CN113462686B - Method for preparing galactose-inducible synthetic promoter with gradient activity, and the prepared promoter and application thereof - Google Patents

Abstract

The invention relates to the field of bioengineering, in particular to a method for preparing galactose-induced synthetic promoters with gradient activity, and the promoters prepared by the same and application thereof. The invention constructs 33 inducible synthetic promoters through the transformation of UAS sequences, quantity, arrangement and combination, and obtains a promoter element library with the activity interval of 9-95%, wherein the activity of part of promoters can reach P _GAL1 Over 80% of activity, the synthetic promoter is very similar to, and even lower than, wild-type P in terms of leakage expression of glucose _GAL1 The strong inducible promoter in Saccharomyces cerevisiae is expanded. The method adopted by the invention has simple operation, short test period and easy realization.

Description

Method for preparing galactose-induced synthetic promoter with gradient activity, and prepared promoter and application thereof

Technical Field

The invention relates to the field of bioengineering, and in particular to a method for preparing a galactose-induced synthetic promoter with gradient activity, and the prepared promoter and application thereof.

Background Art

Saccharomyces cerevisiae is a common eukaryotic model organism. In order to increase the yield of the target synthetic product, it is often necessary to precisely regulate the expression of multiple key genes in the synthetic pathway. The metabolic pathway involves multiple key genes. In order to achieve dynamic balance in the expression of multiple genes, the strength difference between the promoters of the metabolic pathway may reach several orders of magnitude. In order to balance the relationship between metabolic pathways, increase the yield of target metabolites, and reduce interference with the growth of chassis bacteria, it is often necessary to find promoters with appropriate strengths to precisely regulate multiple genes along the metabolic network.

In Saccharomyces cerevisiae, the galactose-inducible promoter P _GAL is recognized as the most active promoter, mainly including P _GAL1 , P _GAL2 , P _GAL7 , and P _GAL10 . Therefore, they are also commonly used promoters in the construction and optimization of metabolic pathways. The galactose-inducible promoter consists of a core promoter sequence, a glucose repression sequence, and a galactose-induced upstream activator sequence (UAS). The UAS of P _GAL is a 17bp sequence with three conservative bases at both ends and a random base in the middle, i.e.

5'-CGGNNNNNNNNNNNCCG-3'. Different P _GALs contain different numbers of UASs, and the sequence of each UAS is different. For example, P _GAL1 contains 4 UASs. The transcription factor Gal4p binds to the UAS and is responsible for inducing activation. Both the number and sequence of UASs affect promoter activity. Due to the characteristics of P _GAL being inhibited by glucose and induced by galactose, it is one of the best candidate promoter types for expressing toxic proteins or enzymes. However, the number of P _GALs is limited and the strength range is small, making it difficult to meet the requirements of precise regulation of metabolic networks. Therefore, the artificial synthesis of galactose-inducible promoters with an activity gradient is an urgently needed strategy for controlling the precise expression of each gene in a complex metabolic pathway.

Hybrid promoter is one of the methods of artificially synthesizing promoters. The basis for constructing hybrid promoters is the structure and function of natural promoters, which can be divided into two parts: upstream regulatory sequence and core promoter. These elements can be split and combined to obtain synthetic promoters with new functions. Blazeck et al. fused the complete UAS regulatory region of P _GAL1 (-457～-148, 309bp) to the core promoter sequences of different yeast promoters to construct a series of promoters induced by galactose. However, the activity strength of these core promoters is different, resulting in different leakage expressions when cultivated under glucose as the carbon source. Therefore, it takes a lot of time and effort to screen the appropriate core promoter sequence.

Summary of the invention

The present invention has been proposed and completed in order to solve the above-mentioned problems.

The object of the present invention is to provide a method for preparing a galactose-inducible synthetic promoter with gradient activity.

Another object of the present invention is to provide a galactose-inducible synthetic promoter.

Another object of the present invention is to provide the application of the above galactose-inducible synthetic promoter.

The embodiment of the present invention provides a method for preparing a galactose-inducible synthetic promoter with gradient activity, the method comprising the step of connecting 1 to 4 UAS regulatory sequences to the upstream of a galactose-inducible core promoter P _GAL1 , wherein the galactose-inducible core promoter P _GAL1 is a promoter that does not contain an endogenous UAS regulatory sequence, and the UAS regulatory sequence is selected from the group including the following UAS regulatory sequences: UAS1 with a nucleotide sequence of 5'-CGGATTAGAAGCCGCCG-3', UAS2 with a nucleotide sequence of 5'-CGGGCGACAGCCCTCCG-3', UAS3 with a nucleotide sequence of 5'-CGGAAGACTCTCCTCCG-3', UAS4 with a nucleotide sequence of 5'-CGCGCCGCACTGCTCCG-3', UAS5 with a nucleotide sequence of 5'-CGGAAAAGCGTCTTCCG-3', UAS6 with a nucleotide sequence of 5'-CGGCGCTCACTCTTCCG-3', UAS7 with a nucleotide sequence of 5'-CGGGGTGGACCACTCCG-3', UAS8 with a nucleotide sequence of 5'-CGGACAACTGTTGACCG-3', and UAS9 with a nucleotide sequence of 5'-CGGGCCGCACTGCTCCG-3.

