In whales, increased muscle myoglobin concentration enables prolonged breath holding - essential for deep diving. Two traits are hypothesized to allow the high solubility of whale myoglobin - increased charge and increased stability - but it is still unknown which trait was the direct target of selection. This study investigates the possible mutational paths taken by whale myoglobin as it evolved to living whales' high concentrations. We explore the set of possible mutations and paths between the common ancestor of whales and hippopotamuses (Ancestral, or A) and the common ancestor of modern whales (Derived, or D). Six amino acid substitutions in the myoglobin protein sequence led to increased protein stability and charge. We aim to understand which trait was the target of selection during adaptation by looking into the space of mutational changes and analyzing the 64 possible intermediates formed by combinations of these six amino acid substitutions. We attempted to express and isolate the myoglobin protein in _E. coli_. Isolation methods were tested on the ancestral and derived myoglobin genes to isolate the protein. We used high-fidelity (HiFi) DNA assembly to efficiently generate 64 intermediate sequences, reducing the time and expense of making an extensive library of intermediate alleles. Primers were designed to PCR amplify the front and back halves of eight synthetic alleles, and HiFi cloning was used to quickly and inexpensively combine different variants to create all 64 possibilities. The myoglobin intermediates were cloned into the pET28a plasmid for growth in _E. coli_. Direct Sanger sequencing confirms this approach successfully created all 64 intermediates. This study provides a cost-effective, efficient method for reconstructing protein sequences for retrospective studies of molecular adaptation. Future work will continue to explore methods to isolate myoglobin and to measure its charge and stability.