阅读说明：本技术 Ires序列、ires序列的应用和多顺反子表达载体 (IRES sequence, use of IRES sequence and polycistronic expression vector ) 是由 戴俊彪 卢俊南 杨炜钐 温栾 林鑫 于 2020-12-11 设计创作，主要内容包括：本申请属于基因工程技术领域,具体涉及一种IRES序列、IRES序列的应用和多顺反子表达载体。所述IRES序列是基于SARS-Cov-2中的ORF8基因片段得到的。该IRES序列可以直接从SARS-Cov-2中分离得到,也可以人工合成。该IRES序列具备对真核生物的起始翻译能力,可以应用于目标基因的表达过程,也可以应用于抗SARS-Cov-2药物的筛选。(The application belongs to the technical field of genetic engineering, and particularly relates to an IRES sequence, application of the IRES sequence and a polycistronic expression vector. The IRES sequence was obtained based on the ORF8 gene fragment in SARS-Cov-2. The IRES sequence can be directly isolated from SARS-Cov-2 or artificially synthesized. The IRES sequence has the ability of starting translation to eukaryote, can be applied to the expression process of target genes, and can also be applied to the screening of anti-SARS-Cov-2 drugs.)

1. An internal ribosome entry site IRES sequence, wherein the IRES sequence is obtained based on ORF8 gene fragment in SARS-Cov-2, and the ORF8 gene fragment is shown as SEQ ID NO. 1.

2. The IRES sequence of claim 1, as set forth in SEQ ID No. 1.

3. The IRES sequence of claim 1, which is a sequence of the ORF8 gene fragment that has been extended, cut, recombined and/or mutated.

4. The IRES sequence of claim 1, wherein the IRES sequence comprises: the ORF8 gene fragment, and the ORF8 gene fragment is in the upstream segment and/or the downstream segment of SARS-Cov-2.

5. The IRES sequence of claim 4, wherein the IRES sequence comprises the ORF8 gene fragment, the upstream segment of 120bp, and the downstream segment of 14bp, and the IRES sequence is set forth in SEQ ID No. 2.

6. The IRES sequence of claim 2, 4, or 5, wherein the IRES sequence is isolated from the SARS-Cov-2.

7. Use of an IRES sequence according to any of claims 1 to 6 for the expression of a gene of interest.

8. Use of an IRES sequence according to any one of claims 1 to 6 in screening for anti-SARS-Cov-2 drugs.

9. A polycistronic expression vector comprising an IRES sequence according to any of claims 1-6, upstream of the start codon of a gene of interest in said polycistronic expression vector.

10. The polycistronic expression vector of claim 9, wherein the polycistronic expression vector is a dual fluorescent protein expression vector comprising the IRES sequence between the stop codon of the GFP and the start codon of the mCherry, GFP and mCherry.

Technical Field

The application belongs to the technical field of genetic engineering, and particularly relates to an Internal Ribosome Entry Site (IRES) sequence, application of the IRES sequence and a polycistronic expression vector.

Background

Translation (Translation) of eukaryotes is an important stage of gene expression, and refers to a process of synthesizing proteins from genetic information on eukaryotic messenger ribonucleic acids (mrnas) obtained by transcription from deoxyribonucleic acids (DNAs). Eukaryotic translation requires the 5' end cap structure of the mRNA to bind to the ribosome and initiate translation. However, for RNA viruses lacking cap constructs, translation can be initiated by recruiting ribosomes into the viral mRNA based on the cis-acting element IRES sequence that regulates translation in the 5' untranslated region (UTR) of the RNA virus.

The IRES sequence is a nucleotide sequence having a cap structure-independent translation initiation ability, and is capable of recruiting ribosomes into the translation initiation site of viral mRNA with the aid of trans-acting factors. Furthermore, it has been shown that by fusing an IRES sequence to a foreign cDNA, IRES can independently initiate translation. Therefore, the study of IRES sequences is of great significance for gene expression.

Disclosure of Invention

The present application provides an IRES sequence, its use, and a polycistronic expression vector, wherein the IRES sequence has an ability to initiate translation in eukaryotes and can be used in an expression process of a target gene.

In a first aspect, the present application provides an IRES sequence obtained based on the ORF8 gene fragment of SARS-Cov-2, wherein the ORF8 gene fragment is shown in SEQ ID NO. 1.

