CN114574490A - Pichia pastoris constitutive promoter and modification method and application thereof - Google Patents

Abstract

The invention discloses a constitutive promoter of pichia pastoris, a modification method and application thereof, wherein the constitutive promoter is P_0887‑GAP、P_0887‑AOX1And P_0887‑GSOne of (1); the promoter P_0887‑GAPThe nucleotide sequence of (A) is shown as SEQ ID NO. 31; the promoter P_0887‑AOX1The nucleotide sequence of (A) is shown as SEQ ID NO. 32; the promoter P_0887‑GSNucleotide sequences of e.g.Shown as SEQ ID NO. 33. The invention replaces promoter P₀₈₈₇The transcription activity of the modified promoter is basically unchanged, and the fluorescence intensity is improved to 37 times of that of the original promoter.

Description

Pichia pastoris constitutive promoter and modification method and application thereof

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a pichia pastoris constitutive promoter, and a modification method and application thereof.

Background

Pichia pastoris is a unicellular eukaryotic microorganism, is a methylotrophic yeast at the same time, is widely used as a host for producing recombinant proteins, has attracted attention as a cell factory for producing chemicals in recent years, has a strictly regulated expression system, has less glycosylation compared with Saccharomyces cerevisiae, can realize high-density fermentation in a basal medium, has a high growth speed, has a eukaryotic post-translational modification mechanism, and can correctly express and secrete exogenous proteins. Although much research work has been done in pichia pastoris, there are fewer promoters in pichia pastoris that can be used for foreign protein production and biosynthetic pathway construction than other model yeasts.

Most inducible promoters in Pichia pastoris are involved in the methanol metabolic pathway and utilize methanol as an inducer, such as P, which is most commonly used for protein production_AOX1And P_FLD1. Compared with these inducible promoters, constitutive promoters do not require expensive, toxic and explosive inducers during production, and are more favorable for controlling production costs and safety issues. P_GAP(glyceraldehyde-3-phosphate dehydrogenase) is the most commonly used constitutive promoter in Pichia pastoris and has a high expression level in glucose and glycerol media but a low expression level when methanol is used as the carbon source. Furthermore, P_TEF1And P_GCW14And are also commonly used for constitutive expression. Recently, Dou et al screened 8 strong endogenous promoters with high transcription levels by RNA-Seq and LacZ reporter systems, and some of them were randomly applied to construct a heterologous beta-carotene synthesis pathway in Pichia pastoris with a yield up to 1.07 mg/L. Meanwhile Dou et al found that the simultaneous use of the same promoter multiple times significantly reduced the transcriptional expression level of the target gene.

Constitutive promoters in Pichia pastoris are not only quite small in number, but are limited to promoters with strong activity. For co-expression of some chaperones or other secretory cofactors, a less transcriptionally active promoter may be required. In addition, the construction of some heterologous metabolic pathways requires a larger promoter pool to avoid competing effects. Therefore, the use of Pichia pastoris for the construction of cell factories requires constitutive promoters of different strengths, which are more stable and suitable for recombinant protein expression.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

The present invention has been made keeping in mind the above and/or other problems occurring in the prior art.

One of the objects of the present invention is to provide a constitutive promoter of Pichia pastoris, by replacing promoter P₀₈₈₇The transcription activity of the modified promoter is basically unchanged, and the fluorescence intensity is improved to 37 times of that of the original promoter.

In order to solve the technical problems, the invention provides the following technical scheme: a constitutive promoter of Pichia pastoris, wherein the constitutive promoter is P_0887-GAP、P_0887-AOX1And P_0887-GSOne of (1);

the promoter P_0887-GAPThe nucleotide sequence of (A) is shown as SEQ ID NO. 31;

the promoter P_0887-AOX1The nucleotide sequence of (A) is shown as SEQ ID NO. 32;

the promoter P_0887-GSThe nucleotide sequence of (A) is shown in SEQ ID NO. 33.

As a preferred embodiment of the constitutive promoter of Pichia pastoris according to the present invention, wherein: the constitutive promoter isP_0887-GS。

Another object of the present invention is to provide a method for modifying constitutive promoter of Pichia pastoris, which comprises using promoter P_GAP5' UTR sequence of (A) or promoter P_AOX15' UTR sequence of (A) or promoter P_GSReplacement of the promoter P by the 5' UTR sequence of (A)₀₈₈₇The 5' UTR sequence of (a);

wherein, the promoter P₀₈₈₇The 5' UTR sequence of (A) is shown in SEQ ID NO. 22;

the promoter P_GSThe 5' UTR sequence of (A) is shown in SEQ ID NO. 25;

the promoter P_GAPThe 5' UTR sequence of (A) is shown in SEQ ID NO. 28;

the promoter P_AOX1The 5' UTR sequence of (A) is shown in SEQ ID NO. 29.

As a preferred embodiment of the method for modifying the constitutive promoter of Pichia pastoris according to the present invention, wherein: the promoter P₀₈₈₇The nucleotide sequence of (A) is shown in SEQ ID NO. 10.

As a preferred embodiment of the method for modifying the constitutive promoter of Pichia pastoris according to the present invention, wherein: the promoter P_GSThe nucleotide sequence of (A) is shown in SEQ ID NO. 14.

As a preferred embodiment of the method for modifying the constitutive promoter of Pichia pastoris according to the present invention, wherein: the promoter P_GAPThe nucleotide sequence of (A) is shown in SEQ ID NO. 1.

As a preferred embodiment of the method for modifying the constitutive promoter of Pichia pastoris according to the present invention, wherein: the promoter P_AOX1The nucleotide sequence of (A) is shown in SEQ ID NO. 30.

It is another object of the present invention to provide the use of the constitutive promoter of Pichia pastoris, as described above, for expression of intracellular and/or secreted proteins.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a method for modifying a constitutive promoter of pichia pastoris, wherein P is P after modification₀₈₈₇The transcription level of the promoter is basically unchanged, and the translation efficiency is improved by 37 times at most. The discovery of the promoters with different activities widens the promoter library of the pichia pastoris, breaks through the limitation of the pichia pastoris in the aspects of recombinant protein production and biosynthesis pathway construction, has good application prospect, enriches synthetic biological elements, and lays a theoretical foundation for the strain in the genetic engineering modification and exogenous gene expression.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:

FIG. 1 shows the expression of fluorescence by a candidate promoter according to the present invention;

FIG. 2 shows the expression of HSA protein by the candidate promoter of the present invention;

FIG. 3 shows fluorescence intensity and transcription level of candidate promoters in YTD medium;

FIG. 4 shows fluorescence intensity and transcription level of the modified promoter in YTD medium.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

EXAMPLE 1 construction of promoter-eGFP and promoter-HAS strains

19 genes were screened initially by transcriptome data (RPS17, RPS8B, RPL35A, RPL10, Eif5a, RPL19A, ILV5, RPL23B, 0887, GSP2, TIF1, RPL14B, GS, SUN, BMH1, TRX1, YPT1, CCP1 and ARC 15). The promoter region of the selected gene (1000 bp upstream of the start codon ATG in the genomic DNA sequence) was amplified by PCR using the primer sequences shown in Table 1. At the same time, P is cloned_GAPThe promoter served as a control.

