BMC Genomics 2006, 7:73 http://www.biomedcentral.com/1471-2164/7/73 non-identical spacer sequences [6,7]. Cre-loxP site promis- cuity is not a new observation; for example initial studies identified another loxP recombination site in the E. coli chromosome, loxB [22]. However, since most applications of Cre-loxP focus on pre-defined spacers there has been limited attention paid to promiscuity. Our data showed that for each spacer, self-recombination was not signifi- cantly greater than recombination with other spacers. Sec- ond, it has been assumed that the wild-type loxP spacer sequence is a favoured substrate for Cre recombinase. Although this assumption has never been explicitly stated, the majority of screens to identify novel spacers focus on point mutations to the wild-type spacer sequence. In the present study the wild-type spacer was detected in only 1 recombination reaction. This was a non-self recombina- SFeigquernec5e logo representing the Cre-loxP consensus site Sequence logo representing the Cre-loxP consensus site. A total of 6,248 spacers that participated in successful recombination reactions were used to generate a sequence "logo" representing the nucleotide preference of Cre recom- binase in the spacer region. Information content (I) as each spacer position was calculated (see Methods for details). Base height (H) represents nucleotide frequency and at each position in the loxP spacer region. assayed since any two spacer sequences could interact in the Cre reaction. This revealed the promiscuity profile of each spacer sequence and identified the spacer sequences that were favoured by the Cre recombinase protein. By applying profiling algorithms typically used to analyze transcription factor binding sites [24], we have generated the sequence profile of the consensus, that is, the spacer most favoured by Cre recombinase (Figure 5). The most notable feature of this "sequence logo" is the abundance of guanine residues. The reason for the strong bias towards guanine content remains unclear. A number of assumptions about Cre-loxP recombination were directly challenged by the present study, the first being that Cre-loxP recombination between identical spacer sequences is favoured over recombination between 100 )90 % ( 80 n o it70 a n i 60 b 50 m o c e40 r m30 u i m20 x a10 M 0 G G G T A G G C EoFfnifgerucetrceoofm6nbuicnlaetoiotindefrleoqcuaetinocny and sequence within the spacer Effect of nucleotide location and sequence within the spacer on recombination frequency. Effect on recombi- nation rate of changing one base at each degenerate spacer position from the sequence of the most promiscuous spacer (i.e. changing the base at position 1 from G to C creates the spacer CGGTAGGC which causes a 88% drop in total recombination relative to that of the most promiscuous spacer GGGTAGGC). Page 9 of 13 (page number not for citation purposes) C G T A