The IRES sequences provided herein have initial translation capability for eukaryotes and can be applied to the expression process of target genes.

Alternatively, the IRES sequence is as shown in SEQ ID NO. 1.

Optionally, the IRES sequence is a sequence obtained by elongation, excision, recombination and/or mutation of the ORF8 gene fragment.

Optionally, the IRES sequence comprises: the ORF8 gene fragment, and the ORF8 gene fragment is in the upstream segment and/or the downstream segment of SARS-Cov-2.

Optionally, the IRES sequence comprises the ORF8 gene fragment, the upstream segment of 120bp and the downstream segment of 14bp, and the IRES sequence is shown as SEQ ID NO. 2.

Alternatively, the IRES sequence is isolated from the SARS-Cov-2.

In a second aspect, the present application provides the use of an IRES sequence according to the first aspect or any alternative form of the first aspect for the expression of a gene of interest.

In a third aspect, the present application provides a use of an IRES sequence as described in the first aspect or any alternative form of the first aspect in a drug screen against SARS-Cov-2.

In a fourth aspect, the present application provides a polycistronic expression vector comprising an IRES sequence as described in the first aspect or any alternative form of the first aspect, said IRES sequence being located upstream of the start codon of a gene of interest in said polycistronic expression vector.

The polycistronic expression vector provided by the application can synchronously express a target gene contained in the polycistronic expression vector.

Optionally, the polycistronic expression vector is a double-fluorescent protein expression vector, the double-fluorescent protein expression vector includes the IRES sequence, GFP and mCherry, and the IRES sequence is located between a stop codon of the GFP and a start codon of the mCherry.

Drawings

FIG. 1 is a map of a dual fluorescent protein expression vector having an IRES sequence provided in the examples herein;

FIG. 2 is an electrophoretogram of a target vector and a target fragment provided in an embodiment of the present application;

FIG. 3 is a map of a dual fluorescent protein expression vector without an IRES sequence as provided in the examples herein;

FIG. 4 is a fluorescence plot of HEK-293T cells at 48 hours post transfection provided in the examples of the present application.

Detailed Description

In this application, the term "and/or" describes an association relationship of associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a is present alone, A and B are present simultaneously, and B is present alone. Wherein A and B can be singular or plural. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.

The terminology used in the embodiments of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in the examples of this application and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Reference throughout this specification to "one embodiment" or "some embodiments," or the like, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in one or more embodiments of the present application. Thus, appearances of the phrases "in one embodiment," "in some embodiments," "in other embodiments," or the like, in various places throughout this specification are not necessarily all referring to the same embodiment, but rather "one or more but not all embodiments" unless specifically stated otherwise. The terms "comprising," "including," "having," and variations thereof mean "including, but not limited to," unless expressly specified otherwise.

The content of the related components mentioned in the description of the embodiments of the present application may not only refer to the specific content of each component, but also represent the proportional relationship of the content among the components, and therefore, the content of the related components is scaled up or down within the scope disclosed in the description of the embodiments of the present application as long as it is scaled up or down according to the description of the embodiments of the present application. Specifically, the volume, the number of nucleotides, the rotation speed, and the time described in the specification of the examples of the present application may be measured units known in the art of genetic engineering, such as μ L, mL, bp, kbp, rpm, s, min, and h.

The IRES sequences, the use of the IRES sequences and polycistronic expression vectors provided herein are exemplified below with reference to specific examples. It should be understood that the specific embodiments described herein are merely illustrative of the principles of the present application and are not intended to limit the present application.

The IRES sequence provided herein is derived based on the ORF8 gene fragment in SARS-Cov-2. Wherein, the ORF8 gene fragment in SARS-Cov-2 is shown in SEQ ID NO. 1.