Promoter P_GAPThe nucleotide sequence of (A) is shown as SEQ ID NO. 1; promoter P_RPS17The nucleotide sequence of (A) is shown as SEQ ID NO. 2; promoter P_RPS8BThe nucleotide sequence of (A) is shown as SEQ ID NO. 3; promoter R_PL35AThe nucleotide sequence of (A) is shown as SEQ ID NO. 4; promoter P_RPL10The nucleotide sequence of (A) is shown as SEQ ID NO. 5; promoter P_Eif5aThe nucleotide sequence of (A) is shown as SEQ ID NO. 6; promoter P_RPL19AThe nucleotide sequence of (A) is shown as SEQ ID NO. 7; promoter P_ILV5The nucleotide sequence of (A) is shown as SEQ ID NO. 8; promoter P_RPL23BThe nucleotide sequence of (A) is shown as SEQ ID NO. 9; promoter P₀₈₈₇The nucleotide sequence of (A) is shown as SEQ ID NO. 10; promoter P_GSP2The nucleotide sequence of (A) is shown as SEQ ID NO. 11; promoter P_TIF1The nucleotide sequence of (A) is shown as SEQ ID NO. 12; promoter P_RPL14BThe nucleotide sequence of (A) is shown as SEQ ID NO. 13; promoter P_GSThe nucleotide sequence of (A) is shown as SEQ ID NO. 14; promoter P_SUNThe nucleotide sequence of (A) is shown as SEQ ID NO. 15; promoter P_BMH1The nucleotide sequence of (A) is shown as SEQ ID NO. 16; promoter P_TRX1The nucleotide sequence of (A) is shown as SEQ ID NO. 17; promoter P_CCP1The nucleotide sequence of (A) is shown as SEQ ID NO. 18; promoter P_ARC15The nucleotide sequence of (A) is shown in SEQ ID NO. 19.

TABLE 1 primers for construction of strains

Replacement of candidate promoter fragments by P in the PGAPZB plasmid backbone (PGAPZB plasmid purchased from Invitrogen, USA) by homologous recombination using MultiF Seamless Assembly kits (ABClonal, China)_GAPAnd inserting the codon optimized eGFP or HSA sequence in the latter to construct the PXXXZB-eGFP or PXXXZB-HSA vector, e.g., by promoter P_GSPGSZB-eGFP or PGSZB-HSA vectors were constructed. The eGFP sequence is shown as SEQ ID NO. 20; the HSA sequence is shown in SEQ ID NO. 21.

These vectors were linearized by PspL I digestion located in the middle of the AOX1 terminator, then passed by electroporation into GS115 competent cells and integrated into the genome by homologous recombination. Positive transformants were selected using bleomycin-containing YTDS plates and verified by genomic PCR. The expression of the protein was carried out as previously reported (Kolacsek O, Pergel E, Varga N, Apati A,&orban TI (2017) Ct shift A novel and available real-time PCR quantification model for direct compliance of differential nucleic acid sequences and its adaptation for translational compliance, Gene 598: 43-49) Single copy strains were selected from the positive clones and named promoter-eGFP strain or promoter-HSA strain, respectively, e.g., promoter P_GSAre respectively corresponding to P_GSeGFP strains and P_GS-a HAS strain.

Example 2 promoter-eGFP Strain fluorescence intensity assay

The promoter-eGFP strain is cultured in the YTD, YTG and YTM medium containing bleomycin at 30 ℃ for 24h, and then the cells are collected by centrifugation. Cells were washed three times with PBS buffer and then diluted to OD₆₀₀Approximately equal to 0.4-0.6 and transferred to a 96-well microplate.

Multimode Plate Reader (Perkinelmer, USA) is used to determine OD value and fluorescence intensity of cells. As fluorescence intensity/OD₆₀₀The values of (A) characterize the promoter strength and the results obtained are shown in FIG. 1.

For better analysis of promoter stability and strength, classical constitutive strong promoter P_GAPWas used as a control. Based on the fluorescence intensity of the promoter-eGFP strain under different conditions, the Coefficient of Variation (CV) of the promoter is calculated, and the promoters with the coefficient of variation smaller than GAP are screened out for further analysis. 12 ratios P selected_GAPMore stable promoters, respectively P₀₈₈₇、P_RPS8B、P_RPL10、P_GS、P_RPL35A、P_CCP1、P_RPS17、P_ILV5、P_RPL23B、P_SUN、P_GSP2、P_YPT1。

EXAMPLE 3 measurement of the concentration of HSA protein secreted by promoter-HSA Strain

The HSA protein was expressed using 12 promoters selected, and the supernatant was subjected to western blotting to analyze the HSA concentration. Culturing promoter-HSA strain at 30 deg.C in YTD, YTG, YTM medium containing bleomycin for 24 hr, collecting fermentation supernatant, adding 5 × loading buffer, boiling, performing SDS gel electrophoresis, transferring protein in gel to

polyvinylidene difluoride (PVDF) membrane (Millipore, USA). Membranes were blocked with TBST buffer containing 5% skim milk for one hour at room temperature and then diluted 1: 10000 of Albumin Monoclonal antibody (Proteintech, USA) was incubated at room temperature for 1H, and after three washes, HRP-conjugated affinity Goat Anti-Mouse IgG (H + L) (Proteintech, USA) was used for incubation at room temperature for 1H. Bound antibodies were detected with High-sig ECL Western Blotting Substrate (Tanon, China). The integral density of the protein bands was calculated by counting with Image J software and obtaining black and white pictures by a chemiluminescence imager. Correlation of the integrated density and concentration according to the standard HSA sampleThe HSA concentration in the culture supernatant was calculated.

The protein bands, HAS concentrations and Coefficient of Variation (CV) obtained by Western Blotting are shown in FIG. 2, and finally 6 constitutive promoters with different strengths, P respectively, were selected₀₈₈₇、P_RPS8B、P_RPL10、P_GS、P_RPL35AAnd P_CCP1Expression intensity distribution in P_GAPThe strength is 27-148%.

Example 4 identification of promoter 5' UTR sequences

Pure total RNA was obtained by Ultrapure RNA kit (CWBIO, China), the integrity of the RNA was judged by agarose nucleic acid electrophoresis, and the purity and concentration of RNA, A of pure RNA, were determined using NanoDrop (Quawell, USA)₂₆₀:A₂₈₀The ratio of (a) to (b) is 2.0. The 5 '-complementary DNA ends of high quality RNA were rapidly amplified using 5' RACE Kits (Sangon, China). Briefly, two rounds of PCR were performed after single-stranded cDNA synthesis. The products were cloned into T-Vector (Takara, Japan) after gel recovery and were Sanger sequenced (Genewiz, China) to determine the transcription start sites of these six promoters as well as the 5' UTR region (Table 3).

