Zip folder containing the raw data and analysis to support the discovery of a novel synthesis pathway for 2’-deoxynucleotide 5’-triphosphates. The .zip file contains the following subfolders. (1) DNA-gels: 15 image files (.tif) of agarose gel electrophoresis of extracted E. coli genomic DNA and polymerase chain reaction (PCR) results. The README file contains detailed descriptions of the samples in each gel image. (2) Enzyme-kinetics: Microsoft excel files containing the raw absorbance measurements at 340 nm output by TECAN plate reader. The activity of nucleotide kinase enzymes was characterized using the double coupled pyruvate kinase-lactate dehydrogenase assay described by Agarwal et al (1978). This assay measures the decrease in absorbance at 340 nm as NADH is converted to NAD+ at a 1:1 molar ratio as the product of the enzyme reaction is generated. Each experiment contains a well plate layout file, a NADH standard curve, and the analyzed reaction velocity files. The initial velocity (mol product/s) is determined by the linear region of the product vs. time curve. Product concentration is determined from the A340 nm standard curve. To determine the Michaelis-Menten parameters, initial velocity was plotted against substrate concentration and fit with a nonlinear regression, generating the KM (uM) and Vmax (umol product/s) values. The specific activity is determined by normalizing the Vmax against the mass of enzyme. Detailed calculations are included in enzyme-kinetics-files.xlsx. (3) HPLC: High pressure liquid chromatography data for commercial nucleotide standards and nucleotides produced via the synthesis pathway proposed in this work. The dataset includes two types of .csv files output by Agilent ChemStation software. Signal files include time (s) and the DAD signal at 259 nm. Integration files contain analysis of the peak signal as calculated by the ChemStation algorithm. See the README file for a detailed description of columns in the integration file. The peak integration of commercial standards at varying concentration was used to generate a standard curve. The signature elution time of commercial standards was used to determine molecular identity of nucleotides in the in-house produced samples. See the Files.xlsx sheet for detailed calculations and sample descriptions. (4) PicoGreen: Microsoft excel files containing the raw fluorescence readings from PicoGreen assays of PCR reactions. Lambda genome DNA diluted in 1X Tris-EDTA buffer was used to generate a standard curve (concentrations in ng/mL). (5) Prism-data-analysis: GraphPad Prism files containing the nonlinear regression analysis of enzyme kinetics (.pzfx). The results of the analysis are also included in enzyme-kinetics-files.xlsx. (6) Protein-gels: sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) images for visualization of recombinantly expressed proteins. The README file contains detailed descriptions of the samples in each gel image. Four (.tif) files, one (.jpg) file, and one Microsoft excel file with image quantification data from analysis in ImageJ software. See the main manuscript for more details.