Jillette, N. et al. Nat. Commun. 10, 4968 (2019).

Selectable markers such as antibiotic resistance genes are important tools for cell genotype screening. Yet it is not easy to select for multiple transgenes because of the limited number of selectable markers. Jillette et al. have developed a system that splits marker genes into different transgenic vectors. In a two-split system, the coding sequence of, for example, an antibiotic resistance gene is split into two fragments. One fragment is cloned upstream of an N-terminal split intein in a vector carrying one transgene and the other fragment is cloned downstream of a C-terminal split intein in the second transgenic vector. Upon delivery of the vectors, only cells expressing both intein-split markers can reconstitute the antibiotic resistance marker via protein trans-splicing. They further demonstrate the approach by sequential selection with two-split markers by reconstituting a six-split hygromycin resistance marker.