This study reveals a novel E3-independent mechanism of ubiquitination catalyzed by the human E2 enzyme UBE2E1, which can specifically recognize and ubiquitinate a SETDB1-derived hexapeptide (KEGYES) without the involvement of an E3 ligase. The structure of UBE2E1 in complex with this peptide was determined, providing insights into the sequence-dependent ubiquitination mechanism. Guided by this structure, the researchers developed an E3-free enzymatic strategy called SUE1 (sequence-dependent ubiquitination using UBE2E1) to efficiently generate ubiquitinated proteins with customized ubiquitination sites, chain linkages, and lengths. This strategy can also be used to create site-specific branched ubiquitin chains or NEDD8-modified proteins. The study not only deepens the understanding of E3-free ubiquitination in human cells but also provides a practical approach for generating ubiquitinated proteins to study the biochemical functions of ubiquitination. The SUE1 strategy can be combined with other chemical enzymatic methods, such as SUMO-conjugating enzyme Ubc9-mediated ligation, to generate different forms of ubiquitination at different sites of the same protein. Additionally, the SUE1 strategy is applicable to NEDD8 modification, which is an unprecedented general enzymatic strategy for introducing NEDD8 modifications into proteins. The study also demonstrates the potential of SUE1 in evaluating proteasomal degradation signals, showing that branched Ub chains can be less efficient in triggering proteasomal degradation compared to K48-linked Ub chains.This study reveals a novel E3-independent mechanism of ubiquitination catalyzed by the human E2 enzyme UBE2E1, which can specifically recognize and ubiquitinate a SETDB1-derived hexapeptide (KEGYES) without the involvement of an E3 ligase. The structure of UBE2E1 in complex with this peptide was determined, providing insights into the sequence-dependent ubiquitination mechanism. Guided by this structure, the researchers developed an E3-free enzymatic strategy called SUE1 (sequence-dependent ubiquitination using UBE2E1) to efficiently generate ubiquitinated proteins with customized ubiquitination sites, chain linkages, and lengths. This strategy can also be used to create site-specific branched ubiquitin chains or NEDD8-modified proteins. The study not only deepens the understanding of E3-free ubiquitination in human cells but also provides a practical approach for generating ubiquitinated proteins to study the biochemical functions of ubiquitination. The SUE1 strategy can be combined with other chemical enzymatic methods, such as SUMO-conjugating enzyme Ubc9-mediated ligation, to generate different forms of ubiquitination at different sites of the same protein. Additionally, the SUE1 strategy is applicable to NEDD8 modification, which is an unprecedented general enzymatic strategy for introducing NEDD8 modifications into proteins. The study also demonstrates the potential of SUE1 in evaluating proteasomal degradation signals, showing that branched Ub chains can be less efficient in triggering proteasomal degradation compared to K48-linked Ub chains.