Construction of the new BAC vector pIndigoBAC536-S and the new BIBAC vector BIBAC-S (diagram is not to scale). A. Schematic outline for the construction of the two new vectors. The Not I fragment of pIndigoBAC536 was amplified by PCR using P1/P2 primers that contained Sal I and I-Sce I recognition sites at the 5' ends and P3/P4 primers that contained Not I and I-Sce I recognition sites at the 5' ends. The PCR products were ligated to the Sal I- digested backbone of pIndigoBAC536 and the Not I-digested backbone of BIBAC2, resulting in the plasmids pIndigoBAC536-S and BIBAC-S, respectively. MCS: multiple cloning sites. Hin dIII, Bam HI and Eco RI are three unique sites in pIndigoBAC536 and pIndigoBAC536-S used for BAC library construction. Bam HI is the unique site in BIBAC2 and BIBAC-S used for BIBAC library construction. The MCS also contains a Sal I site. B. The sequence, orientation and locations of the I-Sce I sites in the pIndigoBAC536-S and BIBAC-S vectors. The two I-Sce I sites are located in an inverted orientation at positions that flank an identical DNA fragment containing the lac Z selection marker and the cloning site. After digestion by I-Sce I, both vectors produce the non-complementary ATAA ends (in bold).