The embodiment of the present invention provides a galactose-inducible synthetic promoter, wherein the synthetic promoter comprises 1 to 4 UAS regulatory sequences and a galactose-inducible core promoter P _GAL1 , wherein the galactose-inducible core promoter P _GAL1 is a promoter that does not contain an endogenous UAS regulatory sequence, and the UAS regulatory sequence is selected from the group including the following UAS regulatory sequences: UAS1 with a nucleotide sequence of 5'-CGGATTAGAAGCCGCCG-3', UAS2 with a nucleotide sequence of 5'-CGGGCGACAGCCCTCCG-3', UAS3 with a nucleotide sequence of 5'-CGGAAGACTCTCCTCCG-3', UAS4 with a nucleotide sequence of 5'-CGCGCCGCACTGCTCCG-3', UAS5 with a nucleotide sequence of 5'-CGGAAAAGCGTCTTCCG-3', UAS6 with a nucleotide sequence of 5'-CGGCGCTCACTCTTCCG-3', UAS7 with a nucleotide sequence of 5'-CGGGGTGGACCACTCCG-3', UAS8 with a nucleotide sequence of 5'-CGGACAACTGTTGACCG-3', and UAS9 with a nucleotide sequence of 5'-CGGGCCGCACTGCTCCG-3.

The present invention provides the galactose-induced synthetic promoter prepared above.

Preferably, the galactose-inducible synthetic promoter of the present invention is used to express heterologous biological substances in yeast.

Preferably, the galactose-inducible synthetic promoter of the present invention is used to express heterologous toxic proteins in yeast.

At least one of the above technical solutions adopted in the embodiments of the present application can achieve the following beneficial effects:

The present invention constructs 33 inducible synthetic promoters through the modification of UAS sequence, quantity and arrangement combination, and obtains a promoter element library with an activity range of 9% to 95%, among which the activity of some promoters can reach more than 80% of the activity of P _GAL1 . The synthetic promoter is very similar to the leakage expression of glucose, even lower than the wild-type P _GAL1 , and expands the strong inducible promoter in saccharomyces cerevisiae. The method adopted by the present invention is simple to operate, has a short test cycle, and is easy to implement.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

Figure 1 is a schematic diagram of the construction of a synthetic promoter, wherein A: UAS4 is mutated to UAS9, B: a single UAS selected from UAS1-UAS9 is combined with scafP _GAL1 and P _CYC1 to construct a synthetic promoter, and C: a combination of 4 UASs is combined with scafP _GAL1 to construct a synthetic promoter.

FIG2 shows the fluorescence intensity of the synthetic promoter in galactose, wherein A: the activity intensity of the synthetic promoter constructed by a single UAS and scafP _GAL1 and P _CYC1 , B: the activity intensity of the synthetic promoter constructed by a combination of four UAS and scafP _GAL1 , and the experimental data were all repeated three times in parallel.

FIG3 shows the fluorescence intensity detection results of the scafP _GAL1 series synthetic promoter library. The fluorescence intensity of the synthetic promoters constructed by a single UAS and four UASs is between 9% and 95% relative to that of P _GAL1 .

FIG4 shows the activity of UAS-scafP _GAL1 in glucose. Compared with the galactose culture condition, the activity of the synthetic promoter in glucose was significantly inhibited.

DETAILED DESCRIPTION

In order to make the purpose, technical solution and advantages of the present application clearer, the technical solution of the present application will be clearly and completely described below in combination with the specific embodiments of the present application and the corresponding drawings. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

The technical solutions provided by various embodiments of the present application are described in detail below in conjunction with the accompanying drawings.

The method for preparing a galactose-inducible synthetic promoter with gradient activity according to the present invention comprises the step of connecting 1 to 4 UAS regulatory sequences to the upstream of the galactose-inducible core promoter P _GAL1 , wherein the galactose-inducible core promoter P _GAL1 is a promoter that does not contain an endogenous UAS regulatory sequence, and the UAS regulatory sequence is selected from the group including the following UAS regulatory sequences: UAS1 with a nucleotide sequence of 5'-CGGATTAGAAGCCGCCG-3', UAS2 with a nucleotide sequence of 5'-CGGGCGACAGCCCTCCG-3', UAS3 with a nucleotide sequence of 5'-CGGAAGACTCTCCTCCG-3', UAS4 with a nucleotide sequence of 5'-CGCGCCGCACTGCTCCG-3', UAS5 with a nucleotide sequence of 5'-CGGAAAAGCGTCTTCCG-3', UAS6 with a nucleotide sequence of 5'-CGGCGCTCACTCTTCCG-3', UAS7 with a nucleotide sequence of 5'-CGGGGTGGACCACTCCG-3', UAS8 with a nucleotide sequence of 5'-CGGACAACTGTTGACCG-3', and UAS9 with a nucleotide sequence of 5'-CGGGCCGCACTGCTCCG-3.

The transcription factor Gal4p binds to UAS (galactose-induced upstream activation sequence) and is responsible for inducing activation. Under glucose culture conditions, the inhibitory factor Gal80p interacts with Gal4p and inhibits Gal4p transcriptional activity. Therefore, P _GAL activity is very weak and protein expression is low; when under galactose culture conditions, Gal80p dissociates from Gal4p and releases its transcriptional activation activity. Therefore, P _GAL begins to enter a highly active state, and its activity after induction is more than 100 times that before induction.

According to a specific embodiment of the present invention, the present invention first selected four UAS sequences of endogenous P _GAL1 of Saccharomyces cerevisiae, namely UAS1, UAS2, UAS3, UAS4, UAS4 mutant UAS9 and one UAS sequence UAS8 of P _GAL7 , and also selected exogenous UAS sequences, namely UAS5, UAS6 and UAS7 from P _GAL2 of Saccharomyces kudriavzevii, and fused them to the sequence of P _GAL1 without UAS (named as the skeleton promoter, scafP _GAL1 ) and the upstream region of the constitutive promoter P _CYC1 without the UAS sequence, respectively. By comparing the fluorescence intensity of the reporter protein, it was found that different UASs had different expression activities, and the activity range of UAS-scafP _GAL1 relative to P _GAL1 was between 9% and 62%; with P _CYC1 as the core promoter, the activity range of UAS-P _CYC1 relative to P _CYC1 was between 106% and 426%. Furthermore, the above nine UASs were combined to replace the four UASs of the PGAL1 promoter. The experimental results showed that the activity range of these recombinant promoters was between 55% and 95%. By regulating the sequence and number of UASs, a promoter element library with an activity range of 9% to 95% can be obtained.

