How does the Gibson Assembly Wizard factor in 5' and 3' overhangs?


The Gibson Assembly Wizard performs assemblies based on the sense strand cuts (rather than overhang directionality) and designs primers accordingly, essentially combining the two sense ends. 

Depending on which kit/exonuclease is used, there would be different exonuclease activity (potentially 5' -> 3' or 3' -> 5'). We picked a common solution to cover both possible scenarios.

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