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Smad and receptor nucleotide sequences were aligned with protein alignment as guide using RevTrans-1.4.
Orthologous coding sequences without signal peptides were aligned following the protein alignment by ClustalW (www.ebi.ac.uk/clustalw/).ac.uk/clustalw/
The DNA sequences were aligned based on the protein alignment.
Protein sequences were aligned using ClustalW [ 23], then protein alignment was backtranslated to obtain codon alignment.
Codons were aligned guided by a clustalw [25] protein alignment (default parameters).
Parsimony analyses were performed using the protein alignment as input.
The optimal models were JTT [35] and gamma distributed across-site rate variation for both the TRPV1/2 protein alignment and the TRPM8 protein alignment.
The entropy at each position in protein alignment was measured with Shannon's entropy [55].
Therefore, the fitness function can be represented as an increasing function of a protein alignment score.
Current protein alignment methods are often slow and require assumptions about relatedness and evolutionary mechanisms [2].
The resulting protein alignment was reviewed and finalized using the multiple alignment sequence editor MASE.
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