The vector pEPI was the first nonviral and episomally replicating vector. Its functional element is an expression unit linked to a chromosomal scaffold/matrix attached region (S/MAR). The vector replicates autonomously with low copy number in various cell lines, is mitotically stable in the absence of selection over hundreds of generations, and was successfully used for the efficient generation of genetically modified pigs. Since it is assumed that establishment of the vector is a stochastic event and strongly depends on the nuclear compartment it reaches after transfection, it is of great interest to identify genomic sequences that guide DNA sequences into certain nuclear compartments. Here we inserted genomic cis-acting sequences into pEPI and examined their impact on transgene expression, long-term stability, and vector establishment. We demonstrated that a ubiquitous chromatin-opening element (UCOE) mediated enhanced transgene expression, while an insulator sequence (cHS4) increased establishment efficiency, presumably via an additional interaction with the nuclear matrix. Thus, besides being a promising alternative to currently used viral vectors in gene therapeutic approaches, pEPI may also serve as a tool to study nuclear compartmentalization; identification of genomic cis-acting sequences that are involved in nuclear organization will contribute to our understanding of the interplay between transgene expression, plasmid establishment, and nuclear architecture.