Design Golden Gate assemblies with optimized 4-bp fusion site overhangs. Potapov 2018 fidelity scoring, Type IIS primer generation (BsaI, BsmBI, SapI, BbsI), CSV and FASTA export. All computation runs client-side.
Try it out
Load example Golden Gate Assembly designer data to see the full workflow
Enter your DNA fragments in the order they should appear in the final construct (5′ → 3′). At least 2 fragments required. The tool will assign high-fidelity overhangs and design primers with Type IIS enzyme extensions.
37 °C incubation | NEB #R3733
Comma-separated 4-bp overhangs for specific junctions
When to use
Do not use for
If any fragment contains the chosen enzyme recognition site (or its reverse complement), digestion will cut your insert. Domesticate fragments by synonymous codon changes before assembly.
Palindromic 4-mers (e.g., AATT, GATC) self-ligate, dramatically reducing assembly efficiency. This tool automatically avoids them.
Use 30–60 cycles of digestion/ligation (e.g., 5 min at 37 °C, 5 min at 16 °C) followed by final digestion and heat inactivation. More cycles improve yield for multi-fragment assemblies.
Type IIS enzymes cut poorly at DNA ends. The 4-bp padding added by this tool ensures efficient cutting. Do not remove it from your primer orders.
Even with high-fidelity overhangs, screen 4–8 colonies by PCR or sequencing. For 10+ fragment assemblies, expect to screen more clones.
Overhang selection: greedy pick from Potapov 2018 curated high-fidelity set, ensuring no pair is reverse-complementary. Fidelity scoring: per-junction from Potapov matrix, overall = product of all junctions. Primer Tm: SantaLucia (1998) nearest-neighbour with salt correction, target 60 ± 3 °C. Primer structure: [padding][enzyme site][spacer][overhang][binding region].
Last validated 2026-04-07. Calculations are designed for planning and documentation support; verify procurement decisions against manufacturer specifications or institutional SOPs.
ConductScience Golden Gate Assembly Designer (v1.19.0). ConductScience, Inc. 2026. Available at: https://conductscience.com/tools/golden-gate-assembly-designer
Potapov V et al. Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly. ACS Synth Biol. 2018;7:2665–2674. doi:10.1021/acssynbio.8b00333
Engler C, Kandzia R, Marillonnet S. A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLoS One. 2008;3:e3647. doi:10.1371/journal.pone.0003647
SantaLucia J Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. PNAS. 1998;95:1460–1465. doi:10.1073/pnas.95.4.1460
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.
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.
This tool selects from the highest-fidelity orthogonal overhang sets, maximizing the probability that every junction assembles correctly.
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.
