Analysis of EcI5 DNA target site EBS3/IBS3 base-pairing interactions by in vivo selection. A selection experiment was done as in Figure 6 with pBRR3-EcI5 randomized at IBS3 position +1 and pACD2-EcI5 randomized at EBS3 position +1. After selecting colonies containing recipient plasmids with integrated introns, the region extending from 75 bp upstream of EBS3 to 500 bp downstream from the inserted intron was amplified by colony PCR using primers EcI5 279s and Rev2-pBRR and sequenced using primer EcI5 297s (see Supplemental Table 2). (A) WebLogo representation of nucleotide frequencies in 79 selected integration events, corrected for biases in the initial pools based on sequences of 59 unselected donor plasmids and 59 unselected recipient plasmids. Nucleotide frequencies (percent) at the randomized position are summarized below. In some cases, percentage totals do not equal 100 due to rounding off. (B) Percentage of introns having Watson–Crick or wobble U•G and G•T base pairs at EBS3/IBS3 in the 79 selected integration events and 59 randomly paired donor and recipient plasmids from the initial pools.
