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asearchtBm, the CpG steps have been replaced with bases that we have shown here were neutral with respect to recombination activity. It is noteworthy that the positions in attB that are critical for recombination other than position 2 i.e. positions 15, 16 were not highlighted as being particularly preferred in the pseudo-attB sites (Figure 1). However all of the pseudo-attB sequences have either the wild-type C at ¨C2 or a wild-type G at +2 (Figure 1) (41,43). It is also noticeable that the regions where there are most identities between attB and attP are not particularly sensitive to mutation (Figure 1). A plausible explanation for both these observations is that the 24 bp of att site DNA from about position 11 to the crossover site is a core sequence that integrase binds to specifically. We propose that the role of positions 15 and 16 in attB revealed by this study is to greatly enhance the efficiency of the reaction and effectively discriminate between the pseudo-sites and the cognate attB site. It is envisaged that this data and a similar analysis with attP will enable an understanding of the optimal sequences to target for continued application of C31 integrase.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
We are grateful for comments on this manuscript from Paul Rowley and Paul Hoskisson. M.G was funded by a scholarship from the University of Aberdeen. This work was funded by the Biotechnology and Biological Sciences Research Council, UK. Funding to pay the open access publication charges for this article was provided by the Biotechnology and Biological Sciences Research Council, UK.
Conflict of interest statement. None declared.
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