Extended Data Fig. 1: MultiSTEP is based on a flexible genomically integrated approach for expressing secreted protein variants.

a, Cartoon depicting integration of a MultiSTEP plasmid construct into a genomically integrated landing pad cassette¹⁶. (Top): Lentivirally integrated landing pad cassette expressing mTagBFP2+ (royal blue) from a tetON inducible promoter. mTagBFP2 is fused to an inducible caspase-9 (iCasp9, orange) and a blasticidin resistance gene (dark yellow) with 2A sequences (dark pink). mTagBFP2-2A-iCasp9-2A-BlastR is expressed from a tetON inducible promoter with a attP serine recombinase recognition site (black) in between. Downstream is a terminator sequence (Term, brown) and tet repressor (tetR, salmon). Bxb1 serine recombinase, expressed from another plasmid, is shown in grey. (Middle): MultiSTEP plasmid construct. Secreted protein coding sequence (Sec Pro, pink) is C-terminally fused to flexible linkers (L1 and L2, teal), Strep II tag (St, green), and CD28 transmembrane domain (TM, medium blue). IRES (purple) drives co-transcription of mCherry (red). Upstream is an attB serine recombinase recognition sequence (goldenrod) and a unique 18 nucleotide degenerate barcode (BC, light yellow). (Bottom): Landing pad following plasmid integration. attP and attB sequences have been recombined, forming attL and attR sequences. b, Sequential flow cytometry gating scheme for detecting and isolating landing pad cells with an integrated MultiSTEP construct. Dot pseudocolor indicates density of cells. FSC: Forward scatter; SSC: side scatter. c, Comparison of negative control 293-F cells (top) with 293-F cells incubated with lentivirus encoding the landing pad cassette (bottom, n > 10,000 cells). d, Comparison of unrecombined landing pad cells (top) with cells transfected with a MultiSTEP plasmid encoding WT FIX (bottom, n > 10,000 cells). e, Comparison of cells transfected with a MultiSTEP construct encoding WT FIX treated with doxycycline (top) or doxycycline and 10 nM AP1903 (bottom, n > 10,000 cells). f, Design iterations of MultiSTEP construct plasmid in (a). L1-Strep MultiSTEP construct does not contain an L2 linker. Flow cytometry of MultiSTEP constructs using a anti-Strep II tag antibody (n ~ 30,000 cells).