Here we report the design, construction and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ~190 kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporated orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enable an orthogonal SCRaMbLE system capable of adjusting tRNA abundance. Following construction, we obtained evidence of a potent selective force once the neochromosome was introduced into yeast cells, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up new opportunities to directly test hypotheses surrounding these essential non-coding RNAs Overall design: To investigate tRNA expression from native chromosomes and neochrosome in Saccharomyces cerevisiae (DSy063, DSy162, DSy177, DSy199), we carried out multiplex small RNA sequencing (MSR-seq) of total RNA isolated from these yeast strains. We then performed sequencing data analysis for mature tRNA and for precursor tRNAs containing the upstream (up) or downstream (down) sequences of these 4 strains of 3 biological replicates each. We compared the expression levels between the mature tRNAs of these 4 strains, and the expression levels of the precursor tRNAs derived from the native chromosomes versus those derived from the neochromosome.