FX cloning: a versatile high-throughput cloning system for characterization of enzyme variants

Methods for the cloning of large numbers of open reading frames (ORFs) into expression vectors are of critical importance for diverse disciplines in biology. Here I describe a system termed FX cloning that facilitates the high-throughput generation of expression constructs. FX cloning combines attractive features of established recombination- and single-strand-annealing-based cloning methods that were thus far not unified in one single method. FX cloning allows the straightforward transfer of a sequence-verified ORF to a variety of expression vectors, and it avoids the common but undesirable feature of significantly extending target ORFs with cloning-related sequences. It leaves a minimal seam of only a single amino acid to either side of the protein. Furthermore, FX cloning is highly efficient and economic in its use. The method is based on a class IIS restriction enzyme and negative selection markers. The full procedure takes place in one pot and does not require intermediate purifications. The method has proven to be very robust and suitable for all common pro- and eukaryotic expression systems.