Animal chromosomes are partitioned into contact domains. Pathogenic domain disruptions can result from chromosomal rearrangements or perturbation of architectural factors. However, such broad-scale alterations are insufficient to define the minimal requirements for domain formation. Moreover, to what extent domains can be engineered is just beginning to be explored. In an attempt to create contact domains, we inserted a 2-kb DNA sequence underlying a tissue-invariant domain boundary-containing a CTCF-binding site (CBS) and a transcription start site (TSS)-into 16 ectopic loci across 11 chromosomes, and characterized its architectural impact. Depending on local constraints, this fragment variably formed new domains, partitioned existing ones, altered compartmentalization and initiated contacts reflecting chromatin loop extrusion. Deletions of the CBS or the TSS individually or in combination within inserts revealed its distinct contributions to genome folding. Altogether, short DNA insertions can suffice to shape the spatial genome in a manner influenced by chromatin context.