Extended Data Fig. 2. Synthetic Trp53 integration via mSwAP-In.

(a) p53 mutation hotspots and the corresponding DNA codons in human and mouse, as well as the recoded codons in synTrp53. (b) SynTrp53 assembly workflow. Red asterisks represent the recoded codons. (c) Restriction enzyme digestion verification of synTrp53 assemblons. XbaI+XhoI and AseI were used for each candidate. Predicted digestion patterns were simulated using Snapgene software. L, 1 kb plus ladder (NEB). (d) Sequencing coverage of synTrp53 payload candidates. Reads were mapped to mm10 reference. Clones 1 and 3 have expected variants reflecting the recoded codons in synthetic Trp53. Clone 2 and clone 4 have additional undesired variants likely introduced by PCR. (e) Marker cassette 1 insertion into the second intron of Wrap53. 20 bp microhomology arms were added to each end of MC1 during plasmid construction. Successful insertion was verified by junction PCR. Primers are indicated as green arrows. (f) Sanger sequencing validation of heterozygous and hemizygous synTrp53 integrants. (g) Trp53 copy number qPCR analysis for the three mESC clones only carrying recoded codons. Trp53 copy number was normalized to Pgk1 gene. Bars represent mean ± SD of three technical replicates. (h) Sequencing coverage for wild-type, hemizygous and heterozygous clones. Sequencing reads were mapped to mm10. Black bar indicates a deletion called by DELLY⁵⁰. (i) RT-qPCR analysis of Trp53 expression level. Bars represent mean ± SD of four technical replicates for cell culture and RNA extraction, as well as three qPCR technical replicates for each reverse transcribed cDNA sample. (j) Venn diagram of upregulated gene numbers in Trp53^wt/wt and Trp53^syn/syn mESCs upon doxorubicin (250 nM for 20 h) treatment. Fold change cutoff is 4, adjusted p value cut off is 0.01. (k) A fold change representation of 17 genes in the p53 signaling pathway in doxorubicin treated (250 nM for 20 h) Trp53^wt/wt and Trp53^syn/syn mESCs.