I performed an InFusion reaction - after digestion with vector of interest in 2 steps with two different restriction enzymes (RE)(BbvCI & BamHI) which cannot be heat-inactivated. I did the digestion in a 20ul volume, using CutSmart buffer. Incubated it 37C for 40 min (BbvCI), and second step for 60min(BamHI-HF).

I performed the InFusion reaction using three gblocks designed to have a 15-25bp overlap with the backbone vector and each of the subsequent fragments. Each fragment was approximately 200-350bp.

I transformed NEB highly competent bacteria.

I picked 6 colonies and sequenced 4 after digestion. Two of those are mostly correct except for a single bp deletion at 5 or 6 bp away from the restriction site of BbvCI.

Since InFusion works based on complementary based pairing, I am unsure how this deletion occurred:

If this would have been a problem with the design of the gblock - I would expect both to have the same deletion. I also checked my gblock orders and they all correspond with my original design.

The point deletion is downstream of the 1st RE digestion site, therefore it seems unlikely that this would be caused by the RE.

Is a particular sequence known for inhibiting or being prone to mistakes in InFusion cloning?

I added a picture of my aligned sequences.