Advances in recombinant DNA technology have increased the demand for novel vectors, particularly those that facilitate the cloning process. In particular, the manipulation of large segments of cloned DNA has been limited by the availability of vectors containing versatile polylinkers. Most of the current plasmid vectors contain a limited number of hexanucleotide restriction sites and one or two rare restriction sites in the polylinker region. Usually, these cloning vectors are highcopynumber plasmids that are built from a pUC (7) backbone that has the lacZ gene fused to the multiple cloning site for blue/white screening. Overlapping extension polymerase chain reaction (OE-PCR) has been used to generate synthetic DNA without the aid of an available template (2, 3, 6). This allows for the construction of computer-designed DNA of known sequence by synthesis of complementary overlapping oligonucleotides and subsequent PCR amplification. Ideally, a polylinker would incorporate several unique sites, both common and rare. This limitation is generally a function of sites absent in the vector backbone. However, in a lacZ fusion polylinker, considerations must also be given to length and maintenance of the open reading frame (ORF). The limitations to length can be overcome by the use of sites with opposite polarity (SWOP) pairs to create a compact multiple cloning site (5). This depends on the creation of adjacent restriction sites that share half or portions of their palindromic recognition sequence. Though many sites can be incorporated into a limited space, some distribution must be allowed for adequate restriction site availability and maintenance of the lacZ ORF. Thus, our goal was to develop a versatile cloning vector with a large polylinker region that would facilitate the manipulation of large segments of cloned DNA. Eight synthetic oligonucleotides and the OE-PCR technique were used to generate a pUC19 (7) based plasmid vector that contained a lacZ-fused polylinker with 44 unique restriction sites, nine of which represent rare-cutter restriction sites. In addition, the multiple cloning site is flanked by the T3 and T7 bacteriophage promoters. Positive transformants can be determined by either disruption of the lacZ gene for blue/white screening or by a characteristic change in colony size from small to large. A 230-bp synthetic polylinker was designed by computer analysis using the software packages MacDNASIS®