Gibson assembly enables seamless joining of multiple DNA fragments using a 5′ exonuclease, DNA polymerase, and DNA ligase in a single isothermal reaction. Fragments must share ≥20 bp homology with adjacent segments (Tm ≥50°C).
Principle: Arrange DNA fragments in desired assembly order. Including vector sequence at both ends streamlines circular construct design.
Primer Design Options
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- Sequences use standard IUB/IUPAC codes:
B=CGT,D=AGT,H=ACT,K=GT,M=AC,N=ACGT,R=AG,S=GC,V=ACG,W=AT,Y=CT. - 3′-end pattern: Use
Nfor any, or specify patterns likeWSS. - Linguistic Complexity (LC%) measures sequence vocabulary richness; 100% = maximum diversity.
Output: Export: Select all (Ctrl+A), copy (Ctrl+C), paste (Ctrl+V)
Quick Help
- Provide fragments in intended order (FASTA). Adjacent fragments must share overlap (≥20 bp, Tm ≥50°C).
- Use vector sequence at both ends for circular assembly support.