Table 3 5' UTR sequences of the promoter sequences screened

Example 5 expression of eGFP by different promoters in YTD Medium translation and transcription levels

Pure total RNA was obtained by Ultrapure RNA kit (CWBIO, china) and then reverse transcribed into cDNA using HiScript III RT SuperMix (nunoprazan, china). Real-time fluorescent quantitative PCR was performed in a StepOneNus RealTime PCR System (Applied Biosystems, USA) using the ChamQ Universal SYBR qPCR Master Mix reagent (Novowed, China). The reference gene was ACT1, and the primers used were as shown in Table 2.

TABLE 2 real-time fluorescent quantitative PCR primers

6 constitutive promoters P of different strengths₀₈₈₇、P_RPS8B、P_RPL10、P_GS、P_RPL35A、P_CCP1And P as a control_GAPThe comparison of the fluorescent intensity (FIG. 3a) and transcription level (FIG. 3b) of the promoter expressing eGFP in YTD medium is shown in FIG. 3.

As can be seen in FIG. 3, P₀₈₈₇The amount of mRNA transcribed by the activated eGFP is the most among seven promoters, but the fluorescence intensity corresponding to the strain is the lowest. This phenomenon may be due to ribosome recruitment during translation of mRNA and translation efficiency.

Example 6 promoter P₀₈₈₇Transformation of

Attempt to pass through replacement of P₀₈₈₇The 5' UTR sequence of (a) to unify its transcription level and translation level. Selected promoter P exhibiting excellent translation level by homologous recombination using MultiF Seamless Assembly kit (ABConal, China)_GSAnd a strong constitutive promoter P commonly used in Pichia pastoris_GAPAnd a strongly inducible promoter P_AOX1The 5' UTR sequence of (A) is replaced to P₀₈₈₇Respectively obtaining the promoter P_0887-GAP、P_0887-AOX1、P_0887-GS。

Strongly inducible promoter P_AOX1The nucleotide sequence of (A) is shown as SEQ ID NO. 30; promoter P_0887-GAPThe nucleotide sequence of (A) is shown as SEQ ID NO. 31; promoter P_0887-AOX1The sequence is shown as SEQ ID NO. 32; promoter P_0887-GSThe sequence is shown in SEQ ID NO. 33.

Testing of the modified promoter P Using the methods of example 2 and example 5_0887-GAP、P_0887-AOX1、P_0887-GSThe fluorescent intensity of eGFP was expressed in YTD medium (fig. 4a) and the transcription level (fig. 4 b).

As can be seen from FIG. 4, P is obtained after the sequence conversion of the 5' UTR₀₈₈₇The relative expression amount of the initiated eGFP is basically unchanged, and the fluorescence intensity is obviously improved. Especially P₀₈₈₇Conversion of 5' UTR to P_GSAfter 5' UTR, the fluorescence intensity increased 37-fold. The results indicate that switching the 5' UTR of the promoter can increase the level of translation of the gene without affecting the level of transcription.

The invention takes pichia pastoris as a research object, clones, screens and transforms a constitutive promoter. By using eGFP as a reporter gene and driving the expression of the eGFP through the selected promoter, the difference of green fluorescence intensity and HSA protein secretion level is analyzed, and finally a series of stable constitutive promoters (P) with different intensities are obtained₀₈₈₇、P_RPS8B、P_RPL10、P_GS、P_RPL35AAnd P_CCP1). The invention also provides a method for modifying the constitutive promoter of the pichia pastoris, wherein P is the modified P₀₈₈₇The transcription level of the promoter is basically unchanged, and the translation efficiency is improved by 37 times. The discovery of the promoters with different activities widens the promoter library of the pichia pastoris, breaks through the limitation of the pichia pastoris in the aspects of recombinant protein production and biosynthesis pathway construction, has good application prospect, enriches synthetic biological elements, and lays a theoretical foundation for the strain in genetic engineering modification and exogenous gene expression.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Sequence listing

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<120> Pichia pastoris constitutive promoter, and transformation method and application thereof

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ttctagagca gttagagaca gcaatgcaag ggaaacgtta gaagctgagt tcccaagaaa 480

aacataccca caatttccag aattcacagg tgaagtgaga agcaaggaac tgggccatac 540

tcaatcaaca attgaccttt acttctcaca ctataggact tcatctggaa aacacagtga 600

ggcccaacaa atgaacagaa tattcaaaca ctacagacca ccctatggac ttgaggcaga 660

ggttattagt gaggatctca gaaagatggg aataaaccca gtaggcaaat aggacgacat 720

gttgacataa gtaaaatcaa tggacactta tctatcttct ccccacaccg ttcatcttcg 780

ttacccggcg taagtctcta tcatgtaaaa ttcagtccgg agtagtgtaa acatcatcac 840

gaagtaaaga aaatcaaaat atcaaaacca aaaattggtt ggtgcacaga gccaacaaga 900

tttctaggcg atcaatgggc aattggctta gaaaaaggca ttcatttttt ttctcatttg 960

ttttttcact tctctttgaa tctccctaac tttaatcaaa 1000

<210> 7

<211> 1000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ctacgacgtt cttttgatta gctcagcaaa ctagtttgaa aaaaaagacc aatacctgct 60

attactgtac aacttttata atatgcttaa tatacacgct acagcaaccc tcatatagca 120

caacactaaa ctaatagaag attaccatgg cttttcgggg gtgaactgtt caaagcccaa 180

acgatgattc cacttttgtc ttcttgatct cttcttaaag ttcttcttgg tcgtcctctt 240

ctcaggcttt ttcaaaggga cagagtctat atctatgtca acttccacag tggagacctt 300

ggcaggttta gagtctttgt cttcgcttgc ctcttccttc tgttttacca gggctgcttg 360

ggacttctca aacagtctct tcctcctttc atcttcgtat ttctggaatt ccaatgacct 420

cttcacagct tttgcttgaa gcttggactt tgatctagag gatttagcca ttactgtggg 480

atatctgtga atgcaaattg ggatcttcct agaagagatg ttattttacc tctcagaaaa 540

tttattttcc tgcaactctc aaaaagtctt ggcccctatc aaagtattgc ttatctaaga 600

agcaaaccgt tattacgtaa catgatctta tcctttacac atcgtgggta aaccctaact 660

ttgataacgt gaccaccttt tgagaatgtg attattggaa gcgaatcact gttccacacg 720

acggaccctc ttacaatttg cccgcgattg ggcccagaaa agggagattt agctcgtgac 780

tacctctttc gctacctttc caagagggaa aaatttacct gtttcaattt cctcttcacc 840

tttttagttt gcccaattac taatttcaaa atgtaagttt gattcccaag cggaagaggt 900

catggagtag ttccattggt aagaccagta caatcagttt caactcgctc aatctgttgt 960

ctttttgttg attgttcatg atcctccagg tattttccct 1000

<210> 8

<211> 1000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atttccagat aatcatgacg catccacctc gttacaatgt acctaaacta aaagacaacg 60