SEQ ID NO.1：

5’-ATGAAATTTCTTGTTTTCTTAGGAATCATCACAACTGTAGCTGCAT TTCACCAAGAATGTAGTTTACAGTCATGTACTCAACATCAACCATATGTA GTTGATGACCCGTGTCCTATTCACTTCTATTCTAAATGGTATATTAGAGT AGGAGCTAGAAAATCAGCACCTTTAATTGAATTGTGCGTGGATGAGGCT GGTTCTAAATCACCCATTCAGTACATCGATATCGGTAATTATACAGTTTC CTGTTTACCTTTTACAATTAATTGCCAGGAACCTAAATTGGGTAGTCTTG TAGTGCGTTGTTCGTTCTATGAAGACTTTTTAGAGTATCATGACGTTCGT GTTGTTTTAGATTTCATCTAA-3’。

SARS-Cov-2, which may also be referred to as a novel coronavirus, is a positive-stranded single-stranded RNA virus with an outer membrane. Coronaviruses belong to the genus coronaviruses of the family coronaviridae of the order Nervirae, and the genus coronaviruses can be subdivided into 4 genera, alpha, beta, gamma and delta coronaviruses, respectively. Among them, SARS-Cov-2 belongs to the genus β coronavirus. SARS-Cov-2 is spherical or elliptical, the diameter of virus particle is 60-200 nm, and the average diameter is 100 nm. The genetic material of SARS-Cov-2 is by far the largest of all RNA viruses. SARS-Cov-2 can infect human, mouse, pig, cat, dog and bird vertebrates, is the 7 th discovered coronavirus capable of infecting human, and has the characteristics of high transmission, long incubation period, high mutation rate and the like.

By cloning and experiment of ORF8 gene fragment of SARS-Cov-2, it can be determined that ORF8 gene fragment has IRES function, i.e. IRES sequence can be obtained based on ORF8 gene fragment, and the IRES sequence is applied in the expression process of target gene.

In the present embodiment, the IRES sequence may be the full-length sequence of ORF8 gene fragment originally, i.e., the IRES sequence is shown in SEQ ID NO. 1. It may also be a sequence obtained by elongation, excision, recombination and/or mutation of the ORF8 gene fragment.

Illustratively, when the ORF8 gene fragment is cleaved, a part of the sequence of the segment can be cleaved at the end of the ORF8 gene fragment to obtain an IRES sequence.

When the ORF8 gene fragment is extended, a partial sequence may be spliced to the end of the ORF8 gene fragment. For example, the ORF8 gene fragment can be extended by the upstream and/or downstream segment of the ORF8 gene fragment in SARS-Cov-2 to obtain an IRES sequence. I.e., the IRES sequence comprises: ORF8 gene fragment, and ORF8 gene fragment in upstream and/or downstream segments of SARS-Cov-2.

For example, as shown in SEQ ID NO.2, the IRES sequence provided herein may include a fragment of ORF8 gene, an upstream segment of 120bp and a downstream segment of 14 bp.

SEQ ID NO.2：

5’-ATTGACTTCTATTTGTGCTTTTTAGCCTTTCTGCTATTCCTTGTTTT AATTATGCTTATTATCTTTTGGTTCTCACTTGAACTGCAAGATCATAATGA AACTTGTCACGCCTAAACGAACATGAAATTTCTTGTTTTCTTAGGAATCA TCACAACTGTAGCTGCATTTCACCAAGAATGTAGTTTACAGTCATGTACT CAACATCAACCATATGTAGTTGATGACCCGTGTCCTATTCACTTCTATTCT AAATGGTATATTAGAGTAGGAGCTAGAAAATCAGCACCTTTAATTGAAT TGTGCGTGGATGAGGCTGGTTCTAAATCACCCATTCAGTACATCGATATC GGTAATTATACAGTTTCCTGTTTACCTTTTACAATTAATTGCCAGGAACCT AAATTGGGTAGTCTTGTAGTGCGTTGTTCGTTCTATGAAGACTTTTTAGA GTATCATGACGTTCGTGTTGTTTTAGATTTCATCTAAACGAACAAACTAA A-3’。

It should be noted that the length of the cut or extended ORF8 gene fragment can be designed based on practical requirements in the case of ensuring the IRES function of the ORF8 gene fragment, and the present application is not limited thereto.

Alternatively, in addition to the above-described modification modes of cutting and elongation, the ORF8 gene fragment may be modified by recombination and/or mutation to obtain an IRES sequence. For example, for the purpose of enhancing the IRES function of the ORF8 gene fragment, the ORF8 gene fragment is recombined and/or mutated to obtain an IRES sequence. Of course, in the case of ensuring the IRES function of the ORF8 gene fragment, the specific manner of recombination and/or mutation of the ORF8 gene fragment can also be designed based on practical requirements, and the present application is not limited thereto.

