Equal partitioning of the multi-copy yeast 2-micron plasmid requires association of plasmid proteins Rep1 and Rep2 with tandem repeats at the plasmid STB locus. To identify sequence elements required for these associations we generated synthetic versions of a 63-bp section of STB, encompassing one repeat. A single copy of this sequence was sufficient for Rep protein association in vivo, while two directly arrayed copies provided partitioning function to a plasmid lacking all other 2-micron sequences. Partitioning efficiency increased with increasing repeat number, reaching that conferred by the native STB repeat array. By altering sequences in synthetic repeats, we identified the TGCA component of a TGCATTTTT motif as critical for Rep protein recognition, with a second TGCA sequence in each repeat also contributing to association. Mutation of TGCATTTTT to TGTATTTT, as found in variant 2-micron STB repeats, also allowed Rep protein association, while mutation to TGCATTAAT impaired inheritance without abolishing Rep protein recognition, suggesting an alternate role for the T-tract. Our identification of sequence motifs required for Rep protein recognition provides the basis for understanding higher-order Rep protein arrangements at STB that enable the yeast 2-micron plasmid to be efficiently partitioned during host cell division.