The PCR amplification conditions for the 0-month Env backbones were 1 cycle of 95uC for 3 min; 35 cycles of 95uC for 1 min, 50uC to 60uC for 30 s (the optimal annealing temperature was determined for each primer set), 72uC for 10 min; 1 cycle of 72uC for 15 min; and storage at 4uC. The amplification conditions for the target domains were the same, except the extension time at 72uC was reduced to 30 s. The 25 ml PCR mixtures contained 50 ng of each primer, 10 ng of the plasmid template, 2.5 mM MgCl 2 , 0.2 mM deoxynucleoside triphosphate, and 16 reaction buffer. PfuUltra II DNA polymerase (Stratagene) was used to generate the blunt-ended PCR amplicons, which were digested with DpnI to remove contaminating template DNA and gel purified from an agarose gel using the QIAquick Gel Extraction Kit (QIAGEN) prior to ligation. Each target domain DNA fragment was then ligated to the purified 0-month env backbone to produce a chimera using T4 DNA ligase (5 U/ml; Roche) at 4uC overnight. The ligation reaction mixture (usually one-third of the volume) was transformed into maximum-efficiency XL2-Blue Ultracompetent cells (1610 9 CFU/mg DNA; stratagene) so that the DNA volume did not exceed 5% of the cell volume. The entire transformation was plated onto LB-ampicillin agar plates, generally resulting in 10 to 50 colonies per ligation reaction.