Design primers for Gibson Assembly and HiFi DNA Assembly cloning. Enter fragment sequences, get primers with optimal binding Tm and overlap extensions. Self-dimer check, IDT CSV export, and shareable URLs.
Try it out
Load example Gibson Assembly primer 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 design forward and reverse primers with overlaps for each junction.
Target Tm: 60 ± 3 °C | ~280x Taq (highest fidelity)
15–40bp (NEB recommends 15–30)
When to use
Do not use for
Shorter overlaps (15–18 bp) work for simple 2-fragment assemblies but fail for 4+ fragments. Use 25–30 bp overlaps for multi-fragment assemblies.
The overlap extension does not anneal during PCR amplification. Set your annealing temperature based on the binding region Tm, not the full primer Tm.
Enter fragments in the order they should appear in the final construct. The tool designs overlaps between adjacent fragments based on this order.
Q5 or Phusion is recommended for amplifying fragments before Gibson Assembly. Taq errors in the overlap region can prevent assembly.
Non-specific PCR products carry incorrect overlaps and create misassemblies. Gel extraction of the correct band before assembly dramatically improves success rates.
Binding region: walks from 15–35 bp to find length with Tm closest to target (SantaLucia 1998 nearest-neighbour, salt-corrected). Overlap: last N bp of previous fragment (forward) or revcomp of first N bp of next fragment (reverse). Self-dimer: heuristic scan for complementary 4-mers, threshold −9 kcal/mol. Gibson Assembly is a method developed by Daniel G. Gibson. NEBuilder HiFi is a trademark of New England Biolabs, Inc. This tool is independent and not affiliated with NEB.
Last validated 2026-04-07. Calculations are designed for planning and documentation support; verify procurement decisions against manufacturer specifications or institutional SOPs.
ConductScience Gibson Assembly Primer Designer (v1.18.0). ConductScience, Inc. 2026. Available at: https://conductscience.com/tools/gibson-assembly-primer-designer
Gibson DG et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods. 2009;6:343–345. doi:10.1038/nmeth.1318
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
Gibson Assembly (Gibson et al., 2009) joins multiple DNA fragments in a single-tube isothermal reaction at 50 °C. Three enzyme activities work in concert:
1. T5 exonuclease chews back 5′ ends, exposing complementary 3′ overhangs 2. Phusion polymerase fills gaps 3. Taq ligase seals nicks
The overlapping ends of adjacent fragments anneal and are seamlessly joined. The reaction typically completes in 15–60 minutes.
Each primer has two parts:
For a forward primer, the overlap comes from the 3′ end of the upstream fragment. For a reverse primer, the overlap is the reverse complement of the 5′ end of the downstream fragment.
This tool uses the SantaLucia (1998) unified nearest-neighbour model:
Where ΔH and ΔS are summed from all dinucleotide steps plus initiation parameters. Salt correction adjusts ΔS:
Default conditions: [Na⁺] = 50 mM, [oligo] = 0.25 µM. Only the binding region Tm is reported (overlap extension does not anneal during PCR).
