Fig. 2

Sequences and recombination frequencies of HK and ΦC31 attB sites. The three ORFs for HK and the six ORFs for ΦC31 were inserted into bla and tested using the conjugation assay as in Fig. 1a. The six open reading frames of the 23 bp attB HK site are shown. As in Fig. 1, black nucleotides represent the 23 bp HK sequence, with the corresponding amino acids also in black. The red nucleotide shows the base changed from the original attB, with the resulting amino acid changes also shown in red. Nucleotides and amino acids in teal represent sequences flanking the 23 bp site. Horizontal arrows indicate the direction of transcription, and asterisks indicate a stop codon. For both a and b, the sequence of recombination exchange is indicated by a horizontal red line. As described in the text, three open reading frames did not have a stop codon and were able to be tested for bla insertion. The recombination frequencies of these open reading frames compared to the 23 bp HK attB site are shown in the bar graph. The open reading frames are also shown in context of the bla sequence flanking the insertion site. Note that to keep the attB site in frame with bla, nucleotides were added to either the 5′ or 3′ end of the site, which changed the expected amino acid residue for ORFs 1 and 3 compared to the original attB. The background colors highlighting the sequence correspond to Fig. 1a. The recombination frequencies of the different ORFs were compared using 1-way ANOVA followed by a Tukey-Kramer test. Each of the HK ORF recombination frequencies are significantly different (p < 0.05). b. None of the six ΦC31 ORFs encode a stop codon. ORFs 1 and 2 recombine at a higher rate than ORFs 3–6 (p < 0.001)