It is understood that when the ORF8 gene fragment is modified, the ORF8 gene fragment may be cut or extended, and recombined and/or mutated. For example, recombination and/or mutation of SEQ ID NO.2 results in an IRES sequence.

Based on actual requirements, the IRES sequence provided by the application can be directly separated from SARS-Cov-2, and can also be artificially synthesized.

The IRES sequences provided herein can be used for expression of a gene of interest. Based on the IRES sequence, a polycistronic expression vector can be constructed, and the synchronous expression of multi-purpose genes can be realized.

The polycistronic expression vector refers to a vector capable of synchronously expressing two or more target genes. Among them, a polycistronic expression vector containing two genes of interest is also generally called a bicistronic expression vector.

The present application provides a polycistronic expression vector comprising an IRES sequence as provided herein upstream of the initiation codon of a gene of interest in the polycistronic expression vector.

The following description will be given by taking a dual-fluorescent protein expression vector as an example, and the polycistronic expression vector, the construction of the polycistronic expression vector and the application of the IRES sequence in the expression of a target gene provided by the present application will be exemplified.

As shown in fig. 1, the dual fluorescent protein expression vector provided by the present application includes a human Cytomegalovirus (CMV) promoter, a Green Fluorescent Protein (GFP), an IRES sequence, a red fluorescent protein (mCherry), and a Bovine Growth Hormone (BGH) tailing signal. Wherein the IRES sequence is located between the stop codon of GFP and the start codon of mCherry. The stop codon of GFP is TAA codon, and the start codon is ATG codon.

Illustratively, the IRES sequence provided by the present application is assumed to be the sequence shown in SEQ ID NO.2, and the target vector is pcDNA3.1-EGFP vector constructed based on pcDNA3.1(+/-) vector. The construction process of the double-fluorescent protein expression vector can be as follows:

step 1, preparing a target carrier.

The pcDNA3.1-EGFP vector plasmid was digested with restriction endonuclease (Xba I) and incubated at 37 ℃ for 3 h. And then carrying out agarose gel electrophoresis detection on the enzyme digestion product, and recovering the linearized vector framework to obtain the target vector. Illustratively, the electrophoretogram of pcDNA3.1-EGFP before and after the cleavage is shown in FIG. 2 (b), and the size of the objective vector after the cleavage is about 6 kb.

And 2, preparing the target fragment.

The target fragment comprises an IRES sequence fragment and a target gene mCherry fragment.

Wherein, when preparing the IRES sequence segment, the plasmid carrying the sequence shown in SEQ ID NO.2 is used as a template to carry out Polymerase Chain Reaction (PCR) amplification. The PCR procedure was: first preheated at 95 deg.C for 30 s. Then 30 amplification cycles were performed, the reaction conditions for each cycle being: denaturation was first carried out at 95 ℃ for 15s, followed by annealing at 50 ℃ for 15s and finally extension at 72 ℃ for 1 min. After the circulation is finished, extension is carried out for 5min at 72 ℃ to obtain an amplification product of the IRES sequence. And homologous sequences of pcDNA3.1-EGFP vector and downstream mCherry fragment are added at the 5' ends of the forward primer and the reverse primer of the amplification product of the IRES sequence to obtain the IRES sequence fragment. Illustratively, the electrophoretogram of the IRES sequence fragment can be as shown in FIG. 2 (a), and the size of the IRES sequence fragment is about 541 bp.

When the mCherry fragment is prepared, PCR amplification is carried out by taking a plasmid carrying the mCherry gene as a template. The PCR procedure was: first preheated at 95 deg.C for 30 s. 33 amplification cycles were then performed, the reaction conditions for each cycle being: denaturation was first carried out at 95 ℃ for 15s, followed by annealing at 50 ℃ for 15s and finally extension at 72 ℃ for 1 min. After the circulation is finished, extension is carried out for 5min at 72 ℃ to obtain an amplification product of mCherry. The 5' end of the reverse primer of the amplification product of mCherry is added with the homologous sequence of pcDNA3.1-EGFP vector to obtain an mCherry fragment. Illustratively, the electrophoretogram of the mCherry fragment may be as shown in fig. 2 (a), with the size of the mCherry fragment being about 732 bp.

And 3, connecting the target fragment and the target vector.

