¹ Clemson University Genomics Institute, Clemson, SC, 29634, USA, http://www.genome.clemson.edu

² Genome Center of Wisconsin, 445 Henry Mall, Genetics Building B19, Madison, WI, 53706, USA

Two series of cloning vectors have been developed for construction of genomic and shotgun library. PCR amplified DNA segments of NPTII from pACYC177, origins of replication from pACYC177 and pBR322, and multicloning site from pUC18, and LacZ including multicloning site from pBluescript SKII(-) were used for construction of series of vectors via in vitro combination. These vectors were successfully used to construct rice whole genomic libraries covering 8.8x rice genome with 11kb insert size and to create shotgun libraries carrying small (2-4kb) and big inserts (6-10kb) from rice and tomato BAC clones. The vectors have the following useful properties: (1) Choice of cloning methods, pCUGI series [pCUGI11(2,242bp), pCUGI21 (2,554bp) and their modifier, pCUGI12 and pCUGI22, by insertion of pBR322 segment between PstI and KpnI sites] for direct cloning using double adaptor methods and pCUGIblu series [pCUGIblu11(2,757bp), pCUGIblu21 (3,069bp), and pCUGIblu31 (2,403bp)] for general cloning method by rapid colour screening for inserts. (2) Choice of relatively small vectors based on origin of replication and copy numbers, pCUGI11 and pCUGIblu11 with p15A ori (medium, 10 copies per cell), pCUGI21 and pCUGIblu21 with pMB1 ori with rop gene (medium, 25 copies per cell), and pCUGIblu31 (2,403bp) with pMB1 without rop gene (high, 500 copies per cell. (3) Unique and rare cutting SwaI (ATTTAAAT) restriction enzyme sites in pCUGI11, pCUGIblu12, pCUGIblu11, and pCUGIblu21 give easy detection of insert size. (4) All vectors carry a kanamycin resistance gene which prevents the chance of contamination with BAC clones carrying CM (chloroamphenicol) resistance gene and the appearance of satellite clones on LB plates unlike ampicilline. (5) All vectors have a multicloning site enabling cloning and rapid sequencing with universal primers, pCUGI series from pUC18 and pCUGIblu series from pBluscriptII.