CN113462686B - Method for preparing galactose-induced synthetic promoter with gradient activity, and prepared promoter and application thereof - Google Patents
Method for preparing galactose-induced synthetic promoter with gradient activity, and prepared promoter and application thereof Download PDFInfo
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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
Technical Field
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.
Background
Saccharomyces cerevisiae (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 a plurality of key genes in the synthetic pathway. Metabolic pathways involve multiple key genes, and the intensity differences between promoters of metabolic pathways may be up to several orders of magnitude in order to achieve dynamic balance of polygenic expression. In order to balance the relationship between metabolic pathways, increase the yield of target metabolites and reduce the interference to the growth of chassis bacteria, a promoter with proper strength is often required to be searched for to accurately regulate and control a plurality of genes along the metabolic network.
In Saccharomyces cerevisiae, galactose induces promoter P GAL Is the most active promoter recognized and mainly comprises P GAL1 ,P GAL2 ,P GAL7 ,P GAL10 Thus, they are also promoters commonly used 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-inducible upstream activation sequence (Upstream activator sequences, UAS). P (P) GAL UAS of (2) is a 17bp sequence with three bases at two ends conserved and random base composition in the middle, namely
5'-CGGNNNNNNNNNNNCCG-3'. Different P GAL Containing a different number of UASs, and each UAS having a different sequence, e.g. P GAL1 Containing 4 UASs. The transcription factor Gal4p binds to UAS and is responsible for inducing activation. Both the number and sequence of UAS affect promoter activity. Due to P GAL Is inhibited by glucose, galactose-induced properties, which is one of the best candidate promoter types for the expression of toxic proteins or enzymes. However, P GAL The number is limited, the intensity range is small, and the requirement of accurately regulating and controlling a metabolic network is difficult to meet, so that the artificial synthesis of the galactose-inducible promoter with the activity gradient is a strategy which needs to be provided for controlling the accurate expression of each gene in a complex metabolic pathway.
Hybrid promoters are one of the methods of artificially synthesizing promoters. The basis of constructing the hybrid promoter is the structure and function of the natural promoter, which can be divided into two parts of elements of an upstream regulatory sequence and a core promoter, and the elements can be split and combined, so that the synthetic promoter with new functions is obtained. Blazeck et al, P GAL1 The complete UAS regulatory regions (-457 to-148,309 bp) are respectively fused to the core promoter sequences of different promoters of yeast to construct a series of promoters induced by galactose, but the different activity intensities of the core promoters lead to different leakage expressions of the core promoters cultured under the condition that glucose is a carbon source, so that great time and energy are required to be spent for screening properIs a core promoter sequence of (a).
Disclosure of Invention
The present invention has been made in order to solve the above-mentioned problems.
The object of the present invention is to provide a method for preparing galactose-inducible synthetic promoters with gradient activity.
It is a further object of the present invention to provide a galactose-inducible synthetic promoter.
It is a further object of the present invention to provide the use of the above galactose-inducible synthetic promoter.
The embodiment of the invention provides a method for preparing a galactose-inducible synthetic promoter with gradient activity, which comprises the steps of connecting 1-4 UAS regulatory sequences to a galactose-inducible core promoter P GAL1 Upstream steps, wherein the galactose induces the core promoter P GAL1 Is a promoter that does not comprise an endogenous UAS regulatory sequence selected from the group consisting of: a UAS1 of nucleotide sequence 5'-CGGATTAGAAGCCGCCG-3', a UAS2 of nucleotide sequence 5'-CGGGCGACAGCCCTCCG-3', a UAS3 of nucleotide sequence 5'-CGGAAGACTCTCCTCCG-3', a UAS5 of nucleotide sequence 5' -CGCGCCGCACTGCTCCG-3' UAS4, a UAS5 of nucleotide sequence 5'-CGGAAAAGCGTCTTCCG-3', a UAS6 of nucleotide sequence 5'-CGGCGCTCACTCTTCCG-3', a UAS7 of nucleotide sequence 5'-CGGGGTGGACCACTCCG-3', a UAS8 of nucleotide sequence 5'-CGGACAACTGTTGACCG-3', and a UAS9 of nucleotide sequence 5' -CGGGCCGCACTGCTCCG-3.
