I'm trying to clone a neomycin sequence with LoxP sites flanking the sequence into an 11 kb backbone. The PCR is not an issue but after the assembly, the colonies that I get are the backbone with a short sequence of the neomycin sequence with only one LoxP site and the rest of the sequence is missing. I have tried the assembly multiple times with the same result. Somehow, due to the tandem repeating sequence of the LoxP sites, the sequence flanking the sites gets excised out. I have tried different primers so that there is a LoxP site from each of the PCR product in the hope to minimise this interaction between the LoxP sites but it has produced the same result. Extending the assembly time from 5 to 1 hour also doesn't help. Any advice regarding this issue is greatly appreciated!