Click DNA ligation promises an alternative to the current enzymatic approaches for DNA assembly, with the ultimate goal of using efficient chemical reactions for the total chemical synthesis and assembly of genes and genomes. Such an approach would enable the incorporation of various chemically modified bases throughout long stretches of DNA, a feat not possible with current polymerase-based methods. An unequivocal requirement for this approach is the biocompatibility of the resulting triazole-linked DNA. The correct function of this unnatural DNA linker in human cells is demonstrated here by using a click-linked gene encoding the fluorescent protein mCherry. Reverse transcription of mRNA isolated from these cells and subsequent sequencing of the mCherry cDNA shows error-free transcription. Nucleotide excision repair (NER) is shown to not play a role in the observed biocompatibility by using a NER-deficient human cell line. This is the first example of a non-natural DNA linker being functional in a eukaryotic cell.