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In the multiple alignment, the N-terminal region of BmrD (Met1-Leu48) is aligned to the elbow helix and the TM1 of other ABC transporters, followed by a heterologous insertion of ∼100 residues.
From the multiple alignment the set of landmarks is then extended to include any additional fully conserved residues not found in the first phase, and residues with less than total conservation.
From the resulting multiple alignment, the fraction amino acid identity between all pairs of proteins was determined and averaged.
At the first stage we find a two-boxed signal via local multiple alignment (the first algorithm, ref. to Response to A.M #2).
For pairwise and multiple alignment the gap opening penalty was set to 10, whereas the gap extension penalties were set to 0.1, and respectively 0.05.
Among the methods for global multiple alignment, the program MAFFT [ 21, 22] performed remarkably well, not only on globally, but also on locally related sequences.
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The traditional steps of building gene trees (constructing multiple alignments, the choice and configuration of inference methods, robustness verification, etc).
The multiple alignment of the Pax gene family of metazoan animals was built based on the full-length amino acid sequences of the proteins using the clustalW2 program36.
The multiple alignment of the conserved Rab family domain were constructed with Clustal X (version 2.0).
DBL5ε homology models were created by submitting the multiple alignment to the HHpred server [38].
The multiple alignment of the protein sequences is then backtranslated to nucleotide space.
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