Advances in DNA synthesis technology have led to rapid growth in the field of synthetic biology, heralding a nascent era of synthetic genomics. Sc2.0 (Saccharomyces cerevisiae version 2.0) is an international consortium with the aim of designing and constructing a fully‐synthetic eukaryotic genome. Fundamental design changes to the synthetic genome include the removal of unstable tRNA genes and their intended collation onto a “tRNA neochromosome”, with the aim of producing a more robust and stable synthetic genome structure. To maintain viability of a synthetic yeast, the tRNA neochromosome is therefore considered an important if not essential aspect of this project. The application of engineering principles is synonymous with synthetic biology, regularly employing the recursive Design‐Build‐Test cycle to improve experimental approach. This doctoral study explores the design, construction and characterisation of a tRNA neochromosome in Saccharomyces cerevisiae. A series of design principles influenced by engineering concepts were used to rationalise the complexities of de novo chromosome engineering, maximise its stability and ensure function in vivo. A methodology based on in vivo homologous recombination was then developed and shown to reliably construct the neochromosome from its constituent parts. Experimental characterisation revealed that genetic elements function as expected, and that the parental strain can tolerate the sole presence of one each of three single‐copy, essential tRNA genes (SUP61, TRT2 and TRR4), although Northern blot revealed potential precursor accumulation of the SUP61 tRNA caused by the presence of a synthetic 5’ flanking sequence. Following the addition of synthetic telomere seed sequences, pulsed‐field gel electrophoresis (PFGE) and deep sequencing revealed complex structure variations in two independent strain backgrounds. Except for these structural variations, successful neochromosome construction demonstrated the applicability of the approaches used and the remarkable ability of the yeast model to support the presence of a 17th chromosome housing an additional 275 tRNA genes. The research in this thesis has for the first time described the design, construction and characterisation of a eukaryotic neochromosome de novo. It is hoped that the findings presented will further our understanding of tRNA biology and enhance the aims of the Sc2.0 project.