## 3 Results
### Structural Characteristics of Regulatory Sequence of Target Gene  
Based on the potential positive correlation between NDR length and gene expression levels [22, 25, 37], we extracted \(\pm 1\) nucleosome sequences along with their corresponding NDR sequences from six _S. cerevisiae_ genes. Then, we utilized these sequences as transcriptional regulatory elements to construct a GFP expression system in _S. cerevisiae_ for investigating the relationship between NDR length and transgene expression level. The GFP expression system in _S. cerevisiae_ is illustrated in Figure 2A. We chose these six genes for the following reasons: firstly, the NDR length is more than 200 bp and the native gene expression is high for the L-NDR model, and the NDR length is shorter than 140 bp and the native gene expression is low for the S-NDR model. Then, the coding regions of the six native genes have no introns. The chromatin structure of the \(\pm 1\) nucleosomes sequences and NDR sequences of the six genes is illustrated in Figure 2B. The lengths of the \(-1\) and \(+1\) nucleosome sequences were set to 150 bp. The transcriptional regulatory sequences started 75 bp upstream from the center position of the \(-1\) nucleosome and extended 75 bp downstream from the center position of the \(+1\) nucleosome. The TSS was designated as position 0 and used as a reference for other locations. Figure 1: Association analysis of the length of the NDR with gene expression levels. **(A)** The length distribution of NDRs in the transcriptional start regions of 1258 _S. cerevisiae_ genes. **(B)** For the 1258 _S. cerevisiae_ genes, the expression levels of the genes were expressed as FPKM. The high expression genes are marked in red lines, and the low expression genes are marked in blue lines. The two NDR groups are cut off by 150 bp in NDR length. L-NDR represents the long NDR group (n = 264), while S-NDR represents the short NDR group (n = 994). **(C)** RT-qPCR validation of RNA-seq data. The genes with extremely low FPKM values (FPKM < 1) were not considered. The 37 genes with L-NDRs (>150 bp) and 49 genes with S-NDRs (<150 bp) were selected randomly. RT-qPCR, reverse transcription-quantitative polymerase chain reaction. The six transcriptional regulatory sequences were categorized into three long NDRs (L-NDR) and three short NDRs (S-NDR), according to the lengths of the NDR sequences. The length of L-NDRs was more than 200 bp, and the length of S-NDRs was in the range of 90-150 bp. For _YBL032W_, _YBL025W_, _YAL030W_, _YAR008W_ and _YBL071W-A_ genes, the translational start site, ATG, was located within the +1 nucleosome. For the _YAR027W_ gene, the ATG was located downstream of the +1 nucleosome. Partial coding sequences of the selected yeast genes were in the transcriptional regulation sequences. The coding sequence of the exogenous gene was connected to the 3'-end of the transcriptional regulation sequence. To ensure that the reading frame of the exogenous gene was not disrupted, we chose the partial coding sequences of the selected yeast genes in multiples of three. In the six selected genes, we did not consider the relative location of TSS and start codon ATG in the transcript region. We focus mainly on the differences in NDR length in the regulation of transgene expression. Information of the six transcriptional regulatory sequences is shown in Table 1. The six transcriptional regulatory sequences are shown in Supplementary File S2.