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ABSTRACT Phylogenies built from multiple genes have become a common component of evolutionary biology studies. Molecular phylogenomic matrices used to build multi-gene phylogenies can be built from either nucleotide or protein matrices. Nucleotide-based analyses are often more appropriate for addressing phylogenetic questions in evolutionarily shallow timescales (i.e., less than 100 million years) while protein-based analyses are often more appropriate for addressing deep phylogenetic questions. PhyloFisher is a phylogenomic software package written in Python3. The manually curated PhyloFisher database contains 240 protein-coding genes from 304 eukaryotic taxa. Here we presentnucl_matrix_constructor.py, an expansion of the PhyloFisher starting database, and an update to PhyloFisher that maintains DNA sequences. This combination will allow users the ability to easily build nucleotide phylogenomic matrices while retaining the benefits of protein-based pre-processing used to identify contaminants and paralogy.