Golden Gate Assembly Designer

How Golden Gate Assembly works

Golden Gate Assembly (Engler et al., 2008) uses Type IIS restriction enzymes that cut outside their recognition sequence. This allows:

1. Enzyme sites are removed after digestion — the final construct has no scar sequences 2. Directional assembly — unique 4-bp overhangs dictate fragment order 3. One-pot reaction — restriction and ligation cycle in a single tube

The cycling protocol (typically 30–60 cycles of 37 °C digestion / 16 °C ligation, followed by heat inactivation) drives the reaction toward the correct product because correctly assembled constructs lack enzyme sites.

Overhang fidelity and the Potapov matrix

Not all 4-bp overhangs ligate with equal fidelity. Potapov et al. (2018) profiled every possible overhang pair for correct vs. mismatch ligation by T4 DNA ligase.

• Some overhangs (e.g., AACT, AATG) ligate with >99% fidelity
• Palindromic overhangs (e.g., AATT, GATC) self-ligate and should be avoided
• Overhangs that are reverse complements of each other create ambiguity

This tool selects from the highest-fidelity orthogonal overhang sets, maximizing the probability that every junction assembles correctly.

Primer design for Golden Gate

Each Golden Gate primer has three parts (5′ to 3′):

1. Padding (4 bp) — ensures efficient enzyme cutting at the end of linear DNA 2. Enzyme recognition site + spacer — e.g., GGTCTC(N)1 for BsaI 3. 4-bp overhang — the fusion site that directs assembly 4. Binding region (15–35 bp) — anneals to template for PCR, optimized for Tm ~60 °C

The forward primer uses the enzyme site in sense orientation; the reverse primer uses the reverse complement. After digestion, the enzyme site and padding are removed, leaving only the overhang + fragment.