Isolation of a 293 clone containing a single copy of the binary expression vector. Genomic DNA (15 μg) prepared from individual 293 cell clones containing an integrated binary expression vector with EGFP in the OFF position, as in Fig 1C. DNA was digested with the restriction enzyme XbaI, which cleaves within the construct, approximately 3.5 kbp 3' to the probe sequence (CMV). Thus each individual insertion should result in a single electrophoretic species whose size is dictated by the nearest genomic XbaI site. Lane 1, 293 parental cells containing no vector; Lanes 2–3, two random clones (X, Y) that contain more than one cross-hybridizing species; Lane 4, one clone (S), showing a single cross-hybridizing species, as predicted for a single insertion. The positions of the DNA markers are indicated at right, in kilobase pairs (kbp).