I need to express a selection marker on a plasmid in human cells (e.g. HEK293). However, I am using a cytoplasmic vector derived from a non-human virus with its own mechanism for transcriptional initiation. This means that the expression level of any protein marker will suck: low copy number, very weak promoter, unstable transcripts - these are things that I cannot change in this precise biological context.
Most selection markers (for instance KanMX) come with their own black-box promoter and so I do not know whether they rely on a high number of proteins being produced.
What would be a marker most likely to be selectable despite a very low expression level? I could not find any resource comparing the protein copy numbers of classic markers.
I need to be sure that the cells that cannot survive the selective pressure died because they did not have the marker, and not because the marker was too weakly expressed to protect cells that have the marker. The standard approach would be to use a regular antibiotic selection and just work with lower concentrations of antibiotics but the number of false positives would most likely be too high.
I can manipulate the host cell to some extent, so I can use antibiotic selection but I can also complement a conditional deletion of an essential gene (auxotrophic marker or non-metabolic genes).