According to a specific embodiment of the present invention, the _PGAL1 -eGFP fragment containing the _eGFP reporter gene and the fusion sequence of each UAS or multiple UASs and the core sequence are amplified from a plasmid containing PGAL1-eGFP by PCR amplification.

The recombinant plasmid was constructed by the Gibson assembly method. The Gibson assembly technique is a simple, fast and efficient DNA directional cloning technique that can clone the insert fragment PCR product to any site of any vector. The vector is linearized at the cloning site, and the linearized cloning vector terminal sequence is introduced at the 5' end of the insert fragment PCR primer, so that the 5' and 3' ends of the insert fragment PCR product respectively carry sequences that are completely consistent with the two ends of the linearized cloning vector (15bp to 20bp). After the PCR product with the vector terminal sequence at both ends and the linearized cloning vector are mixed in a certain proportion, the conversion can be carried out under the catalysis of nuclease exonuclease, DNA polymerase and ligase in just 15 minutes to complete the directional cloning.

According to a specific embodiment of the present invention, a combination of four UASs replaces UAS1, UAS2, UAS3, and UAS4, and constructs a synthetic promoter with scafP _GAL1 . For example, UAS1239 refers to the composition of UAS1, UAS2, UAS3, and UAS9 from the 5'-3' direction, and UAS1, UAS2, UAS3, and UAS4 are replaced by 1239.

Example 1

As shown in Figure 1, a schematic diagram of the construction of a synthetic promoter is shown. The present invention obtains upstream activation sequences (UAS) from Saccharomyces cerevisiae P _GAL1 and P _GAL7 , respectively, and names them UAS1, UAS2, UAS3, UAS4 and UAS8, wherein UAS4 is mutated to UAS9, as shown in Figure 1 A. In addition, exogenous UAS5, UAS6 and UAS7 are obtained from the promoter of Saccharomyces kudriavzevii P _GAL2 to obtain UAS1 to UAS9, and the sequences of the above UASs are shown in Table 1.

The schematic diagram of the fusion of a single UAS with a promoter is shown in Figure 1 B. Using the existing plasmid POT2-P _GAL1 -eGFP as a template, primers 1 and 2 were used for PCR to amplify the plasmid backbone fragment 1 (SEQ ID NO: 1) containing the eGFP reporter gene. The resulting plasmid backbone is a single copy and contains the URA auxotrophic marker and the ampicillin resistance marker. Primer 3 was then used as a common primer and used with primers 4 to 12 to amplify the UAS-scafP _GAL1 synthetic promoter containing UAS1 to UAS9 (SEQ ID NO: 2 to SEQ ID NO: 10). Correspondingly, using POT2-P _CYC1 -eGFP as a template, primer 13 was used as a common primer and used with primers 14 to 22 to amplify the UAS-P _CYC1 synthetic promoter containing UAS1 to UAS9 (SEQ ID NO: 11 to SEQ ID NO: 19). The above synthetic promoter fragment and plasmid backbone fragment 1 (SEQ ID NO: 1) were used to construct POT2-UAS-scafP _GAL1 -eGFP and POT2-UAS-P _CYC1 -eGFP recombinant plasmids respectively by Gibson Assembly method.

Table 1 UAS names and sequences

name sequence UAS1 5’-CGGATTAGAAGCCGCCG-3’ UAS2 5’-CGGGCGACAGCCCTCCG-3’ UAS3 5’-CGGAAGACTCTCCTCCG-3’ UAS4 5’-CGCGCCGCACTGCTCCG-3’ UAS5 5’-CGGAAAAGCGTCTTCCG-3’ UAS6 5’-CGGCGCTCACTCTTCCG-3’ UAS7 5’-CGGGGTGGACCACTCCG-3’ UAS8 5’-CGGACAACTGTTGACCG-3’ UAS9 5'-CGGGCCGCACTGCTCCG-3

The schematic diagram of the fusion of four UASs with the scafP _GAL1 promoter is shown in Figure 1 C. Using POT2-P _GAL1 -eGFP as a template, primers 1 and 23 were used to amplify the plasmid backbone fragment 20 (SEQ ID NO: 20) containing scafP _GAL1 . Then, PCR amplification was performed using primers 24/25 (UAS1239), 26/27 (UAS2222), 28/29 (UAS2223), 30/31 (UAS2224), 32/33 (UAS2229), 34/35 (UAS2233), 36/37 (UAS2244), 38/39 (UAS2333), 40/41 (UAS2444), 42/43 (UAS333), 44/45 (UAS4222), 46/47 (UAS7234), 48/49 (UAS7238), 50/51 (UAS7239), and 52/53 (UAS4444) as templates to obtain the amplified fragment, i.e., the fusion fragment of the four UASs shown in the above brackets. The above amplified product and plasmid backbone fragment 20 were then assembled into POT2-4×UAS-scafP _GAL1 -eGFP recombinant plasmid by Gibson Assembly method. The primers involved are shown in Table 2.

