
3. Assembly topology: Determine if the assembly is linear or circular (circular plasmids require the last overhang to match the first)
4. Fragment order: Identify the correct order of fragments in the final assembly
5. Reading frame considerations: Note which fragments require start codons, stop codons, or neither

Phase 2: Overhang Design