In one example, the objective fragment and the objective vector are incubated for 15min at 50 ℃ by using a recombination kit (cat # C115) of Novozan biotechnology, and homologous recombination and ligation are performed to obtain a homologous recombination product. The homologous recombination product was then transformed into E.coli DH 5. alpha. competent cells. The conversion step can be as follows:

s1, 50. mu.L of DH 5. alpha. competent cells were thawed on ice.

S2, adding 5 mu L of homologous recombination product into the thawed DH5 alpha competent cells, and uniformly mixing to obtain a mixed product.

S3, the mixed product is ice-cooled for 30min and then heat shocked for 90S at 42 ℃. After the heat shock was finished, the ice bath was further carried out for 5 min. Then, 1mL of non-resistant LB (Luria-Bertani) medium was added thereto, and the mixture was cultured for 1 hour with resuscitation at 37 ℃ and 220rpm, to obtain a resuscitated culture. During the culture, LB plates containing antibiotics were left at room temperature for further use.

S4, 200. mu.L of the resuscitative culture was spread on LB plates containing antibiotics and cultured in an inverted state at 37 ℃ for about 12 hours. Then selecting a sample from the cultured colony for sequencing to obtain a clone sequence with a correct sequencing result. The selected sample can be sent to a gene sequencing company based on experimental needs, and the correctness of the cultured clone sequence is verified by the gene sequencing company.

And 4, preparing a plasmid to obtain the dual-fluorescent protein expression vector.

After obtaining the correct cloning sequence, the plasmid preparation can be carried out by adopting an endotoxin-free plasmid extraction kit to obtain the dual-fluorescent protein expression vector containing the IRES sequence. For example, a kit for producing a plasmid with a small quantity of a meiji low endotoxin plasmid (cat # P1112-02) can be selected, and the specific procedures can be performed according to the instructions of the kit.

It is worth mentioning that, in the above process of constructing the dual-fluorescent protein expression vector, the target vector, the endonuclease, the recombination kit and the plasmid extraction kit are all exemplified schemes. At present, a plurality of products available on the market can be selected based on actual experiment requirements, and the application is not limited.

After the double-fluorescent protein expression vector is constructed, the gene expression function of the double-fluorescent protein expression vector can be further verified.

In one embodiment, three experimental groups may be set, including a blank control group (Mock), a negative control group (Vector), and a positive control group (IRES).

The blank control group (Mock) is a control group of untransfected expression vectors, the negative control group is a control group of transfected double-fluorescent protein expression vectors without IRES sequences, and the positive control group is an experimental group of transfected double-fluorescent protein expression vectors containing IRES sequences (namely the double-fluorescent protein expression vectors provided by the application).

The dual fluorescent protein expression vector without IRES sequence can be shown in fig. 3, and includes GFP, IRES sequence, mCherry and BGH tailed signal. In this case, an IRES sequence was not inserted between the stop codon of GFP and the start codon of mCherry. The dual-fluorescent protein expression vector without the IRES sequence does not need to prepare IRES sequence fragments in the construction process, and homologous sequences of a target vector need to be added to the 5' ends of a reverse primer and a forward primer of an amplification product of mCherry when the mCherry fragment is prepared, so that the mCherry fragment and the target vector can be conveniently subjected to homologous recombination connection. The remaining process is similar to that of a dual fluorescent protein expression vector containing an IRES sequence.

In the functional verification, the negative control group and the positive control group need to be transfected with cells first, so that the expression of GFP and mCherry can be observed by the fluorescence phenomenon. In the cell transfection of expression vectors, appropriate well plates, flasks or dishes are first selected and inoculated 24h in advance. For example, 12-well HEK-293T cells are selected for plating. Cell transfection was performed when the conjugation rate reached about 80%.

Illustratively, cell transfection can be performed using electroporation, or can be performed using commercially available transfection reagents. For example, electrotransformation can be carried out using a Celetrix electrotransfer instrument, a commercial transfection assayThe preparation can be Lipo8000 of Biyunyan^TMA reagent (cat # C0533FT), or Lipofectamine 3000 reagent (cat # L3000015) by Saimer Feishal. If cell transfection is performed using commercial transfection reagents, the amounts of reagents may be used in reference to the amounts of reagents described in the instructions for commercial transfection reagents for 12-well plates.

After cell transfection was performed on the negative control group and the positive control group, fluorescence observation was performed after 48 hours. As shown in fig. 4, normally, the blank control group was not transfected with vectors containing GFP and mCherry, and thus green fluorescence and red fluorescence were not observed in the blank control group.