Table 2 Primer names and sequences for plasmid construction

After the plasmid is constructed, it is transferred into the competent state of Saccharomyces cerevisiae to verify the activity of the synthetic promoter. First, prepare the competent state of yeast: pick the wild-type yeast strains, add 5mL YPD (1% yeast extract, 2% peptone, 2% glucose) liquid culture medium respectively, and place it in a 30℃ shaker at 250rpm for overnight culture for 12 hours. The activated strains are transferred to 50mL YPD liquid culture medium respectively, and the initial OD ₆₀₀ is adjusted to 0.2, and then placed in a 30℃ shaker at 250rpm for about 5 hours to make its OD ₆₀₀ between 0.7-1.0. After culture, take it out, centrifuge it at 3000rpm for 5min, and remove the supernatant. Add 50mL of pure water, centrifuge it at 3000rpm for 5min, and remove the supernatant. Then add 20mL of 100mM lithium acetate (LiAC), mix well, centrifuge at 3000rpm for 5min, remove the supernatant, and finally add 400μL of 100mM LiAC to resuspend the cells, and the yeast competent state is obtained. For a transformation example, take 30uL of yeast competent state in a 1.5mL centrifuge tube, add 240μL of 50% PEG3350; 36μL of 1M LiAC; 5μL of 10mg/mL single-stranded fish sperm DNA (before use, boil at 99℃ for 5-10min, and put on ice immediately after taking it out); 500ng-1μg of the constructed recombinant plasmid; 70ul pure water. After mixing the above transformation solution, first place it at 30℃ for 30min, and then place it at 42℃ for 25-40min. Centrifuge the transformation solution after heat shock at 5000rpm for 1min, and remove the supernatant. After removing the supernatant, 100 μL of water was added and mixed, and then evenly spread on SC-URA (formula see Table 3) solid culture medium, and finally cultured at 30° C. for 48 hours.

Table 3 SC-URA medium formula

The synthetic promoter activity test method is as follows: take a 96-well plate, add 300 μL of SC-URA liquid medium containing 2% glucose to each well, then pick the yeast colony containing the recombinant plasmid, pick three replicates in a 96-well plate containing the medium, and place it at 30°C for activation for 24 hours. After the yeast is activated, measure the OD ₆₀₀ absorbance, centrifuge at 5000rpm for 5min, and remove the supernatant. Then add 400 μL of pure water to mix, centrifuge at 5000rpm for 5min, remove the supernatant, and add 200 μL of pure water to resuspend. After resuspension, the yeast is transferred to a 96-well plate containing SC-URA liquid medium containing 2% galactose, with a final concentration of OD ₆₀₀ of about 0.2 and a final volume of 300 μL. After culturing for 24 hours, the sample is diluted 10 times and the eGFP fluorescence intensity is measured by flow cytometry to characterize the synthetic promoter activity. A bar graph was drawn based on the measured fluorescence intensity (as shown in Figures 2 and 3).

The specific activity ratio values of each promoter in Figures 2 and 3 relative to P _GAL1 and P _CYC1 are shown in the following Table 4.

Table 4 Activity of synthetic promoters relative to the original promoter.

Synthetic promoter Relative intensity of P _GAL1 Relative intensity of P _CYC1 UAS1-scafP _GAL1 /P _CYC1 twenty two% 169% UAS2-scafP _GAL1 /P _CYC1 46% 426% UAS3-scafP _GAL1 /P _CYC1 49% 384% UAS4-scafP _GAL1 /P _CYC1 9% 106% UAS5-scafP _GAL1 /P _CYC1 55% 238% UAS6-scafP _GAL1 /P _CYC1 39% 291% UAS7-scafP _GAL1 /P _CYC1 36% 332% UAS8-scafP _GAL1 /P _CYC1 48% 377% UAS9-scafP _GAL1 /P _CYC1 54% 409% UAS2223-scafP _GAL1 55% UAS4444-scafP _GAL1 60% UAS2333-scafP _GAL1 62% UAS4222-scafP _GAL1 68% UAS7239-scafP _GAL1 74% UAS2233-scafP _GAL1 75% UAS2244-scafP _GAL1 78% UAS2222-scafP _GAL1 81% UAS3333-scafP _GAL1 82% UAS1239-scafP _GAL1 83% UAS7234-scafP _GAL1 84% UAS2229-scafP _GAL1 85% UAS2224-scafP _GAL1 85% UAS7238-scafP _GAL1 90% UAS2444-scafP _GAL1 95%

The experimental results are shown in Figures 2 and 3. As can be seen from Figure A in Figure 2, different UAS sequences have different effects on promoter activity. The activity range of UAS-scafP _GAL1 synthetic promoter relative to P _GAL1 is between 9% and 62%, and these synthetic promoters are very similar in glucose leakage expression, even lower than the wild-type P _GAL1 (as shown in Figure 4); the activity range of synthetic promoter UAS-P _CYC1 relative to P _CYC1 is between 106% and 426%. Figure B in Figure 2 shows that increasing the number of strong inducible promoters can further improve promoter activity, and the random arrangement of different UASs will also have different effects on promoter activity. After scafP _GAL1 is fused with 4 UASs, the activity range is between 55% and 95%. The above results show that the present invention obtains a gradient galactose-inducible promoter library with a large activity range.

The present invention provides a new method for constructing a synthetic promoter, which has high potential value in the application of synthetic biology and also has certain guiding significance for the modification of promoters.

The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Sequence Listing

<110> Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences

<120> Method for preparing a galactose-induced synthetic promoter with gradient activity, and the prepared promoter and its application

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agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 4020

cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 4080

tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 4140

ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 4200

tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 4260

cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 4320

actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 4380

ggtttattgc tgataaatct ggagccggtg agcgtggtag tcgcggtatc attgcagcac 4440

tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 4500

ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 4560

aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 4620

ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 4680

agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 4740

ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 4800

tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 4860

cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact 4920

ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 4980

gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 5040

ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 5100

aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 5160

cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 5220

ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 5280

gatttttgtg atgctcgtca gggggcgga gcctatggaa aaacgccagc aacgcggcct 5340

ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 5400

ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 5460

gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac 5520

cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 5580

ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta cctcactcat taggcacccc 5640