acagccccct tggttgtgca gatcatcatc tcctatcaaa cagcacacaa aaaactgggt 120

aataagttta gaacgagtta caaaatgtct tcctcctttt gcaattctaa tctacgcgga 180

atctgtcacc ctcttaggtt tattctctta cagtacttcc cctagaatcc cgacaagagc 240

taaacaaaaa cttaggccag aaagcaaagt tcccttagca tataatttac ctagctttgt 300

taggctattt cgaacttgat tccgttcaat cgcccactcc acttcatctt cgacattatc 360

ttccatcaat tctccttcta cagaaacata ggctgaccca tctaaagaag atctttcagt 420

aatgtcttgt ttcttttgtt gcagtggtga gccattttga cttcgtgaaa gtttctttag 480

aatagttgtt tccagaggcc aaacattcca cccgtagtaa agtgcaagcg taggaagacc 540

aagactggca taaatcaggt ataagtgtcg agcactggca ggtgatcttc tgaaagtttc 600

tactagcaga taagatccag tagtcatgca tatggcaaca atgtaccgtg tggatctaag 660

aacgcgtcct actaaccttc gcattcgttg gtccagtttg ttgttatcga tcaacgtgac 720

aaggttgtcg attccgcgta agcatgcata cccaaggacg cctgttgcaa ttccaagtga 780

gccagttcca acaatctttg taatattaga gcacttcatt gtgttgcgct tgaaagtaaa 840

atgcgaacaa attaagagat aatctcgaaa ccgcgacttc aaacgccaat atgatgtgcg 900

gcacacaata agcgttcata tccgctgggt gactttctcg ctttaaaaaa ttatccgaaa 960

aaattttctt cccttctctt ccaaatatcg tctccacaaa 1000

<210> 9

<211> 1000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aggatgcaag gtatctcttt gtggacgggg aactgagccg taaacgagaa tgctctgtag 60

ttggggtctt ctgggtaggg gcgcttcact tcgctatctg cgcatcttca aatgcaccag 120

tccattccat gacatcactt tgcgacacaa actacaatgc ccctgctggg actgagcatt 180

attaggttta gagcttgcca tggctctata gatacaattg gcacaaaata tatgaccaca 240

tggcgtgact accgcttcgt tgatggcgtc aaaacaaata ggacattctg tcatcttgaa 300

cagcttttga ttatcctttc tctttgcaga cgcctctaac agttctatag tcgatttgtt 360

agcagctgga tctttttcgc catcggatcc cagcacaata acatcttcct cctcttcctc 420

ctcctcctcc tctatctcgt ctccactaga cacctcagta tctgacccgt cgggtacttc 480

caaaacagaa tgctttctct tattggtgcg atctctttgc tcatcgtcat taatcacgat 540

ctgctgattc tcctcttgtt cagatccagt atccacaaca tcgcttagag ggctactgct 600

gccatcaatt ggtgtaatga actcctggtc agaatcctca accggagtaa aaagtgaatc 660

ttcctgtgca ttcattcttt aacttgacat tcttgaataa gctctaattt acaagcagtt 720

gatgatccag tgctgatgga aaccctgctt atccgatgta gtcttaatgt attcagaggg 780

gaggtagagg caggatagat aagaggaaca gtcttagtaa tcatcacgtg cacttgttca 840

gggctctctt cgggttttgt ttcaaagatt cgcgaactgc acctattgtc ctacatttca 900

aacgaaccca gagcagcaat ttttgattca tcgcacgaca gtaattagtt ttcgttaaaa 960

aaaatatcca acttttacct caataacata atcatcaact 1000

<210> 10

<211> 1000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

aaactgttta gagttgtgac gatgatcatg tccaataact attgatttac cgccagcttt 60

tgacatatag ttggccagtc cctgagatgc ttgtattatc gtgagatcat tcattagact 120

aaaaccagca cccattctac ctcgaagtcc cgctgttcca aaagtaattc ttcttcgtaa 180

gcgttcttca agaactttaa acttttcatt ctcaagaagt ttgagaatct cagatctagt 240

ggatggatta cgatcaatgc taagccattg atgcgctaac gccttaattg atggggacat 300

gatgtcagat gtaatacctt gtttaccgaa atttagctat ctcacgtagc ctagaatggc 360

cgctgctaat cggcctatat ctaaaacggc atatcgagga tcgatgaagt ttacacgtga 420

cgcattcagt gtaagtaaat tacatgcagt gccggctact tgactaaagt aagaaaaaac 480

tcagaaatca aagaaataac agaagctgta cgacacaacg aaataaatac aaactttctt 540

aggatgcaat ttaacaagaa tcgtcttatt gtctctcgga aacattcaag aatttcagcc 600

cattatccac attcagtcat ggcgcattat ttagattgtg ttgtttgata aaaaaagaat 660

acagcaagta acaacgagta gtcagttact gagatatttt ttagtaccgt cgatccggtt 720

acggggaatt taactgcaat atatcaaacg ttaaactgga tcgagttcaa aaaaacaagc 780

tctaactcag tcaagcttta tgtatagttt cctaatcagg taatttttta tgatcttgtc 840

tgtaggtttg attaaatttc cattacatct aaaaatagta acccagccat ctgaaaaatg 900

gatctttcgg gattcaatat tcacagtata aaaggaagac ttctcttagg attctgaccc 960

gttcatactc attgctaaac atattaaata ctgagaaatg 1000

<210> 11

<211> 1000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

aggtaaatgt tagtacttgt tcatcgtgtc tgtcgtctag aattttttca ctcctgattg 60

atacttactt cttctggctg gggcagcaga gaaagctcta acttgagttc tcagggcctg 120

aatatgttag tcaagttaaa cattgaaaaa cccaccgaat tgagtggtga aggctagact 180

tacggatgat ctgataacgg caaacattgt gtgtgtgtct aagggttgat ttagatggtt 240

cctccttgac ttggacattt cgagcgattt cgttggttgg caagaagacg ggtaatcgag 300

gagtaatggt tgggacacta tcagaggacc attgagagaa gaggtaagca agattaagat 360

taatggcaaa gtattattgt gaatattgca attgtgagtg tgaaccgtaa ttgatcgtcg 420

ccaaatggat cttactaaca agtttagcct acttgaccca tgactctttg agtgtcagaa 480

aatctcattt gattggccga aaccatctca gaaagtattg cgattactac gaaggtaaga 540

catcttgtga ttcgcccacc acagggccat actatcataa agactactaa cagttctaga 600

aaaggctaaa gagcttggga tttgggactc caacgattta ccatatgaaa taactgaatc 660

cgatctctat gcgaatattc caggtagacc actgtcatta gaaactcctt tggaaacatt 720

tccgccgccg ccaagtctgc caaacctacc aaaccctcca ccagccatat accactacaa 780

caccacagaa gagaaagagc tcatatcatc cgtcatgaga gagtaccagc acagaaatac 840

agtcaagtaa aactagtatg caagcattac gtaataatag caactttatg acaaatcatt 900

ccattttttt ccactggagc gtgcactgcg taaatcattc tctttggaag gcaagggaag 960

aacaacaaaa tttttccttc cgttatacaa acattgaatc 1000

<210> 12

<211> 1000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ctcttctcta tccggccctc tagataaata tacttgagaa ataattgtac aacttatcag 60

