I've encountered an issue following an LR reaction intended to construct a gene within a pENTR-3C (KanR) vector and combine it with a pCDH (AmpR) vector. Despite expectations, the protein was not detectable in Western Blot assays. Sequencing the product of transformation with the LR reaction revealed that rather than recombining with pCDH, two pENTR vectors had recombined with each other. This puzzling outcome raises several questions: Firstly, the competent cells formed colonies on ampicillin-containing agar, which is confusing since pENTR doesn't carry an AmpR resistance gene. How could this growth have occurred? Secondly, I was under the impression that attL sites would exclusively recombine with attR sites. What could explain the self-recombination observed between the attL sites? Any insights or advice on these matters would be greatly appreciated. Thank you!