aggctttaca ctttatgctt ccggctccta tgttgtgtgg aattgtgagc ggataacaat 5700

ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa 5760

gggaacaaaa gctggagctc caccgcggtg gcggccgcga attccccggg tctagaggtc 5820

tcggttggca gtgactcggt ctctacctgg ct 5852

<210> 2

<211> 383

<212> DNA

<213> Saccharomyces cerevisiae

<400> 2

gtgactcggt ctctacctgg ctcggattag aagccgccga acaataaaga ttctacaata 60

ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa 120

attaacgaat caaattaaca accataggat gataatgcga ttagtttttt agccttatattt 180

ctggggtaat taatcagcga agcgatgatt tttgatctat taacagatat ataaatggaa 240

aagctgcata accactttaa ctaatacttt caacattttc agtttgtatt acttcttatt 300

caaatgtcat aaaagtatca acaaaaaatt gttaatatac ctctatactt taacgtcaag 360

gaggatgggt aagggagaag aac 383

<210> 3

<211> 383

<212> DNA

<213> Saccharomyces cerevisiae

<400> 3

gtgactcggt ctctacctgg ctcgggcgac agccctccga acaataaaga ttctacaata 60

ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa 120

attaacgaat caaattaaca accataggat gataatgcga ttagtttttt agccttatattt 180

ctggggtaat taatcagcga agcgatgatt tttgatctat taacagatat ataaatggaa 240

aagctgcata accactttaa ctaatacttt caacattttc agtttgtatt acttcttatt 300

caaatgtcat aaaagtatca acaaaaaatt gttaatatac ctctatactt taacgtcaag 360

gaggatgggt aagggagaag aac 383

<210> 4

<211> 383

<212> DNA

<213> Saccharomyces cerevisiae

<400> 4

gtgactcggt ctctacctgg ctcggaagac tctcctccga acaataaaga ttctacaata 60

ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa 120

attaacgaat caaattaaca accataggat gataatgcga ttagtttttt agccttatattt 180

ctggggtaat taatcagcga agcgatgatt tttgatctat taacagatat ataaatggaa 240

aagctgcata accactttaa ctaatacttt caacattttc agtttgtatt acttcttatt 300

caaatgtcat aaaagtatca acaaaaaatt gttaatatac ctctatactt taacgtcaag 360

gaggatgggt aagggagaag aac 383

<210> 5

<211> 383

<212> DNA

<213> Saccharomyces cerevisiae

<400> 5

gtgactcggt ctctacctgg ctcgcgccgc actgctccga acaataaaga ttctacaata 60

ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa 120

attaacgaat caaattaaca accataggat gataatgcga ttagtttttt agccttatattt 180

ctggggtaat taatcagcga agcgatgatt tttgatctat taacagatat ataaatggaa 240

aagctgcata accactttaa ctaatacttt caacattttc agtttgtatt acttcttatt 300

caaatgtcat aaaagtatca acaaaaaatt gttaatatac ctctatactt taacgtcaag 360

gaggatgggt aagggagaag aac 383

<210> 6

<211> 383

<212> DNA

<213> Saccharomyces cerevisiae

<400> 6

gtgactcggt ctctacctgg ctcggaaaag cgtcttccga acaataaaga ttctacaata 60

ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa 120

attaacgaat caaattaaca accataggat gataatgcga ttagtttttt agccttatattt 180

ctggggtaat taatcagcga agcgatgatt tttgatctat taacagatat ataaatggaa 240

aagctgcata accactttaa ctaatacttt caacattttc agtttgtatt acttcttatt 300

caaatgtcat aaaagtatca acaaaaaatt gttaatatac ctctatactt taacgtcaag 360

gaggatgggt aagggagaag aac 383

<210> 7

<211> 383

<212> DNA

<213> Saccharomyces cerevisiae

<400> 7

gtgactcggt ctctacctgg ctcggcgctc actcttccga acaataaaga ttctacaata 60

ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa 120

attaacgaat caaattaaca accataggat gataatgcga ttagtttttt agccttatattt 180

ctggggtaat taatcagcga agcgatgatt tttgatctat taacagatat ataaatggaa 240

aagctgcata accactttaa ctaatacttt caacattttc agtttgtatt acttcttatt 300

caaatgtcat aaaagtatca acaaaaaatt gttaatatac ctctatactt taacgtcaag 360

gaggatgggt aagggagaag aac 383

<210> 8

<211> 383

<212> DNA

<213> Saccharomyces cerevisiae

<400> 8

gtgactcggt ctctacctgg ctcggggtgg accactccga acaataaaga ttctacaata 60

ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa 120

attaacgaat caaattaaca accataggat gataatgcga ttagtttttt agccttatattt 180

ctggggtaat taatcagcga agcgatgatt tttgatctat taacagatat ataaatggaa 240

aagctgcata accactttaa ctaatacttt caacattttc agtttgtatt acttcttatt 300

caaatgtcat aaaagtatca acaaaaaatt gttaatatac ctctatactt taacgtcaag 360

gaggatgggt aagggagaag aac 383

<210> 9

<211> 383

<212> DNA

<213> Saccharomyces cerevisiae

<400> 9

gtgactcggt ctctacctgg ctcggacaac tgttgaccga acaataaaga ttctacaata 60

ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa 120

attaacgaat caaattaaca accataggat gataatgcga ttagtttttt agccttatattt 180

ctggggtaat taatcagcga agcgatgatt tttgatctat taacagatat ataaatggaa 240

aagctgcata accactttaa ctaatacttt caacattttc agtttgtatt acttcttatt 300

caaatgtcat aaaagtatca acaaaaaatt gttaatatac ctctatactt taacgtcaag 360

gaggatgggt aagggagaag aac 383

<210> 10

<211> 383

<212> DNA

<213> Saccharomyces cerevisiae

<400> 10

gtgactcggt ctctacctgg ctcgggccgc actgctccga acaataaaga ttctacaata 60

ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa 120

attaacgaat caaattaaca accataggat gataatgcga ttagtttttt agccttatattt 180

ctggggtaat taatcagcga agcgatgatt tttgatctat taacagatat ataaatggaa 240

aagctgcata accactttaa ctaatacttt caacattttc agtttgtatt acttcttatt 300

caaatgtcat aaaagtatca acaaaaaatt gttaatatac ctctatactt taacgtcaag 360

gaggatgggt aagggagaag aac 383

<210> 11

<211> 308

<212> DNA

<213> Saccharomyces cerevisiae

<400> 11

agtgactcgg tctctacctg gctcggatta gaagccgccg ctcgagcaga tccgccaggc 60

gtgtatatat agcgtggatg gccaggcaac tttagtgctg acacatacag gcatatatat 120

atgtgtgcga cgacacatga tcatatggca tgcatgtgct ctgtatgtat ataaaactct 180

tgttttcttc ttttctctaa atattctttc cttatacatt aggacctttg cagcataaat 240

tactatactt ctatagacac gcaaacacaa atacacacac taagatgggt aagggagaag 300

