Synthetic genomics is an emerging field of study and a sub-discipline of synthetic biology. The construction of the first synthetic prokaryotic genome, that of Mycoplasma myocoides in 2010, has inspired the creation of many new synthetic genomes. Recently, the synthesis of all seventeen synthetic Saccharomyces cerevisiae chromosomes has been completed as part of the Sc2.0 project. Particular alterations have been made to the synthetic sequence. One such modification involves the elimination of redundant DNA sequences. The reduction of genomes has demonstrated the potential for engineering a simplified organism, thereby reducing the expenditure of energy on non-essential genes. A notable distinction in the Sc2.0 sequence from the wild-type genome involves the integration of symmetrical loxP (loxPsym) sites, positioned 3 base pairs downstream of all non-essential genes. These sites function as recognition sequences for the Cre recombinase, which catalyzes inversions, duplications, translocations or deletions between two loxPsym sites in the synthetic chromosome (referred to as SCRaMbLE). It has been demonstrated in prior studies that the Cre recombinase can be utilized to compact a synthetic yeast chromosome. However, there are still bottlenecks in the system. In addition to its role in gene deletion, the Cre recombinase can induce inversions, duplications and translocations, thereby generating complex restructured genomes. In this thesis, I expored a comprehensive manner for integrating deletions in synthetic yeast chromosomes in a top-down manner as well as a novel screening method based on quantitative Polymerase-Kettenreaktion (qPCR) technology. In order to increase the amount of structural variations than can be detected, we developed a set of primers called loxTags. qTagGer, was developed to design genotyping primers that amplify the area around each loxPsym site. These tags facilitate the identification of deletion, inversions and translocations. LoxTags were validated by applying an optogenetically controlled Cre recombinase (L-SCRaMbLE) to a synthetic yeast chromosome. Then, a semi-automated pipeline was adapted to dispense miniaturized qPCR reactions in high-throughput. Strains showing high amounts of recombination using qPCR were corroborated using Nanopore sequencing. The findings obtained through qPCR show all different forms of structural variations, and an increase in structural variations for ring chromosomes. Following an initial presentation on the functionality of loxTags in the context of genotyping, the thesis proposes an expansion of the available tools through the application of the same design principles for screening of cloned plasmid libraries. Identification of modular golden gate libraries is achieved by amplifying the area around each cloning site, allowing for high-throughput screening of colonies. In Chapter III, a pipeline for the reduction of synthetic yeast chromosomes is presented. As an alterative to SCRaMbLE-based genome compaction, in the pipeline, a URA3 gene is removed through an endonuclease. First, homologous recombination (HR) is used to insert the marker into a synthetic yeast chromosome using the loxPsym sites as sequences for homology. Specificity of the insertion was investigated and confirmed through transposon-based sequencing techniques. Next, the marker is enzymatically removed. For this, we perform a systematic comparison of the Cre recombinase, Cas9, Cas3 and I-SceI. Through the comparison, I confirm the use of Cas9 as the most effective way for removing URA3 without inducing additional structural variations. Furthermore, we show single iterations of genome reductions creates deletions of over 10kbp, depending on variables such as the location of the integration of the marker. In this thesis, we propose a series of tools designed to expedite the identification of non-essential DNA. The development of an alternative to classic transposon insertions, which specifically targets synthetic chromosomes and knocks out the full region between two loxPsym sites, was achieved through the introduction and subsequent removal of the URA3 gene. The implementation of a qPCR pipeline that utilizes loxTags facilitates the straightforward identification of mutation sizes. The qPCR pipeline presented in this thesis can be applied for pre-screening or verification of any type high-throughput DNA recombination or cloning approach. The genome reduction pipeline can be used to create combinatorial deletions in semi-high throughput. Screening of many candidates can lead to the identification and better understanding on non-essential gene space in synthetic S. cerevisiae chromosomes.