## 5 Conclusions  
In conclusion, this study introduced an innovative design strategy for regulating transgene expression. By modulating the length of NDRs, it is possible to control the expression levels of transgenes. Future research should aim to elucidate the molecular mechanisms underlying the differential nucleosome response by investigating the roles of histone variants, the spectrum of histone modifications, and their interplay with DNA methylation in these regions. Furthermore, a genome-wide analysis could determine whether this phenomenon is widespread and functionally significant throughout the yeast genome. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app142311339/s1](https://www.mdpi.com/article/10.3390/app142311339/s1)]([https://www.mdpi.com/article/10.3390/app142311339/s1](https://www.mdpi.com/article/10.3390/app142311339/s1)), Supplementary File S1, Table S1: Primers used for isothermal amplification PCR; Table S2: Primers for Absolute qPCR and Copy Numbers Assay; Table S3: Primers used for quantitative RT-PCR; Table S4: Copy number of GFP gene; Table S5: Differences for mRNA and protein expression level of GFP in L-NDR and S-NDR; Table S6: The numerical data of Figure 6; Table S7: The numerical data of Figure S2; Table S8: The numerical data of Figure 7A,B; Table S9: The numerical data of Figure 7D,E; Table S10: \(p\) value of Figure 7A,D; Table S11: \(p\) value of Figure 7B,E. Supplementary File S2, the six transcriptional regulatory sequences. Supplementary File S3, Figure S1: Schematic illustration of experimental design and workflow; Figure S2: GFP gene expression in recombinant _S. cerevisiae_ BY4741. Conceptualization, H.L. and S.W. ; methodology, S.W. ; software, S.W. ; validation, H.L., S.W. and X.L. ; formal analysis, S.W. ; investigation, S.W. ; resources, H.L. and S.W. ; data curation, H.L. and S.W. ; writing--original draft preparation, S.W. ; writing--review and editing, H.L., S.W. and X.L. ; visualization, S.W. ; supervision, H.L. ; project administration  
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