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)
Don't use for
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).