ttctctatca cgtgacagtg gactttccgc agtcacagag cattgctcca aatggattcg 120

agcctttcgg agggaccttg tagcatctac gcggaaatat acgggcacag agccttcgcg 180

aagcagtgac cacctaacac cacaattctt cgagctcgca aaacgtctga aaaatttaac 240

ctatcttcaa cctgttagct tcaactacaa ctatacaaaa tggaaaacga caacggtgaa 300

ttggtatgta tcccgaacta cacgagaaat ggtgatgtgt agcataaaat gaagctcaga 360

aaatctgtag ccacagtgaa gttatacggc catttgcgtt gttagttggt caagttcgaa 420

aaatactaac atttcaaggt tgaactttac gttccacgca agtgctctgc caccaacaga 480

attatcaagg ctaaagacca cgcctccgtt caaattaaca ttgctaacgt tgatgaggaa 540

ggacgtgcca tcagaggaga caacaccact ttcgctctct ccggttacgt tagatccaga 600

ggtgaggctg acgactcttt gaacagattg gctcaacaag cgggtctgtt gaagaacgtc 660

tggtcctact cccgttaagc actcaaaaag atgtttttgt acctttaatt tagcccgtca 720

acattattag tgtattacac caggttgtaa aatgcatcca tacaccgtag tatttgcgcg 780

cctcctcctg ggtgttttct caactgccgc ccccaactcg ccccccccct tttgcacttc 840

ttgccacctt ggagatcaga gattgtggaa ccttgctcaa aatctggcat gcacactacc 900

tcctcaaaaa aattttcctt gccttgttgc aaggcgctca aaaatcaaaa gtttttctac 960

aacttttgtt cccaacaaat caccttttta cttacccatt 1000

<210> 13

<211> 1000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

acagaaggac aaacaggagc tagagtcgca gttaaggaag acgacgactt acaggcacgc 60

ttggacgatc tccggagata aacgctgcgt ctcccacttg tgagattatt atcaatagcc 120

taacaggtca tcgtgatcat ttggaccatc tacccgagca gagaagctcg tagctctgtt 180

tttggtaacg atcttagtac aggctactca ttttgcgaat atttagctta cttagtgtca 240

tttctgcctt gcacttcttt ggacctgcca aactaccacc cagcagagca acttggctca 300

acaactttac caggcgcaca gagtctatgt aacaactatc aatgccatag ataacaagct 360

cattgaaaga ttcactgtga acctgacgta agtcctgaag cgcaactagc accaagtata 420

aggcagtcac ttcatctggc ttcaaaaata gaaacttagc tccaccggaa atcagatgtt 480

cctcaatggt atcaactata gcaactgagg ggcaattcac acatatcaat accaccaggg 540

ttttcctttt atttatacct tccataatca tcttgaaatc aaccttgtcg tcatcattgc 600

tagacatcga tcataggggc aaaactgatt acctttaaag ttagataatc aagttctaga 660

ataaaaagcc atcctttatt cagaaagaaa tttatcgcat ttcgaaaaga gaacacattg 720

aaactaggca tgtttcgaac cttttctctc aatcgcaaat tatttcatca taagaaagat 780

aaaaatagat tagcaactta ctaaaagact ctgacaatca cacgagcctg aacatcgtct 840

tttttcctgt agaacaaaca cttattgaaa cgagattcga gactctcaaa gcgaatattc 900

tctagggctc aaccctggtc atgtgattgg ttcttgcgcg cttgtttttt gcagtacaac 960

aaaaaaaatt attgtccttc ttctatcaac caaagtaaag 1000

<210> 14

<211> 1000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gctggtgcgt caggttcggt ccccgggagg agtctcgacc aatggctagg tagtgataac 60

aatcaaagtt tatgagacca gtagtttgac acatagtgca tttattggaa atatggggac 120

actaccttcc gccaatagga gacgagaata cgactagacg tatctcgtaa agtccctccg 180

cctcgatcta gaactgccac catatttgag gatcaaagac cagaagaagt gtcgtgccag 240

tttttcagat aaatcattcg ccgagatcag tcgatcatca gcgttcagtc catgagaaca 300

aggtgctggc ttgacgtatc ttcaggcagc cagcagcttt ttgtttcagc ctatcatctt 360

tgaggcttat catcttactg ataagactat gggaaaaaac ccaagaaaat gagatgtttt 420

atgtgaacga ccatttactt ttttagatag gcaatcttct gccccggtga gtctcctcat 480

tgggcaatgt tgtaattaca cgactaacaa atcagaggcg gtaaacgcta gtctcggcag 540

cgtcatcgag gtcagtgcat actcaacaat agaaggcgcg caatcattag acaacttgat 600

ccatagcccg cagcgtttct tctccagaaa gttcactata attacccgta gaggattggt 660

tcaacataca agaagatcag caaatcacct tcagtattcg gcgacaagat ttatttttca 720

ctgaggtttc aaagcttttc aaatttcaat ataatacttc cctattaatc atgcatgcac 780

cctacaagat ttcgcattct tttttttact acaggaatta ctgaccaggt gactctaaaa 840

tagctttgag gtatataaat cacaaaggcg ctgttagcag acagtttttt tctttttcgt 900

ttcttacctt cggtcaacaa tattaattcg cttcaagcta cttagttctc tccattataa 960

gagaatctta atataaacac ctatacatta caattacaat 1000

<210> 15

<211> 1000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

acatctatga taactaccta aacaacgagt tcatctacat gagatggaag gaaatgtttt 60

tgattccaga tgccaaagtt agagatataa agggggcatc gtttgctgga ttttactacg 120

tatgtttcag ccaactaact ggatcaatta gcgggctata tttccacaag tactctgaaa 180

agttccagca gctagaattg tgccatgctc cagacggcgg gtgtagctcc tacttcaagc 240

tagcttagag agagagagga gatagacggc gacacattgt aggcaaggag gagccattca 300

ccctcaagag ctggtctttt gggcgccatg tgtgcacacg cggtttaatc gcttaccgga 360

ggtgttctag tgttttggca gggatataat tctttattaa tagagtgtat gaattaaatt 420

aggtacctat cgcctatgcg atatggccaa aaagggaagt gtaactactt gcaccatgtt 480

gccttgaaca ccggtaacgt ggtaagtgca cgactgtgtg ttaaagacgg aatgtatgga 540

atgtgggtgt ttctcacaag gagaatctaa aacgaacctt caaggactca aacgctaagc 600

cacgcgttat tggccttgtg taggcggcac tgctgcacac tgacggccct caattgcact 660

ggaggtttcc atctctgcct cctgagaagg ccctcatcgc cagtcccact tgatcggatc 720

aatgttgcta gtatgtagat cggtatttat gttaatctga gattgccaga tgcggcaata 780

aatctagatt tatcatgaaa ctttttcttc agactcttgc tcgcccgaat atatagttta 840

acaaatccag ctcgatctga cgttcgttag aaacaaacct ttgatcactc caactacaag 900

agaaacttgc ctactcactg ccttataggt acttacattc attccaagtt ctttttgttc 960

agtctttcct tacattcatt ttttaaccgt gcttctcaat 1000

<210> 16

<211> 1000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cgagatcacc aacagacgtt tatgtgttac aaaacctatt caaaccgctc gattcatccc 60