The embodiment of the invention provides a galactose-inducible synthetic promoter, which comprises 1-4 UAS regulatory sequences and a galactose-inducible core promoter P GAL1 The galactose-inducible core promoter P GAL1 Is a promoter that does not comprise an endogenous UAS regulatory sequence selected from the group consisting of: UAS1 of nucleotide sequence 5'-CGGATTAGAAGCCGCCG-3', UAS2 of nucleotide sequence 5'-CGGGCGACAGCCCTCCG-3', UAS3 of nucleotide sequence 5'-CGGAAGACTCTCCTCCG-3', UAS4 of nucleotide sequence 5' -CGCGCCGCACTGCTCCG-3', UAS4 of nucleotide sequence 5' -CGGAAAAGCGTCTTCCUAS5 of G-3', UAS6 of nucleotide sequence 5'-CGGCGCTCACTCTTCCG-3', UAS7 of nucleotide sequence 5'-CGGGGTGGACCACTCCG-3', UAS8 of nucleotide sequence 5'-CGGACAACTGTTGACCG-3', and UAS9 of nucleotide sequence 5' -CGGGCCGCACTGCTCCG-3.
The invention provides the galactose-induced synthesis promoter prepared by the method.
Preferably, the galactose-induced synthesis initiation of the present invention is used for expression of heterologous biological substances in yeast.
Preferably, galactose-induced synthesis of the present invention initiates expression of heterologous toxic proteins in yeast.
The above-mentioned at least one technical scheme that this application embodiment adopted can reach following beneficial effect:
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.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute an undue limitation to the application. In the drawings:
FIG. 1 is a schematic diagram of the construction of a synthetic promoter, wherein A: UAS4 was mutated to UAS9, B: now a single UAS selected from UAS1-UAS9 with a scafP GAL1 And P CYC1 Constructing a synthetic promoter, C:4 UAS combinations and scafP GAL1 A synthetic promoter was constructed.
FIG. 2 shows the fluorescence intensity of synthetic promoters in galactose, wherein A: single UAS and scafP GAL1 And P CYC1 Construction of the Activity intensity of the synthetic promoter, B:4 UAS combinations and scafP GAL1 Construction of a compositeThe activity strength of the promoters was obtained, and experimental data were repeated in triplicate.
FIG. 3 shows a scafP GAL1 Fluorescence intensity detection results of series of synthetic promoter libraries, synthetic promoters constructed by single UAS and 4 UAS, fluorescence intensity versus P GAL1 Between 9% and 95%.
FIG. 4 shows a UAS-scafP GAL1 Activity in glucose the activity of the synthetic promoter in glucose was significantly inhibited compared to galactose culture conditions.
Detailed Description
For the purposes, technical solutions and advantages of the present application, the technical solutions of the present application will be clearly and completely described below with reference to specific embodiments of the present application and corresponding drawings. It will be apparent that the described embodiments are only some, but not all, of the embodiments of the present application. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.
The following describes in detail the technical solutions provided by the embodiments of the present application with reference to the accompanying drawings.
The method for preparing the galactose-inducible synthetic promoter with gradient activity according to the present invention comprises ligating 1 to 4 UAS regulatory sequences to a galactose-inducible core promoter P GAL1 Upstream steps, wherein the galactose induces the core promoter P GAL1 Is a promoter that does not comprise an endogenous UAS regulatory sequence selected from the group consisting of: a UAS1 of nucleotide sequence 5'-CGGATTAGAAGCCGCCG-3', a UAS2 of nucleotide sequence 5'-CGGGCGACAGCCCTCCG-3', a UAS3 of nucleotide sequence 5'-CGGAAGACTCTCCTCCG-3', a UAS5 of nucleotide sequence 5' -CGCGCCGCACTGCTCCG-3' UAS4, a UAS5 of nucleotide sequence 5'-CGGAAAAGCGTCTTCCG-3', a UAS6 of nucleotide sequence 5'-CGGCGCTCACTCTTCCG-3', a UAS7 of nucleotide sequence 5'-CGGGGTGGACCACTCCG-3', a UAS8 of nucleotide sequence 5'-CGGACAACTGTTGACCG-3', and a UAS9 of nucleotide sequence 5' -CGGGCCGCACTGCTCCG-3.
