This is the information supplied by BACPAC Resources about the new vector pBACe3.6 that was created by them to improve the BAC vector system:
In the course of our work, we experienced feedback from users of our PAC clones at several major sequencing centers. A minor disadvantage was the size of the PAC vector (16 kb left in recombinants) as compared to the size of the BAC vector (7 to 8 kb, depending on the vector variant). For the use of BACs or PACs with inserts in the range of 100-150 kb, this means that a larger fraction of the clones in shotgun sequence libraries (M13 or pUC) will be derived from the PAC/BAC vector sequence, hence resulting in a few percent increase in the cost per base pair of final sequence.
In view of these observations and because the existing BAC libraries were of insufficient size, we have improved the BAC vector (Shizuya H. et. al. 1992, Proc. Natl. Acad. Sci. USA 89:8794-8797) to include some of the retrofitting options included in the pPAC4 vector. The new vector has been named pBACe3.6. Specifically, we maintained the wildtype loxP site, added an additional mutant loxP511 site, a site for the intron encoded nuclease PI-SceI and the Tn7att site.
In addition, we changed the cloning area to allow positive selection for inserts containing BAC clones through inclusion of the sacBII gene from Nat Sternberg's P1 vector (Pierce et al. 1992, Proc. Natl. Acad. Sci. USA 89:2056-2060) and disrupted the BamHI cloning site with a fragment containing a pUC plasmid.
The presence of this pUC plasmid serves dual functions: high copy number of the vector for preparing large quantities and appropriate disruption of the sacBII gene to increase viability of the vector containing strain. In addition to the BNamHI site, 5 additional sites can be used for preparing BAC libraries: SacI, SacII, MluI, EcoRI, and AvaIII. The EcoRI site is particularly important in view of the use of this site in the RARE cleavage procedure, thus opening the possibility for selective cloning of similar fragments from different DNA donors. pBACe3.6 clones have chloramphenicol antibiotic resistance. Clones should be grown in LB containing 20ug chloramphenicol/ml.
Roswell Park Cancer Institute, Human Genetics Department, Pieter J. de Jong - Principal Investigator, Elm and Carltin Streets, Buffalo, New York 14263-0001
Bacpac Resources (http://bacpac.chori.org)