attgaaggtt gtattggaag cttctaatgc cacaggattg gatggttacg acgcagccaa 120

cccaactttg ccaggcttag aaacacccct accaaggctg ggaatttcca gcttgaaact 180

taagtcactt ttgaaaaagt ctattccaat tgacgttgcc agagaatgct ctacaatcct 240

tctacctgaa caacaaccac caatcaggct gacaggaggt agcacctcca ggcccagtta 300

cgtccattca aactacgaat tagatggtcc ttacaaacag gcgatctacg aacttgtcaa 360

actgaccgaa gactccaagt tcggactcaa tatccaagag tgtcctccat tctagttaaa 420

agaccatgta ataatagaga atacacattg cacctaaaca ccaccatatt ttcgtaaagc 480

ggttttattt tatctccctc cgggtaacgg tggctgtcga aatttcttat ttctttctga 540

tgatttccat agaggtgtac caatcaaaac aatatgggcc tttgttgaac ctcagattcg 600

tttgcgaata ccttcacacg tacttattat caaaggttct aaagaattat tttttaattg 660

agtaactacg gattaatttg agttttaaag tgagtagttc atgtgtaagg tgagaaggat 720

gattggactt ttgagaaaca agtgaactta ctctgatcca tcgtactacc gggtggcgaa 780

gtagtataaa caaacatagc aaacgggaga tttcgtctat tggctacaaa gacacgattt 840

gaaatttgct acgatccaag cgaaatgacc gttaagcaat gaagtggtga ttttccgatg 900

attgcttcga ttttttaagt tccagtattt cttccttcct ttttttcccc attttttttt 960

tttttgtccc gaagcctcct ttactaacat tacctttagc 1000

<210> 17

<211> 1000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

cgaaatttgg ctcggacacg tcttaaaagg ttatgtaagg aggtgatgtt agatagagac 60

gaagctgtgg atgagctgga gaaccttggt aatctgaacg gatctaaaga acaatcatca 120

ctttgggcct agaggtttac agaggtttgg gcaattttta ctacattacc tggtgttgga 180

gacacccaaa tgctgagaca tttgtacatg tcgtcgggga gattgtttga acgaactgac 240

gagaattgga atgcccattg caggatcata actgatggga tggaacattt atcatgtgca 300

gtaagcaaaa gtcgtttctt agtcaatcat atatctttta acagatacta agtaaccata 360

ggagatgtgt tatcttagta gaagaatgat ttatatatta ataatattat atctttttcc 420

tagatcgcat tggtcaactt tgaccaacaa ctatgggtgc ttcaatccta acaggcttat 480

accactgaag ttacagtaat tcttcctagt gaaactgatt gtgtccttac attaccatgt 540

ttttccaagc ttcgttcctc atggcccaat ggtcaaggcg tctggctacg attagccaac 600

tccgagtaaa ctcggggtaa taggaaacca gaagattcca ggttcgactc ctggtgggga 660

agcttctttt tttctgagtt gtattgggga aaaactgagg cgcattaatc tattcccaac 720

aaattccaca ccaactacgt tactcatgtg agagcctctt tgcagtttca gtacaattac 780

cttccattaa tcatctggca tccagtagtt actaaacacc ctattgatga ccaaatattc 840

actccacgtt acctgattac ctaataatag ttagcgtgat tagtaactaa agaatctcga 900

acaaccgatg gccaacatgc ttataagcgg cacagcatcc cactcctgaa ttctcaattc 960

aatttcctgt tttataccac agtgctgaaa ttaacaaaca 1000

<210> 18

<211> 1000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

cattcggaga ctgtgatctc tcaaggggaa caggcgtgtt tgttattgac attttgcctg 60

aggttaggaa caaacaaaaa ggttgattga tttcagagag attcgtgtac tctgattatt 120

cgagggtgag ttcgggggtt ggttggtgaa cgaaaaaaaa ttctgaacgg ctgagattat 180

tagggatgaa tcaggtggtt atagagccgg agatggactg gtatagatag tggcacaact 240

gagagggaga tgttttagcc aaagtattac gtctttatcc tagataagta gtaattatcc 300

ggaggcattg tgagatacca ggtaccataa ccatacaaca ggttcaatgt ggttacagga 360

tacgatagaa ggattcatag gagctgcatt acgactctgt tccactactg ggcgtttcaa 420

ggtgaatgga aaataaacca aggaagtgct ctctggcttt ctggaaaact ttaagtcatc 480

agagatttaa gtccctcggt tctgcgtggg cgctaaaaac gatcccccac tagctagctc 540

tgctctactt cagtcatgtc tcatgatcac gatcatgatt tggtcatatt cgtgcaccac 600

ataattgtgt ccaatcagga atgaataatt tcaaagacgg ttgtaatgag gcagatgaca 660

taatcaatcg actgtaaacc tagtgagaga cttttaccca attggcgtcg aaatagccgt 720

gaaacctagc tagattccag acagaaaacg tctagggaat cgtagagaat ataaggcttt 780

gaacgtctgt acagtttcag ctcaaatctg cggaattaaa tgatcgtcta gactgtagta 840

gattacagtt atgtatcccc gcgccatctg cggaactata ttattcatcg gctcacttct 900

agaatgcagg aaaattaggt ctcctctgtg ggaagcggag aactataaat actgccaata 960

tccacatatt gattcttcct aggctattat actttttaac 1000

<210> 19

<211> 1000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

agaactgtga aaaattggaa acctcggcgt cttggaggtg gtttaggtgg tcgtcattac 60