The transcription factor Gal4p binds to UAS (galactose-induced upstream activating sequence) and is responsible for inducing activation. Under the condition of glucose culture, the inhibitor Gal80P interacts with Gal4P to inhibit the transcriptional activity of Gal4P, thus, P GAL The activity is very weak, and the protein expression quantity is low; under galactose culture conditions Gal80P dissociates from Gal4P, releasing its transcriptional activation activity, thus P GAL The high activity state is started, and the activity after induction is more than 100 times that before induction.
According to a specific embodiment of the invention, the invention first selects endogenous P of Saccharomyces cerevisiae GAL1 Is UAS1, UAS2, UAS3, UAS4 and UAS4 mutant UAS9 and P, respectively GAL7 UAS8 of the sequence, an exogenous UAS sequence derived from Saccharomyces cerevisiae (Saccharomyces kudriavzevii) P was also selected GAL2 UAS5, UAS6 and UAS7, respectively, fused to P GAL1 UAS-free sequences (designated the backbone promoter, scafP GAL1 ) And a constitutive promoter P with UAS sequence removed CYC1 An upstream region. As a result of comparison of fluorescence intensities of the reporter proteins, it was found that different UAS had different expression activities, UAS-scafP GAL1 Relative to P GAL1 The activity range of (2) is 9-62%; with P CYC1 UAS-P as core promoter CYC1 Relative to P CYC1 The activity range of (2) is between 106% and 426%. Furthermore, the above 9 UASs were combined to replace four UASs of the PGAL1 promoter, and the experimental results showed that the activity of these recombinant promoters ranged between 55% and 95%. Through the regulation and control of UAS sequences and quantity, a promoter element library with an activity interval of 9% -95% can be obtained.
According to a specific embodiment of the invention, the amplification from the P-containing by PCR GAL1 Plasmid amplification of eGFP containing the eGFP reporter Gene P GAL1 An eGFP fragment, and fusion sequences of the respective UAS or UASs with the core sequence.
Recombinant plasmids were constructed by the Gibson assembly method. The Gibson assembly technique is a simple, rapid and efficient DNA directed cloning technique that can direct the insertion PCR product to any site in any vector. Linearizing the vector at cloning site, introducing end sequence of linearizing cloning vector to 5' end of inserted segment PCR primer, and making 5' and 3' end of inserted segment PCR product have completely identical sequences (15-20 bp) corresponding to two ends of linearizing cloning vector. The PCR product with the carrier terminal sequences at the two ends and the linearization cloning carrier are mixed according to a certain proportion, and the PCR product is converted only by reacting for 15min under the catalysis of exonuclease, DNA polymerase and ligase, so that the directional cloning is completed.
According to a specific embodiment of the invention, 4 UAS combinations replace UAS1, UAS2, UAS3, UAS4, and scafP GAL1 Construction of a synthetic promoter, for example, UAS1239 refers to the composition of UAS1, UAS2, UAS3, UAS9 from the 5'-3' direction, with UAS1, UAS2, UAS3, UAS4 replaced with 1239.
Example 1
As shown in FIG. 1, a schematic diagram of the construction of a synthetic promoter is shown. The invention respectively uses Saccharomyces cerevisiae P GAL1 And P GAL7 Is designated as UAS1, UAS2, UAS3, UAS4, and UAS8, respectively, wherein UAS4 is mutated to UAS9, as shown in FIG. 1, panel A. In addition, from Saccharomyces cerevisiae P GAL2 Exogenous UAS5, UAS6 and UAS7 were obtained from the promoters, and UAS1 to UAS9 were obtained, the sequences of which are shown in Table 1.
