## 3 Results
### Construction of the NDR\(-\) and NDR\(+\) Sequences  
To verify the impact of NDR length on gene expression, we manipulated the NDR length of six original transcriptional regulatory sequences and developed a GFP expression system employing these sequences as transcriptional regulatory elements. For long NDRs, an 80 bp DNA fragment was truncated near the 5' end, reducing the NDR length to less than 150 bp, named NDR\(-\). This reduction decreases the physical space for transcription factor binding to the DNA sequence, prolonging the transcription factor binding time and thereby reducing gene transcription efficiency. For short NDRs, an additional 80 bp was added to each original NDR sequence near the 5' end, increasing the NDR length to more than 150 bp (NDR\(+\)). This extension expands the physical space for transcription factor binding and shortens the binding time, thereby enhancing the gene transcription efficiency. The length of the NDR is directly related to the accessibility of DNA. A shorter NDR means that the chromatin state of the region is tighter, making it difficult for transcription factors to bind to the target NDR. In contrast, a longer NDR provides an open chromatin environment that allows transcription factors to bind more easily and quickly, resulting in a relatively shorter binding time [22; 37; 38; 39]. The structures of transcriptional regulatory sequences with altered NDR lengths are shown in Figure 4. The NDR\(-\) and NDR\(+\) lengths are listed in Table 1. The details of NDR sequence modifications are as follows.  
Figure 4: Schematic illustration of the structure of three NDR\(+\) and three NDR- transcriptional regulatory sequences. The short NDRs were lengthened by 80 bp in the transcriptional regulatory sequences of the _YAL030W_, _YAR008W_, and _YAR027W_ genes (named NDR\(+\)). The long NDRs were shortened by 80 bp in the transcriptional regulatory sequences of the _YBL071W-A_, _YBL032W_, and _YBL025W_ genes (named as NDR\(-\)). The bars with oblique lines indicate that the sequence fragments were inserted into the NDR regions. The bars with crossed lines indicate that the sequence fragments were excised from the NDR regions.  
The NDR\(-\) transcriptional regulatory sequences are _YBL071W-A_, _YBL03 ZW_, and _YBL025W_. In the _YBL071W-A_ NDR sequence, an 80 bp DNA fragment was excised upstream of the \(-47\) bp site, and the remaining NDR sequences were spliced to create a 128 bp NDR\(-\) sequence. For _YBL032W_, an 80 bp fragment was removed upstream of the \(-72\) bp site, resulting in a 138 bp NDR\(-\) sequence after splicing. Similarly, in _YBL025W_, an 80 bp fragment excised upstream of the \(-134\) bp site yielded a 134 bp NDR\(-\) sequence upon splicing.  
The NDR+ transcriptional regulatory sequences are _YAL030W_, _YAR008W_, and _YAR027W_. In the _YAL030W_ NDR sequence, an 80 bp DNA fragment spanning from \(-46\) bp to \(-125\) bp was inserted at the \(-126\) bp position, extending the NDR+ sequence to 214 bp. For _YAR008W_, an 80 bp fragment from \(-36\) bp to \(-115\) bp was inserted at the \(-116\) bp site, resulting in a 203 bp NDR+ sequence. In _YAR027W_, an 80 bp fragment between \(-19\) bp and \(-98\) bp was inserted at \(-99\) bp, expanding the NDR+ sequence to 175 bp.