acaaaacgtc aaatagaaaa acttaccaga caggctccaa gatatgatag atatcctccc 120

ccagagagat actccacaag ttcaaatcgt ggggcatata gaggaggggc aggctccgga 180

ggctttcgaa gccatggtgg tggaggcaga gagggacaag gtaatttcag atcttctgat 240

caacgagata accgtagagg aggatggaat ggtcaaagaa gatactagta agatgctgta 300

gagatgaagc tatcttcatt gacatccaac atccatcgcg gttagtattt ttttttcttt 360

tttgcccttt tttccttcgt atcactttct taaagcaaaa ccactatggc ctccaataaa 420

gaggactatc cagttccgga acaagatgag ttagaaaatg attttagaat aacgctggtg 480

gttcagtcca aagaaatacc agatggtgag cttcacgaac gatttctttc gaatatctcc 540

acgtttactg gaatgaacca gtttttcgac atttttgatg aaaaagtggc ttatcctaat 600

gaaggacagt tgaaatatga agtaggaagt gacggcttgg tcgtgatgct agtgaccaat 660

agggatatca gagaccaatg tttagacttt attgacaaat acattgagga catagagccc 720

aaagaatcgg ccagagggga cgatgagcaa gctgagacta gtagtaatga tagtgagaat 780

gacagtgacc acacagagag agtatcgtct aagaagagta gaaaataaag catacataca 840

taatacactt gcactttaca catttgtaag ccctcccctc gatcagagat tgatccggat 900

ctctgtgatt atataagctg tatccgtttt tctacgcatt gtcttctcgg ttatataatc 960

agcactttct ccgccccttt acgacaaaag taacgagacc 1000

<210> 20

<211> 720

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720

<210> 21

<211> 1794

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

atgaggggtg tgtttcgtcg agatgcacac aagagtgagg ttgctcatcg atttaaagat 60

ttgggagaag aaaatttcaa agccttggtg ttgattgcct ttgctcagta tcttcagcag 120

tgtccatttg aagatcatgt aaaattagtg aatgaagtaa ctgaatttgc aaaaacatgt 180

gttgctgatg agtcagctga aaattgtgac aaatcacttc ataccctttt tggagacaaa 240

ttatgcacag ttgcaactct tcgtgaaacc tatggtgaaa tggctgactg ctgtgcaaaa 300

caagaacctg agagaaatga atgcttcttg caacacaaag atgacaaccc aaacctcccc 360

cgattggtga gaccagaggt tgatgtgatg tgcactgctt ttcatgacaa tgaagagaca 420

tttttgaaaa aatacttata tgaaattgcc agaagacatc cttactttta tgccccggaa 480

ctccttttct ttgctaaaag gtataaagct gcttttacag aatgttgcca agctgctgat 540

aaagctgcct gcctgttgcc aaagctcgat gaacttcggg atgaagggaa ggcttcgtct 600

gccaaacaga gactcaagtg tgccagtctc caaaaatttg gagaaagagc tttcaaagca 660

tgggcagtag ctcgcctgag ccagagattt cccaaagctg agtttgcaga agtttccaag 720

ttagtgacag atcttaccaa agtccacacg gaatgctgcc atggagatct gcttgaatgt 780

gctgatgaca gggcggacct tgccaagtat atctgtgaaa atcaagattc gatctccagt 840

aaactgaagg aatgctgtga aaaacctctg ttggaaaaat cccactgcat tgccgaagtg 900

gaaaatgatg agatgcctgc tgacttgcct tcattagctg ctgattttgt tgaaagtaag 960

gatgtttgca aaaactatgc tgaggcaaag gatgtcttcc tgggcatgtt tttgtatgaa 1020

tatgcaagaa ggcatcctga ttactctgtc gtgctgctgc tgagacttgc caagacatat 1080

gaaaccactc tagagaagtg ctgtgccgct gcagatcctc atgaatgcta tgccaaagtg 1140

ttcgatgaat ttaaacctct tgtggaagag cctcagaatt taatcaaaca aaattgtgag 1200

ctttttgagc agcttggaga gtacaaattc cagaatgcgc tattagttcg ttacaccaag 1260

aaagtacccc aagtgtcaac tccaactctt gtagaggtct caagaaacct aggaaaagtg 1320

ggcagcaaat gttgtaaaca tcctgaagca aaaagaatgc cctgtgcaga agactatcta 1380

tccgtggtcc tgaaccagtt atgtgtgttg catgagaaaa cgccagtaag tgacagagtc 1440

accaaatgct gcacagaatc cttggtgaac aggcgaccat gcttttcagc tctggaagtc 1500

gatgaaacat acgttcccaa agagtttaat gctgaaacat tcaccttcca tgcagatata 1560

tgcacacttt ctgagaagga gagacaaatc aagaaacaaa ctgcacttgt tgagctcgtg 1620

aaacacaagc ccaaggcaac aaaagagcaa ctgaaagctg ttatggatga tttcgcagct 1680

tttgtagaga agtgctgcaa ggctgacgat aaggagacct gctttgccga ggagggtaaa 1740

aaacttgttg ctgcaagtca agctgcctta ggccatcatc atcatcatca ttaa 1794

<210> 22

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

atactcattg ctaaacatat taaatactga gaa 33

<210> 23

<211> 93

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

tttccttaaa catcaagtaa agtaagtaaa ttaccagaac ttgcgaaatc aagtcattgc 60

tccattcaac ttcatcggct aacagttatt aga 93

<210> 24

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ccgtactaac aagttcagac aaa 23

