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Many diseases such as DiGeorge syndrome, leukemia and likely all cancers display abnormal replication timing, the temporal order in which segments of chromosomes are replicated. However, the elements that regulate normal replication timing are poorly understood. DNA replicates during S phase of the cell cycle and does so in a spatio-temporal pattern. DNA replicating early in S phase localizes within the nuclear interior and is highly correlated with transcriptionally active euchromatin. On the other hand, DNA replicating late in S phase localizes at the nuclear and nucleolar periphery and is highly correlated with transcriptionally inactive heterochromatin. Taken together, this pattern of replication shows that DNA replication is likely involved in higher chromosome structure and function. To continue progressing within the field, replication timing regulatory elements must be identified. Uncovering these elements will allow a greater understanding of higher chromosome structure and function organization as well as elucidating the relationship between abnormal replication timing and disease. In order to show necessity of suspected candidate regulatory elements, each must be removed and a dramatic effect on replication timing must be observed. This thesis focuses on two means of showing element necessity: the development of a targeting vector to generate small deletions of DNA that harbor suspected regulatory elements and the confirmation that puromycin reconstitution to generate and identify large deletions is feasible.