Fig. 6

SV40 poly-A spacer sequences mitigate gene leakage caused by A2UCOE. A. Schematic of genetic circuit architecture with various AT-rich spacer sequences between A2UCOE and the TRE-FOXN1 promoter. Spacer sequences were designed randomly with 238 bp lengths and varied AT content: about 35% (AT35), 50% (AT50), and 65% (AT65), as well as a 65% AT-rich spacer containing the 122 bp SV40 poly-A termination sequence (SV40). The dark green and light green bars represent the sequence composition, with A and T shown in dark green and C and G in light green. The SV40 poly-A sequence is highlighted with an orange box. B. Quantification of FOXN1 gene leakage in bulk cell populations following the integration of gene circuits with different AT-rich spacers by RT-qPCR (n = 3). C. Quantification of FOXN1 gene induction in the bulk cell populations after 5 days of culture with blasticidin, followed by doxycycline treatment (n = 4). D. Flow cytometry analysis of RFP-positive (mScarlet+) cells in the SV40 poly-A spacer group after 30 days of culture without blasticidin (n = 3). P-values were calculated using one-way analysis of variance with Tukey’s honestly significant difference test. The data are presented as mean ± SEM. For detailed data, statistical analyses, and exact p-values, see source data file