A schematic of a single UAS fused to a promoter is shown in FIG. 1, panel B. With the existing plasmid POT2-P GAL1 The eGFP is used as a template, and PCR is carried out by using the primer 1 and the primer 2, so that a plasmid skeleton fragment 1 (SEQ ID NO: 1) containing the eGFP reporter gene is amplified, and the obtained plasmid skeleton is single copy and contains a URA auxotroph marker and an ampicillin resistance marker. Then using the primer 3 as a common primer to amplify UAS-scafP containing UAS1-UAS9 with the primers 4-12 respectively GAL1 Synthetic promoters (SEQ ID NO: 2-SEQ ID NO: 10). Correspondingly, POT2-P CYC1 eGFP as template, and primers 13 as common primer to amplify UAS-P containing UAS1-UAS9 with primers 14-22, respectively CYC1 Synthetic promoters (SEQ ID NO: 11-SEQ ID NO: 19). The above synthetic promoter fragment and plasmid boneFrame fragment 1 (SEQ ID NO: 1), POT2-UAS-scafP was constructed by the method of Gibson Assembly, respectively GAL1 eGFP and POT2-UAS-P CYC1 -eGFP recombinant plasmid.
TABLE 1 UAS names and sequences
| Name of the name | Sequence(s) |
| 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 |
4 UAS and scafP GAL1 The promoter fusion schematic is shown in FIG. 1, panel C. With POT2-P GAL1 eGFP as template, amplified with primer 1 and primer 23, respectively, to contain scafP GAL1 Plasmid backbone fragment 20 (SEQ ID NO: 20). Then, PCR was performed using the primers 24/25 (UAS 1239), 26/27 (UAS 2222), 28/29 (UAS 2223), 30/31 (UAS 2224), 32/33 (UAS 2229), 34/35 (UAS 2233), 36/37 (UAS 2244), 38/39 (UAS 2333), 40/41 (UAS 2444), 42/43 (UAS 333), 44/45 (UAS 4222), 46/47 (UAS 7234), 48/49 (UAS 7238), 50/51 (UAS 7239), 52/53 (UAS 4444) as templates to obtain amplified fragments, i.e., fusion fragments of the four UASs shown in the brackets. The amplified product and plasmid backbone fragment 20 were then constructed as POT2-4 XUAS-scafP by the method of Gibson Assembly GAL1 -eGFP recombinant plasmid. The primers involved are shown in Table 2.
TABLE 2 primer names and sequences for plasmid construction
After the plasmid is constructed, the plasmid is transferred into saccharomyces cerevisiae competence to verify the activity of the synthetic promoter. First, yeast competence is prepared: selecting wild type yeast strain, adding 5mL YPD (1% yeast extract, 2% peptone, 2% glucose) liquid culture medium, respectively, and culturing at 250rpm overnight in shaking table at 30deg.C for 12 hr. The activated strains were transferred to 50mL of YPD liquid medium, respectively, and the starting OD was determined 600 Adjusting to 0.2, culturing in a shaking table at 30deg.C at 250rpm for about 5 hr to make OD 600 Between 0.7 and 1.0. After culturing, the cells were removed and centrifuged at 3000rpm for 5min to remove the supernatant. Further, 50mL of purified water was added thereto, and the mixture was centrifuged at 3000rpm for 5 minutes to remove the supernatant. Then 20mL of lithium acetate (LiAC) with the concentration of 100mM is added, after uniform mixing, the mixture is centrifuged at 3000rpm for 5min to remove supernatant, and finally 400 mu L of LiAC with the concentration of 100mM is added to resuspend cells, thus obtaining the yeast competence. Taking one transformation example, 30uL of yeast competence was placed in a 1.5mL centrifuge tube, and 240 uL of 50% PEG3350 was added; 36. Mu.L of LiAC at a concentration of 1M; 5 mu L of single-stranded fish sperm DNA (boiled at 99 ℃ C. For 5-10min before use, immediately placed on ice after removal); 500ng-1 μg of the constructed recombinant plasmid; 70ul of pure water. Mixing the above solutions, culturing at 30deg.C for 30min, and heat-shocking at 42deg.C for 25-40min. The heat-shocked conversion solution was centrifuged at 5000rpm for 1min, and the supernatant was removed. After removing the supernatant, 100. Mu.L of water was added and mixed uniformly, and then the mixture was spread on a solid medium of SC-URA (the formulation is shown in Table 3), and finally, the mixture was allowed to stand at 30℃for 48 hours.