<210> 25

<211> 95

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

accttcggtc aacaatatta attcgcttca agctacttag ttctctccat tataagagaa 60

tcttaatata aacacctata cattacaatt acaat 95

<210> 26

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

accttacatc aataactaat ataaa 25

<210> 27

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

aggctattat actttttaac 20

<210> 28

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

ttgaacaact atcaaaacac a 21

<210> 29

<211> 114

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

atcattatta gcttactttc ataattgcga ctggttccaa ttgacaagct tttgatttta 60

acgactttta acgacaactt gagaagatca aaaaacaact aattattcga aacg 114

<210> 30

<211> 936

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

gatctaacat ccaaagacga aaggttgaat gaaacctttt tgccatccga catccacagg 60

tccattctca cacataagtg ccaaacgcaa caggagggga tacactagca gcagaccgtt 120

gcaaacgcag gacctccact cctcttctcc tcaacaccca cttttgccat cgaaaaacca 180

gcccagttat tgggcttgat tggagctcgc tcattccaat tccttctatt aggctactaa 240

caccatgact ttattagcct gtctatcctg gcccccctgg cgaggttcat gtttgtttat 300

ttccgaatgc aacaagctcc gcattacacc cgaacatcac tccagatgag ggctttctga 360

gtgtggggtc aaatagtttc atgttcccca aatggcccaa aactgacagt ttaaacgctg 420

tcttggaacc taatatgaca aaagcgtgat ctcatccaag atgaactaag tttggttcgt 480

tgaaatgcta acggccagtt ggtcaaaaag aaacttccaa aagtcgccat accgtttgtc 540

ttgtttggta ttgattgacg aatgctcaaa aataatctca ttaatgctta gcgcagtctc 600

tctatcgctt ctgaaccccg gtgcacctgt gccgaaacgc aaatggggaa acacccgctt 660

tttggatgat tatgcattgt ctccacattg tatgcttcca agattctggt gggaatactg 720

ctgatagcct aacgttcatg atcaaaattt aactgttcta acccctactt gacagcaata 780

tataaacaga aggaagctgc cctgtcttaa accttttttt ttatcatcat tattagctta 840

ctttcataat tgcgactggt tccaattgac aagcttttga ttttaacgac ttttaacgac 900

aacttgagaa gatcaaaaaa caactaatta ttcgaa 936

<210> 31

<211> 985

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

aaactgttta gagttgtgac gatgatcatg tccaataact attgatttac cgccagcttt 60

tgacatatag ttggccagtc cctgagatgc ttgtattatc gtgagatcat tcattagact 120

aaaaccagca cccattctac ctcgaagtcc cgctgttcca aaagtaattc ttcttcgtaa 180

gcgttcttca agaactttaa acttttcatt ctcaagaagt ttgagaatct cagatctagt 240

ggatggatta cgatcaatgc taagccattg atgcgctaac gccttaattg atggggacat 300

gatgtcagat gtaatacctt gtttaccgaa atttagctat ctcacgtagc ctagaatggc 360

cgctgctaat cggcctatat ctaaaacggc atatcgagga tcgatgaagt ttacacgtga 420

cgcattcagt gtaagtaaat tacatgcagt gccggctact tgactaaagt aagaaaaaac 480

tcagaaatca aagaaataac agaagctgta cgacacaacg aaataaatac aaactttctt 540

aggatgcaat ttaacaagaa tcgtcttatt gtctctcgga aacattcaag aatttcagcc 600

cattatccac attcagtcat ggcgcattat ttagattgtg ttgtttgata aaaaaagaat 660

acagcaagta acaacgagta gtcagttact gagatatttt ttagtaccgt cgatccggtt 720

acggggaatt taactgcaat atatcaaacg ttaaactgga tcgagttcaa aaaaacaagc 780

tctaactcag tcaagcttta tgtatagttt cctaatcagg taatttttta tgatcttgtc 840

tgtaggtttg attaaatttc cattacatct aaaaatagta acccagccat ctgaaaaatg 900

gatctttcgg gattcaatat tcacagtata aaaggaagac ttctcttagg attctgaccc 960

gttcttgaac aactatcaaa acaca 985

<210> 32

<211> 1078

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

aaactgttta gagttgtgac gatgatcatg tccaataact attgatttac cgccagcttt 60

tgacatatag ttggccagtc cctgagatgc ttgtattatc gtgagatcat tcattagact 120

aaaaccagca cccattctac ctcgaagtcc cgctgttcca aaagtaattc ttcttcgtaa 180

gcgttcttca agaactttaa acttttcatt ctcaagaagt ttgagaatct cagatctagt 240

ggatggatta cgatcaatgc taagccattg atgcgctaac gccttaattg atggggacat 300

gatgtcagat gtaatacctt gtttaccgaa atttagctat ctcacgtagc ctagaatggc 360

cgctgctaat cggcctatat ctaaaacggc atatcgagga tcgatgaagt ttacacgtga 420

cgcattcagt gtaagtaaat tacatgcagt gccggctact tgactaaagt aagaaaaaac 480

tcagaaatca aagaaataac agaagctgta cgacacaacg aaataaatac aaactttctt 540

aggatgcaat ttaacaagaa tcgtcttatt gtctctcgga aacattcaag aatttcagcc 600

cattatccac attcagtcat ggcgcattat ttagattgtg ttgtttgata aaaaaagaat 660

acagcaagta acaacgagta gtcagttact gagatatttt ttagtaccgt cgatccggtt 720

acggggaatt taactgcaat atatcaaacg ttaaactgga tcgagttcaa aaaaacaagc 780

tctaactcag tcaagcttta tgtatagttt cctaatcagg taatttttta tgatcttgtc 840

tgtaggtttg attaaatttc cattacatct aaaaatagta acccagccat ctgaaaaatg 900

gatctttcgg gattcaatat tcacagtata aaaggaagac ttctcttagg attctgaccc 960

gttcatcatt attagcttac tttcataatt gcgactggtt ccaattgaca agcttttgat 1020

tttaacgact tttaacgaca acttgagaag atcaaaaaac aactaattat tcgaaacg 1078

<210> 33

<211> 1059

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

aaactgttta gagttgtgac gatgatcatg tccaataact attgatttac cgccagcttt 60

tgacatatag ttggccagtc cctgagatgc ttgtattatc gtgagatcat tcattagact 120

aaaaccagca cccattctac ctcgaagtcc cgctgttcca aaagtaattc ttcttcgtaa 180

gcgttcttca agaactttaa acttttcatt ctcaagaagt ttgagaatct cagatctagt 240

ggatggatta cgatcaatgc taagccattg atgcgctaac gccttaattg atggggacat 300

gatgtcagat gtaatacctt gtttaccgaa atttagctat ctcacgtagc ctagaatggc 360

cgctgctaat cggcctatat ctaaaacggc atatcgagga tcgatgaagt ttacacgtga 420

cgcattcagt gtaagtaaat tacatgcagt gccggctact tgactaaagt aagaaaaaac 480

tcagaaatca aagaaataac agaagctgta cgacacaacg aaataaatac aaactttctt 540

aggatgcaat ttaacaagaa tcgtcttatt gtctctcgga aacattcaag aatttcagcc 600

cattatccac attcagtcat ggcgcattat ttagattgtg ttgtttgata aaaaaagaat 660

acagcaagta acaacgagta gtcagttact gagatatttt ttagtaccgt cgatccggtt 720

acggggaatt taactgcaat atatcaaacg ttaaactgga tcgagttcaa aaaaacaagc 780

tctaactcag tcaagcttta tgtatagttt cctaatcagg taatttttta tgatcttgtc 840

tgtaggtttg attaaatttc cattacatct aaaaatagta acccagccat ctgaaaaatg 900

gatctttcgg gattcaatat tcacagtata aaaggaagac ttctcttagg attctgaccc 960

gttcaccttc ggtcaacaat attaattcgc ttcaagctac ttagttctct ccattataag 1020

agaatcttaa tataaacacc tatacattac aattacaat 1059

Claims (8)

1. A Pichia pastoris constitutive promoter, wherein: the constitutive promoter is P_0887-GAP、P_0887-AOX1And P_0887-GSOne of (1);

the promoter P_0887-GAPThe nucleotide sequence of (A) is shown as SEQ ID NO. 31;

the promoter P_0887-AOX1The nucleotide sequence of (A) is shown as SEQ ID NO. 32;

the promoter P_0887-GSThe nucleotide sequence of (A) is shown in SEQ ID NO. 33.

2. The pichia pastoris constitutive promoter of claim 1, wherein: the constitutive promoter is P_0887-GS。

3. The method of engineering a constitutive promoter of pichia pastoris according to claim 1 or 2, wherein: comprising the use of a promoter P_GAP5' UTR sequence of (A) or promoter P_AOX15' UTR sequence of (A) or promoter P_GSReplacement of the promoter P by the 5' UTR sequence of (A)₀₈₈₇The 5' UTR sequence of (a);

wherein, the promoter P₀₈₈₇The 5' UTR sequence of (b) is shown in SEQ ID NO. 22;

the promoter P_GSThe 5' UTR sequence of (A) is shown as SEQ ID NO. 25;

the promoter P_GAPThe 5' UTR sequence of (A) is shown in SEQ ID NO. 28;

the promoter P_AOX1The 5' UTR sequence of (A) is shown in SEQ ID NO. 29.

4. The method of engineering a constitutive promoter of pichia pastoris according to claim 3, wherein: the promoter P₀₈₈₇The nucleotide sequence of (A) is shown in SEQ ID NO. 10.

5. The method of engineering a constitutive promoter of pichia pastoris according to claim 3, wherein: the promoter P_GSThe nucleotide sequence of (A) is shown in SEQ ID NO. 14.

6. The method of engineering a constitutive promoter of pichia pastoris according to claim 3, wherein: the promoter P_GAPThe nucleotide sequence of (A) is shown in SEQ ID NO. 1.

7. The method of engineering a constitutive promoter of pichia pastoris according to claim 3, wherein: the promoter P_AOX1The nucleotide sequence of (A) is shown in SEQ ID NO. 30.

8. Use of a constitutive promoter of pichia pastoris according to claim 1 or 2, for expression of intracellular proteins and/or secreted proteins.

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