![]() ![]() The In‑Fusion enzyme mix generates single-stranded 5' overhangs at the termini of the cloning insert and linearized cloning vector. ![]() 15-bp complementary regions must be located at the termini of adjacent DNA fragments or they will not be joined by In‑Fusion Cloning. Translational reading frame continuity of a fusion construct can be adjusted by adding nucleotides between the insert-specific sequence and 15-nt overlap. Homologous overlaps shorter than 12 nt or longer than 21 nt are not recommended. These 15-bp homologous overlaps can be generated by PCR amplification or oligo synthesis of either of the cloning components. For multiple-insert cloning, we recommend increasing the overlap to 20 bp. In‑Fusion Cloning requires 15 bp of overlap at the termini of the cloning insert and linearized cloning vector, or adjacent cloning inserts if multiple inserts are to be joined simultaneously. ![]()
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