Table 3 SC-URA Medium formulation
The synthetic promoter activity test method is as follows: a96-well plate was used, 300. Mu.L of SC-URA liquid medium containing 2% glucose was added to each well, then yeast colonies containing recombinant plasmids were picked, three were picked separately and repeated in the 96-well plate containing the medium, and the plates were placed at 30℃for activation for 24 hours. OD measurement of activated Yeast 600 The absorbance was centrifuged at 5000rpm for 5min and the supernatant removed. Then adding 400 μl of pure water, mixing, centrifuging at 5000rpm for 5min, removing supernatant, adding 200 μl of pure waterAnd (5) re-suspending by water. After resuspension, the yeast was then transferred to 96-well plates containing SC-URA liquid medium with 2% galactose, final concentration OD 600 Around 0.2, the final volume was 300 μl. After 24 hours of incubation, samples were diluted 10-fold and the synthetic promoter activity was characterized by measuring the eGFP fluorescence intensity by flow cytometry. A histogram is plotted (as shown in fig. 2 and 3) based on the measured fluorescence intensity.
The respective promoters in FIGS. 2 and 3 were relative to P GAL1 And P CYC1 Specific activity ratio values of (2) are shown in the following Table 4.
Table 4 Activity of synthetic promoters relative to the original promoters.
| Synthetic promoters | Relative P GAL1 Strength of (2) | Relative P CYC1 Strength of (2) |
| UAS1-scafP GAL1 /P CYC1 | 22％ | 169％ |
| UAS2-scafP GAL1 /P CYC1 | 46％ | 426％ |
| UAS3-scafP GAL1 /P CYC1 | 49％ | 384％ |
| UAS4-scafP GAL1 / |
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 fig. 2 and 3. As can be seen from FIG. 2A, the effect of different UAS sequences on promoter activity is also different, UAS-scafP GAL1 Synthetic promoter relative to P GAL1 The activity of these synthetic promoters ranges between 9% and 62%, and the leaky expression of glucose is very similar, even lower than that of the wild-type P GAL1 (shown in FIG. 4); synthetic promoter UAS-P CYC1 Relative to P CYC1 The activity range of (2) is between 106% and 426%. FIG. 2B shows that increasing the number of strongly inducible promoters further increases promoter activity,and the random arrangement of different UAS can also have different effects on the activity of the promoter, in the scafP GAL1 After 4 UAS fusion, the activity range was between 55% and 95%. The results show that the gradient galactose inducible promoter library with a larger activity interval is obtained.
The invention provides a new method for constructing the synthetic promoter, which has high potential value in the application of synthetic biology and has certain guiding significance on the transformation of the promoter.
The foregoing is merely exemplary of the present application and is not intended to limit the present application. Various modifications and changes may be made to the present application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc. which are within the spirit and principles of the present application are intended to be included within the scope of the claims of the present application.
Sequence listing
<110> Shenzhen advanced technology research institute of China academy of sciences
<120> method for preparing galactose-induced synthetic promoter having gradient activity, promoter prepared thereby, and use thereof
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5852
<212> DNA
<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 1
atgggtaagg gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60
gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120
aaacttaccc ttaaatttat ttgcactact ggaaagcttc ctgttccttg gccaacactt 180
gtcactactc ttacttatgg tgttcaatgc ttttcaagat acccagatca tatgaagcgg 240
cacgacttct tcaagagcgc catgcctgag ggatacgtgc aggagaggac catcttcttc 300
