Modern synthetic biology depends on the manufacture of large DNA constructs from libraries of
genes, regulatory elements or other genetic parts. Type IIS restriction enzyme-dependent DNA
assembly methods (e.g., Golden Gate) enable rapid one-pot, ordered, multi-fragment DNA assembly,
facilitating the generation of high-complexity constructs. The order of assembly of genetic parts is
determined by the ligation of flanking Watson-Crick base-paired overhangs. The ligation of
mismatched overhangs leads to erroneous assembly, and the need to avoid such pairings has
typically been accomplished by using small sets of empirically vetted junction pairs, limiting the
number of parts that can be joined in a single reaction. Here, we report the use of a comprehensive
method for profiling end-joining ligation fidelity and bias to predict highly accurate sets of connections
for ligation-based DNA assembly methods. This data set allows quantification of sequence-dependent
ligation efficiency and identification of mismatch-prone pairings. The ligation profile accurately
predicted junction fidelity in ten-fragment Golden Gate assembly reactions, and enabled efficient
assembly of a lac cassette from up to 24-fragments in a single reaction. Application of the ligation
fidelity profile to inform choice of junctions thus enables highly flexible assembly design, with >20
fragments in a single reaction.

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