aacttttc 308

<210> 12

<211> 308

<212> DNA

<213> Saccharomyces cerevisiae

<400> 12

agtgactcgg tctctacctg gctcgggcga cagccctccg ctcgagcaga tccgccaggc 60

gtgtatatat agcgtggatg gccaggcaac tttagtgctg acacatacag gcatatatat 120

atgtgtgcga cgacacatga tcatatggca tgcatgtgct ctgtatgtat ataaaactct 180

tgttttcttc ttttctctaa atattctttc cttatacatt aggacctttg cagcataaat 240

tactatactt ctatagacac gcaaacacaa atacacacac taagatgggt aagggagaag 300

aacttttc 308

<210> 13

<211> 308

<212> DNA

<213> Saccharomyces cerevisiae

<400> 13

agtgactcgg tctctacctg gctcggaaga ctctcctccg ctcgagcaga tccgccaggc 60

gtgtatatat agcgtggatg gccaggcaac tttagtgctg acacatacag gcatatatat 120

atgtgtgcga cgacacatga tcatatggca tgcatgtgct ctgtatgtat ataaaactct 180

tgttttcttc ttttctctaa atattctttc cttatacatt aggacctttg cagcataaat 240

tactatactt ctatagacac gcaaacacaa atacacacac taagatgggt aagggagaag 300

aacttttc 308

<210> 14

<211> 308

<212> DNA

<213> Saccharomyces cerevisiae

<400> 14

agtgactcgg tctctacctg gctcgcgccg cactgctccg ctcgagcaga tccgccaggc 60

gtgtatatat agcgtggatg gccaggcaac tttagtgctg acacatacag gcatatatat 120

atgtgtgcga cgacacatga tcatatggca tgcatgtgct ctgtatgtat ataaaactct 180

tgttttcttc ttttctctaa atattctttc cttatacatt aggacctttg cagcataaat 240

tactatactt ctatagacac gcaaacacaa atacacacac taagatgggt aagggagaag 300

aacttttc 308

<210> 15

<211> 308

<212> DNA

<213> Saccharomyces cerevisiae

<400> 15

agtgactcgg tctctacctg gctcggaaaa gcgtcttccg ctcgagcaga tccgccaggc 60

gtgtatatat agcgtggatg gccaggcaac tttagtgctg acacatacag gcatatatat 120

atgtgtgcga cgacacatga tcatatggca tgcatgtgct ctgtatgtat ataaaactct 180

tgttttcttc ttttctctaa atattctttc cttatacatt aggacctttg cagcataaat 240

tactatactt ctatagacac gcaaacacaa atacacacac taagatgggt aagggagaag 300

aacttttc 308

<210> 16

<211> 308

<212> DNA

<213> Saccharomyces cerevisiae

<400> 16

agtgactcgg tctctacctg gctcggcgct cactcttccg ctcgagcaga tccgccaggc 60

gtgtatatat agcgtggatg gccaggcaac tttagtgctg acacatacag gcatatatat 120

atgtgtgcga cgacacatga tcatatggca tgcatgtgct ctgtatgtat ataaaactct 180

tgttttcttc ttttctctaa atattctttc cttatacatt aggacctttg cagcataaat 240

tactatactt ctatagacac gcaaacacaa atacacacac taagatgggt aagggagaag 300

aacttttc 308

<210> 17

<211> 308

<212> DNA

<213> Saccharomyces cerevisiae

<400> 17

agtgactcgg tctctacctg gctcggggtg gaccactccg ctcgagcaga tccgccaggc 60

gtgtatatat agcgtggatg gccaggcaac tttagtgctg acacatacag gcatatatat 120

atgtgtgcga cgacacatga tcatatggca tgcatgtgct ctgtatgtat ataaaactct 180

tgttttcttc ttttctctaa atattctttc cttatacatt aggacctttg cagcataaat 240

tactatactt ctatagacac gcaaacacaa atacacacac taagatgggt aagggagaag 300

aacttttc 308

<210> 18

<211> 308

<212> DNA

<213> Saccharomyces cerevisiae

<400> 18

agtgactcgg tctctacctg gctcggacaa ctgttgaccg ctcgagcaga tccgccaggc 60

gtgtatatat agcgtggatg gccaggcaac tttagtgctg acacatacag gcatatatat 120

atgtgtgcga cgacacatga tcatatggca tgcatgtgct ctgtatgtat ataaaactct 180

tgttttcttc ttttctctaa atattctttc cttatacatt aggacctttg cagcataaat 240

tactatactt ctatagacac gcaaacacaa atacacacac taagatgggt aagggagaag 300

aacttttc 308

<210> 19

<211> 308

<212> DNA

<213> Saccharomyces cerevisiae

<400> 19

agtgactcgg tctctacctg gctcgggccg cactgctccg ctcgagcaga tccgccaggc 60

gtgtatatat agcgtggatg gccaggcaac tttagtgctg acacatacag gcatatatat 120

atgtgtgcga cgacacatga tcatatggca tgcatgtgct ctgtatgtat ataaaactct 180

tgttttcttc ttttctctaa atattctttc cttatacatt aggacctttg cagcataaat 240

tactatactt ctatagacac gcaaacacaa atacacacac taagatgggt aagggagaag 300

aacttttc 308

<210> 20

<211> 6177

<212> DNA

<213> Saccharomyces cerevisiae

<400> 20

aacaataaag attctacaat actagctttt atggttatga agaggaaaaa ttggcagtaa 60

cctggcccca caaaccttca aattaacgaa tcaaattaac aaccatagga tgataatgcg 120

attagttttt tagccttatttctggggtaa ttaatcagcg aagcgatgatttttgatcta 180

ttaacagata tataaatgga aaagctgcat aaccacttta actaatactt tcaacatttt 240

cagtttgtat tacttctttat tcaaatgtca taaaagtatc aacaaaaaat tgttaatata 300

cctctatact ttaacgtcaa ggaggatggg taagggagaa gaacttttca ctggagttgt 360

cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg tcagtggaga 420

gggtgaaggt gatgcaacat acggaaaact tacccttaaa tttatttgca ctactggaaa 480

gcttcctgtt ccttggccaa cacttgtcac tactcttact tatggtgttc aatgcttttc 540

aagataccca gatcatatga agcggcacga cttcttcaag agcgccatgc ctgagggata 600

cgtgcaggag aggaccatct tcttcaagga cgacgggaac tacaagacac gtgctgaagt 660

caagtttgag ggagacaccc tcgtcaacag aatcgagctt aagggaatcg atttcaagga 720

ggacggaaac atcctcggcc acaagttgga atacaactac aactcccaca acgtatacat 780

catggcagac aaacaaaaga atggaatcaa agttaacttc aaaattagac acaacattga 840

agatggaagc gttcaactag cagaccatta tcaacaaaat actccaattg gcgatggccc 900

tgtcctttta ccagacaacc attacctgtc cacacaatct gccctttcga aagatcccaa 960

cgaaaagaga gaccacatgg tccttcttga gtttgtaaca gctgctggga ttacacatgg 1020

catggatgaa ctatacaaat aatagccgaa tttcttatga tttatgattt ttattattaa 1080

ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg ttttaaaacg 1140

aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc aggtatagca 1200

tgaggtcgct cttattgacc acacctctac cggcctcagg cagagaccca agacactgcg 1260

gatcgagacc actagtaccg gtctgcagct cgaggggggg cccggtaccc aattcgccct 1320

atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa 1380

accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 1440

atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat 1500

ggcgcgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg 1560

tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc 1620

tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 1680

gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta 1740

gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 1800

atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg 1860

atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa 1920

aatttaacgc gaattttaac aaaatattaa cgtttacaat ttcctgatgc ggtattttct 1980

ccttacgcat ctgtgcggta tttcacaccg catagggtaa taactgatat aattaaattg 2040

aagctctaat ttgtgagttt agtatacatg catttactta taatacagtt ttttagtttt 2100

gctggccgca tcttctcaaa tatgcttccc agcctgcttt tctgtaacgt tcaccctcta 2160

ccttagcatc ccttcccttt gcaaatagtc ctcttccaac aataataatg tcagatcctg 2220

tagacaccac atcatccacg gttctatact gttgacccaa tgcgtcaccc ttgtcatcta 2280

aacccacacc gggtgtcata atcaaccaat cgtaaccttc atctcttcca cccatgtctc 2340

tttgagcaat aaagccgata acaaaatctt tgtcgctctt cgcaatgtca acagtaccct 2400

tagtatattc tccagtagat agggagccct tgcatgacaa ttctgctaac atcaaaaggc 2460

ctctaggttc ctttgttaact tcttctgccg cctgcttcaa accgctaaca atacctgggc 2520

ccaccacacc gtgtgcattc gtaatgtctg cccattctgc tattctgtat acacccgcag 2580

agtactgcaa tttgactgta ttaccaatgt cagcaaattt tctgtcttcg aagagtaaaa 2640

aattgtactt ggcggataat gcctttagcg gcttaactgt gccctccatg gaaaaatcag 2700

tcaagatatc cacatgtgtt tttagtaaac aaattttggg acctaatgct tcaactaact 2760

ccagtaattc cttggtggta cgaacatcca atgaagcaca caagtttgtt tgcttttcgt 2820

gcatgatatt aaatagcttg gcagcaacag gactaggatg agtagcagca cgttccttat 2880

atgtagcttt cgacatgatt tatcttcgtt tcctgcaggt ttttgttctg tgcagttggg 2940

ttaagaatac tgggcaattt catgtttctt caacactaca tatgcgtata tataccaatc 3000

taagtctgtg ctccttcctt cgttcttcct tctgttcgga gattaccgaa tcaaaaaaat 3060

ttcaaagaaa ccgaaatcaa aaaaaagaat aaaaaaaaaa tgatgaattg aattgaaaag 3120

ctgtggtatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc 3180

gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 3240

acagacaagc tgtgacaatc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 3300

cgaaacgcgc gagattaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga 3360

taataatggt ttcttaggac ggatcgcttg cctgtaactt acacgcgcct cgtatctttt 3420

aatgatggaa taatttggga atttactctg tgtttattta tttttatgtt ttgtatttgg 3480

attttagaaa gtaaataaag aaggtagaag agttacggaa tgaagaaaaa aaaataaaca 3540

aaggtttaaa aaatttcaac aaaaagcgta ctttacatat atatttatta gacaagaaaa 3600

gcagattaaa tagataca ttcgattaac gataagtaaa atgtaaaatc acaggatttt 3660

cgtgtgtggt cttctacaca gacaagatga aacaattcgg cattaatacc tgagagcagg 3720

aagagcaaga taaaaggtag tatttgttgg cgatccccct agagtctttt acatcttcgg 3780

aaaacaaaaa ctattttttc tttaatttct ttttttactt tctattttta atttatatat 3840

ttatattaaa aaatttaaat tataattatt tttatagcac gtgatgaaaa ggacccaggt 3900

ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 3960

aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 4020

aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 4080

cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 4140

ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 4200

cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 4260

ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 4320

gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 4380

gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 4440

acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 4500

cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 4560

acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 4620

ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 4680

ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 4740

ggtagtcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 4800

atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 4860

ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 4920

attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcctttttgataat 4980

ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 5040

aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 5100

aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 5160

ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg 5220

tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 5280

ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 5340

cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 5400

agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 5460

gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 5520

ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 5580

tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 5640

tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 5700

cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 5760

tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 5820

gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc 5880

agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg 5940

agttacctca ctcattaggc accccaggct ttacacttta tgcttccggc tcctatgttg 6000

tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc 6060

aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctccaccg cggtggcggc 6120

cgcgaattcc ccgggtctag aggtctcggt tggcagtgac tcggtctcta cctggct 6177

Claims (4)