aaggacgacg ggaactacaa gacacgtgct gaagtcaagt ttgagggaga caccctcgtc 360
aacagaatcg agcttaaggg aatcgatttc aaggaggacg gaaacatcct cggccacaag 420
ttggaataca actacaactc ccacaacgta tacatcatgg cagacaaaca aaagaatgga 480
atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac 540
cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac 600
ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt 660
cttgagtttg taacagctgc tgggattaca catggcatgg atgaactata caaataatag 720
ccgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta 780
tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttattctt gagtaactct 840
ttcctgtagg tcaggttgct ttctcaggta tagcatgagg tcgctcttat tgaccacacc 900
tctaccggcc tcaggcagag acccaagaca ctgcggatcg agaccactag taccggtctg 960
cagctcgagg gggggcccgg tacccaattc gccctatagt gagtcgtatt acgcgcgctc 1020
actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg 1080
ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg 1140
cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc gacgcgccct gtagcggcgc 1200
attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 1260
agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 1320
tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 1380
ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 1440
ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 1500
aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 1560
ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 1620
attaacgttt acaatttcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 1680
caccgcatag ggtaataact gatataatta aattgaagct ctaatttgtg agtttagtat 1740
acatgcattt acttataata cagtttttta gttttgctgg ccgcatcttc tcaaatatgc 1800
ttcccagcct gcttttctgt aacgttcacc ctctacctta gcatcccttc cctttgcaaa 1860
tagtcctctt ccaacaataa taatgtcaga tcctgtagac accacatcat ccacggttct 1920
atactgttga cccaatgcgt cacccttgtc atctaaaccc acaccgggtg tcataatcaa 1980
ccaatcgtaa ccttcatctc ttccacccat gtctctttga gcaataaagc cgataacaaa 2040
atctttgtcg ctcttcgcaa tgtcaacagt acccttagta tattctccag tagataggga 2100
gcccttgcat gacaattctg ctaacatcaa aaggcctcta ggttcctttg ttacttcttc 2160
tgccgcctgc ttcaaaccgc taacaatacc tgggcccacc acaccgtgtg cattcgtaat 2220
gtctgcccat tctgctattc tgtatacacc cgcagagtac tgcaatttga ctgtattacc 2280
aatgtcagca aattttctgt cttcgaagag taaaaaattg tacttggcgg ataatgcctt 2340
tagcggctta actgtgccct ccatggaaaa atcagtcaag atatccacat gtgtttttag 2400
taaacaaatt ttgggaccta atgcttcaac taactccagt aattccttgg tggtacgaac 2460
atccaatgaa gcacacaagt ttgtttgctt ttcgtgcatg atattaaata gcttggcagc 2520
aacaggacta ggatgagtag cagcacgttc cttatatgta gctttcgaca tgatttatct 2580
tcgtttcctg caggtttttg ttctgtgcag ttgggttaag aatactgggc aatttcatgt 2640
ttcttcaaca ctacatatgc gtatatatac caatctaagt ctgtgctcct tccttcgttc 2700
ttccttctgt tcggagatta ccgaatcaaa aaaatttcaa agaaaccgaa atcaaaaaaa 2760
agaataaaaa aaaaatgatg aattgaattg aaaagctgtg gtatggtgca ctctcagtac 2820
aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc 2880
gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga caatctccgg 2940
gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagat taaagggcct 3000
cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt aggacggatc 3060
gcttgcctgt aacttacacg cgcctcgtat cttttaatga tggaataatt tgggaattta 3120
ctctgtgttt atttattttt atgttttgta tttggatttt agaaagtaaa taaagaaggt 3180
agaagagtta cggaatgaag aaaaaaaaat aaacaaaggt ttaaaaaatt tcaacaaaaa 3240
gcgtacttta catatatatt tattagacaa gaaaagcaga ttaaatagat atacattcga 3300
ttaacgataa gtaaaatgta aaatcacagg attttcgtgt gtggtcttct acacagacaa 3360
gatgaaacaa ttcggcatta atacctgaga gcaggaagag caagataaaa ggtagtattt 3420
gttggcgatc cccctagagt cttttacatc ttcggaaaac aaaaactatt ttttctttaa 3480
tttctttttt tactttctat ttttaattta tatatttata ttaaaaaatt taaattataa 3540
ttatttttat agcacgtgat gaaaaggacc caggtggcac ttttcgggga aatgtgcgcg 3600
gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 3660
aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 3720
gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 3780
cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 3840
tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 3900
tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 3960
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 ggggggcgga 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 (Saccharomyces cerevisiae)
<400> 2
gtgactcggt ctctacctgg ctcggattag aagccgccga acaataaaga ttctacaata 60
ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa 120
attaacgaat caaattaaca accataggat gataatgcga ttagtttttt agccttattt 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 (Saccharomyces cerevisiae)
<400> 3
gtgactcggt ctctacctgg ctcgggcgac agccctccga acaataaaga ttctacaata 60
ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa 120
attaacgaat caaattaaca accataggat gataatgcga ttagtttttt agccttattt 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 (Saccharomyces cerevisiae)
<400> 4
gtgactcggt ctctacctgg ctcggaagac tctcctccga acaataaaga ttctacaata 60
ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa 120
attaacgaat caaattaaca accataggat gataatgcga ttagtttttt agccttattt 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 (Saccharomyces cerevisiae)
<400> 5
gtgactcggt ctctacctgg ctcgcgccgc actgctccga acaataaaga ttctacaata 60
ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa 120
attaacgaat caaattaaca accataggat gataatgcga ttagtttttt agccttattt 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 (Saccharomyces cerevisiae)
<400> 6
gtgactcggt ctctacctgg ctcggaaaag cgtcttccga acaataaaga ttctacaata 60
ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa 120
attaacgaat caaattaaca accataggat gataatgcga ttagtttttt agccttattt 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 (Saccharomyces cerevisiae)
<400> 7
gtgactcggt ctctacctgg ctcggcgctc actcttccga acaataaaga ttctacaata 60
ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa 120
attaacgaat caaattaaca accataggat gataatgcga ttagtttttt agccttattt 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 (Saccharomyces cerevisiae)
<400> 8
gtgactcggt ctctacctgg ctcggggtgg accactccga acaataaaga ttctacaata 60
ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa 120
attaacgaat caaattaaca accataggat gataatgcga ttagtttttt agccttattt 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 (Saccharomyces cerevisiae)
<400> 9
gtgactcggt ctctacctgg ctcggacaac tgttgaccga acaataaaga ttctacaata 60
ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa 120
attaacgaat caaattaaca accataggat gataatgcga ttagtttttt agccttattt 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 (Saccharomyces cerevisiae)
<400> 10
gtgactcggt ctctacctgg ctcgggccgc actgctccga acaataaaga ttctacaata 60
ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa 120
attaacgaat caaattaaca accataggat gataatgcga ttagtttttt agccttattt 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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (Saccharomyces cerevisiae)
<400> 20
aacaataaag attctacaat actagctttt atggttatga agaggaaaaa ttggcagtaa 60
cctggcccca caaaccttca aattaacgaa tcaaattaac aaccatagga tgataatgcg 120
attagttttt tagccttatt tctggggtaa ttaatcagcg aagcgatgat ttttgatcta 180
ttaacagata tataaatgga aaagctgcat aaccacttta actaatactt tcaacatttt 240
cagtttgtat tacttcttat 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 ctttgttact 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 tagatataca 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 tgaagatcct ttttgataat 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 galactose-inducible synthetic promoter with gradient activity, which comprises ligating 4 UAS regulatory sequences to galactose-inducible core promoter P GAL1 An upstream step, wherein,
the galactose-inducible core promoter P GAL1 Is a promoter that does not contain endogenous UAS regulatory sequences;
