FAQ: For Golden Gate Assembly, how many base pairs should my amplicon inserts have flanking the PaqCI restriction site?

Amplicon inserts must posses 5' flanking bases and PaqCI restriction sites at both ends of the amplicon in the proper orientation. We recommend adding 6 flanking bases at the 5' ends of the primers for optimal PaqCI binding, cleavage, and overall Golden Gate Assembly efficiency. This 6 base pair recommendation supersedes other known end length preferences for the enzyme due to Golden Gate Assembly protocols requiring maximum enzyme activity for efficient assembly.

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