1. A method for preparing a galactose-inducible synthetic promoter with gradient activity, characterized in that the method comprises the step of connecting four UAS regulatory sequences to the upstream of the galactose-inducible core promoter _PGAL1 , wherein, The galactose-inducible core promoter _PGAL1 is a promoter that does not contain an endogenous UAS regulatory sequence; The UAS regulatory sequence is selected from the group comprising the following UAS regulatory sequences: UAS1 with a nucleotide sequence of 5'-CGGATTAGAAGCCGCCG-3', UAS2 with a nucleotide sequence of 5'-CGGGCGACAGCCCTCCG-3', and a nucleotide sequence of 5'-CGGAAGACTTCTCCTCCG-3'UAS3, the nucleotide sequence is 5'-CGCGCCGCACTGCTCCG-3'UAS4, the nucleotide sequence is 5'-CGGGGTGGACCACTCCG-3'UAS7, the nucleotide sequence is 5'-CGGACAACTGTTGACCG-3 'UAS8', and UAS9 whose nucleotide sequence is 5'-CGGGCCGCACTGCTCCG-3; The galactose-inducible synthetic promoter is the following promoter, UAS2223-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 3 UAS2 and 1 UAS3, UAS4444-scafPGAL1, 4 UAS regulatory sequences from 5'-3' direction are 4 UAS4, UAS2333-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 1 UAS2 and 3 UAS3, UAS4222-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 1 UAS4 and 3 UAS2, UAS7239-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 1 UAS7, 1 UAS2, 1 UAS3 and 1 UAS9, UAS2233-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 2 UAS2 and 2 UAS3, UAS2244-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 2 UAS2 and 2 UAS4, UAS2222-scafPGAL1, 4 UAS regulatory sequences from 5'-3' direction are 4 UAS2, UAS3333-scafPGAL1, 4 UAS regulatory sequences from 5'-3' direction are 4 UAS3, UAS1239-scafPGAL1, 4 UAS regulatory sequences from 5'-3' direction are 1 UAS1, 1 UAS2, 1 UAS3 and 1 UAS9 UAS7234-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 1 UAS7, 1 UAS2, 1 UAS3 and 1 UAS4, UAS2229-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 3 UAS2 and 1 UAS9, UAS2224-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 3 UAS2 and 1 UAS4, UAS7238-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 1 UAS7, 1 UAS2, 1 UAS3 and 1 UAS8, UAS2444-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 1 UAS2 and 3 UAS4. 2. A galactose-induced synthetic promoter, characterized in that the synthetic promoter comprises 4 UAS regulatory sequences and a galactose-induced core promoter _PGAL1 , wherein the galactose-induced core promoter _PGAL1 is not A promoter comprising an endogenous UAS regulatory sequence; The UAS regulatory sequence is selected from the group comprising the following UAS regulatory sequences: UAS1 with a nucleotide sequence of 5'-CGGATTAGAAGCCGCCG-3', UAS2 with a nucleotide sequence of 5'-CGGGCGACAGCCCTCCG-3', and a nucleotide sequence of 5'-CGGAAGACTTCTCCTCCG-3'UAS3, the nucleotide sequence is 5'-CGCGCCGCACTGCTCCG-3'UAS4, the nucleotide sequence is 5'-CGGGGTGGACCACTCCG-3'UAS7, the nucleotide sequence is 5'-CGGACAACTGTTGACCG-3 'UAS8', and UAS9 whose nucleotide sequence is 5'-CGGGCCGCACTGCTCCG-3; The galactose-inducible synthetic promoter is the following promoter, UAS2223-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 3 UAS2 and 1 UAS3, UAS4444-scafPGAL1, 4 UAS regulatory sequences from 5'-3' direction are 4 UAS4, UAS2333-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 1 UAS2 and 3 UAS3, UAS4222-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 1 UAS4 and 3 UAS2, UAS7239-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 1 UAS7, 1 UAS2, 1 UAS3 and 1 UAS9, UAS2233-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 2 UAS2 and 2 UAS3, UAS2244-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 2 UAS2 and 2 UAS4, UAS2222-scafPGAL1, 4 UAS regulatory sequences from 5'-3' direction are 4 UAS2, UAS3333-scafPGAL1, 4 UAS regulatory sequences from 5'-3' direction are 4 UAS3, UAS1239-scafPGAL1, 4 UAS regulatory sequences from 5'-3' direction are 1 UAS1, 1 UAS2, 1 UAS3 and 1 UAS9 UAS7234-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 1 UAS7, 1 UAS2, 1 UAS3 and 1 UAS4, UAS2229-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 3 UAS2 and 1 UAS9, UAS2224-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 3 UAS2 and 1 UAS4, UAS7238-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 1 UAS7, 1 UAS2, 1 UAS3 and 1 UAS8, UAS2444-scafPGAL1, the 4 UAS regulatory sequences from the 5'-3' direction are 1 UAS2 and 3 UAS4. 3. The application of the galactose-induced synthetic promoter as claimed in claim 2 in expressing heterologous biological substances in yeast. 4. The application according to claim 3, wherein the heterologous biological substance is a toxic protein.

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