In the negative control group, since the GFP is linked to the CMV promoter, the 3' UTR provided by the CMV promoter will direct the ribosome to enter into initiation of translation, thereby allowing GFP expression, and green fluorescence can be observed. Then, ribosome translated to the stop codon of GFP and detached from mRNA, so that the downstream gene mCherry of GFP could not be translated and red fluorescence could not be observed. Then, under normal conditions, green fluorescence can be finally observed after the superposition (merge) of the expression results of GFP and mCherry in the negative control group.

In the positive control group, since the IRES sequence is present between the stop codon of GFP and the start codon of mCherry, even if the ribosome is separated from mRNA after passing through the stop codon of GFP, the IRES sequence can recruit the ribosome again to enter the gene mCherry downstream of the IRES sequence and initiate translation, so that mCherry is expressed and red fluorescence can be observed. Then, under normal conditions, when the expression results of GFP and mCherry in the positive control group are superimposed (merged), a mixed light of green fluorescence and red fluorescence can be finally observed.

According to observation, fluorescence expression of three experimental groups at 50 μm under a microscope scale is shown in the merged column in fig. 4, and all the results are normally shown, namely the experimental results are shown in the following table 1:

TABLE 1

Experiments prove that the IRES sequence provided by the application has high-efficiency non-cap structure-dependent initial translation capability, can be applied to the expression process of a target gene, and improves the expression efficiency of the target gene.

In addition, because the IRES sequence is obtained based on ORF8 gene fragment in SARS-Cov-2, the IRES sequence can also be used as a drug target to screen drugs against SARS-Cov-2. For example, the bi-fluorescent protein expression vector shown in fig. 1 is used, the drug molecules to be screened are added after the bi-fluorescent protein expression vector transfects cells, and then the ratio of the red fluorescence intensity to the green fluorescence intensity is analyzed, so that the inhibition effect of the corresponding drug molecules on the IRES function can be rapidly known. This IRES is responsible for the translation of Nucleocapsid protein (Nucleocapsid protein), which is one of the core structural proteins of SARS-Cov-2, at least during the initial stages of virus invasion in SARS-Cov-2. In this way, it is expected that effective IRES inhibitors can be screened, which inhibit the function of SARS-Cov-2 by inhibiting the translation of the nucleocapsid protein of SARS-Cov-2.

The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

SEQUENCE LISTING

<110> IRES sequence, use of IRES sequence and polycistronic expression vector

<120> Shenzhen advanced technology research institute of Chinese academy of sciences

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 366

<212> DNA

<213> SARS-Cov-2

<400> 1

atgaaatttc ttgttttctt aggaatcatc acaactgtag ctgcatttca ccaagaatgt 60

agtttacagt catgtactca acatcaacca tatgtagttg atgacccgtg tcctattcac 120

ttctattcta aatggtatat tagagtagga gctagaaaat cagcaccttt aattgaattg 180

tgcgtggatg aggctggttc taaatcaccc attcagtaca tcgatatcgg taattataca 240

gtttcctgtt taccttttac aattaattgc caggaaccta aattgggtag tcttgtagtg 300

cgttgttcgt tctatgaaga ctttttagag tatcatgacg ttcgtgttgt tttagatttc 360

atctaa 366

<210> 2

<211> 500

<212> DNA

<213> SARS-Cov-2

<400> 2

attgacttct atttgtgctt tttagccttt ctgctattcc ttgttttaat tatgcttatt 60

atcttttggt tctcacttga actgcaagat cataatgaaa cttgtcacgc ctaaacgaac 120

atgaaatttc ttgttttctt aggaatcatc acaactgtag ctgcatttca ccaagaatgt 180

agtttacagt catgtactca acatcaacca tatgtagttg atgacccgtg tcctattcac 240

ttctattcta aatggtatat tagagtagga gctagaaaat cagcaccttt aattgaattg 300

tgcgtggatg aggctggttc taaatcaccc attcagtaca tcgatatcgg taattataca 360

gtttcctgtt taccttttac aattaattgc caggaaccta aattgggtag tcttgtagtg 420

cgttgttcgt tctatgaaga ctttttagag tatcatgacg ttcgtgttgt tttagatttc 480

atctaaacga acaaactaaa 500