the UAS regulatory sequences are selected from the group consisting of: UAS1 of nucleotide sequence 5'-CGGATTAGAAGCCGCCG-3', UAS2 of nucleotide sequence 5'-CGGGCGACAGCCCTCCG-3', UAS3 of nucleotide sequence 5'-CGGAAGACTCTCCTCCG-3', UAS4 of nucleotide sequence 5'-CGCGCCGCACTGCTCCG-3', UAS7 of nucleotide sequence 5'-CGGGGTGGACCACTCCG-3', UAS8 of nucleotide sequence 5'-CGGACAACTGTTGACCG-3', and UAS9 of nucleotide sequence 5' -CGGGCCGCACTGCTCCG-3;
the galactose-inducible synthetic promoter is a promoter,
UAS2223-scafpGAL1, 4 UAS regulatory sequences from the 5'-3' direction are 3 UAS2 and 1 UAS3,
UAS4444-scafpGAL1, 4 UAS regulatory sequences 4 UAS4 from the 5'-3' direction,
UAS2333-scafpGAL1, 4 UAS regulatory sequences from 5'-3' direction are 1 UAS2 and 3 UAS3,
UAS4222-scafpGAL1, 4 UAS regulatory sequences from 5'-3' direction are 1 UAS4 and 3 UAS2,
UAS7239-scafpGAL1, 4 UAS regulatory sequences from 5'-3' direction are 1 UAS7, 1 UAS2, 1 UAS3 and 1 UAS9,
UAS2233-scafpGAL1, 4 UAS regulatory sequences from 5'-3' direction are 2 UAS2 and 2 UAS3,
UAS2244-scafpGAL1, 4 UAS regulatory sequences from 5'-3' direction are 2 UAS2 and 2 UAS4,
UAS2222-scafpGAL1, 4 UAS regulatory sequences from the 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, 4 UAS regulatory sequences from 5'-3' direction are 1 UAS7, 1 UAS2, 1 UAS3 and 1 UAS4,
UAS2229-scafpGAL1, 4 UAS regulatory sequences from 5'-3' direction are 3 UAS2 and 1 UAS9,
UAS2224-scafpGAL1, 4 UAS regulatory sequences from the 5'-3' direction are 3 UAS2 and 1 UAS4,
UAS7238-scafpGAL1, 4 UAS regulatory sequences from 5'-3' direction are 1 UAS7, 1 UAS2, 1 UAS3 and 1 UAS8,
UAS2444-scafpGAL1, 4 UAS regulatory sequences from 5'-3' are 1 UAS2 and 3 UAS4.
2. A galactose-inducible synthetic promoter comprising 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 endogenous UAS regulatory sequences;
the UAS regulatory sequences are selected from the group consisting of: UAS1 of nucleotide sequence 5'-CGGATTAGAAGCCGCCG-3', UAS2 of nucleotide sequence 5'-CGGGCGACAGCCCTCCG-3', UAS3 of nucleotide sequence 5'-CGGAAGACTCTCCTCCG-3', UAS4 of nucleotide sequence 5'-CGCGCCGCACTGCTCCG-3', UAS7 of nucleotide sequence 5'-CGGGGTGGACCACTCCG-3', UAS8 of nucleotide sequence 5'-CGGACAACTGTTGACCG-3', and UAS9 of nucleotide sequence 5' -CGGGCCGCACTGCTCCG-3;
the galactose-inducible synthetic promoter is a promoter,
UAS2223-scafpGAL1, 4 UAS regulatory sequences from the 5'-3' direction are 3 UAS2 and 1 UAS3,
UAS4444-scafpGAL1, 4 UAS regulatory sequences 4 UAS4 from the 5'-3' direction,
UAS2333-scafpGAL1, 4 UAS regulatory sequences from 5'-3' direction are 1 UAS2 and 3 UAS3,
UAS4222-scafpGAL1, 4 UAS regulatory sequences from 5'-3' direction are 1 UAS4 and 3 UAS2,
UAS7239-scafpGAL1, 4 UAS regulatory sequences from 5'-3' direction are 1 UAS7, 1 UAS2, 1 UAS3 and 1 UAS9,
UAS2233-scafpGAL1, 4 UAS regulatory sequences from 5'-3' direction are 2 UAS2 and 2 UAS3,
UAS2244-scafpGAL1, 4 UAS regulatory sequences from 5'-3' direction are 2 UAS2 and 2 UAS4,
UAS2222-scafpGAL1, 4 UAS regulatory sequences from the 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, 4 UAS regulatory sequences from 5'-3' direction are 1 UAS7, 1 UAS2, 1 UAS3 and 1 UAS4,
UAS2229-scafpGAL1, 4 UAS regulatory sequences from 5'-3' direction are 3 UAS2 and 1 UAS9,
UAS2224-scafpGAL1, 4 UAS regulatory sequences from the 5'-3' direction are 3 UAS2 and 1 UAS4,
UAS7238-scafpGAL1, 4 UAS regulatory sequences from 5'-3' direction are 1 UAS7, 1 UAS2, 1 UAS3 and 1 UAS8,
UAS2444-scafpGAL1, 4 UAS regulatory sequences from 5'-3' are 1 UAS2 and 3 UAS4.
3. Use of the galactose-inducible synthetic promoter according to claim 2 for expressing heterologous biological substances in yeast.
4. The use according to claim 3, wherein the heterologous biological substance is a toxic protein.
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