pINS plasmids are introduced always in the same orientation. Upper part. Schematic representation of the Cre-mediated recombination between Insertion vector pINS-Puro and Target vector phrGFP. Recombination intermediate and the sizes of the plasmids are shown. Lower part. Orientation of the pINS-Puro insert in the product of recombination (Puro × GFP) was analyzed by EcoRI. The sizes of the digestion products were analyzed on the agarose gel. We observed the fragments specific to only one orientation of the pINS-Puro in the product of recombination. This orientation is determined by the orientation of loxP sites.