CN112553240A - Recombinant expression vector system, recombinant engineering bacterium, preparation method and application thereof - Google Patents

Recombinant expression vector system, recombinant engineering bacterium, preparation method and application thereof Download PDF

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CN112553240A
CN112553240A CN201911174901.1A CN201911174901A CN112553240A CN 112553240 A CN112553240 A CN 112553240A CN 201911174901 A CN201911174901 A CN 201911174901A CN 112553240 A CN112553240 A CN 112553240A
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hpv
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丁珊
潘婷
姬美彤
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Shanghai Bowei Biotechnology Co ltd
Chongqing Bloomer Bio Pharmaceutical Co ltd
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Abstract

The invention provides a recombinant expression vector system, a recombinant engineering bacterium, a preparation method and application thereof, wherein the recombinant expression vector system at least comprises two recombinant expression vectors, each recombinant expression vector contains one or more nucleic acid expression cassettes, each nucleic acid expression cassette contains a target gene and a promoter operably connected with the target gene, each target gene is respectively and independently selected from one of HPV L, HPV L or HPV L, and all the target genes are different from each other. The recombinant expression vector system is transformed into engineering bacteria to obtain recombinant engineering bacteria. The recombinant engineering bacteria can express a plurality of HPV L1 proteins at the same time and is used for preparing HPV vaccines.

Description

Recombinant expression vector system, recombinant engineering bacterium, preparation method and application thereof
Technical Field
The invention relates to a bioengineering technology, in particular to a recombinant expression vector system, recombinant engineering bacteria, a preparation method and application thereof.
Background
Human Papilloma Virus (HPV) mainly infects Human epithelial cells to cause epithelial cell damage, thereby causing a series of diseases including genital warts, cervical cancer and the like, and in recent years, more and more researches show that HPV infection can also cause other anogenital related cancers, such as vaginal cancer, anal cancer, penile cancer and the like, and even is related to oropharyngeal cancer. The cervical cancer, which is the third largest female cancer in the world and has the highest attention and the most clear HPV pathogenic mechanism, takes 25 million people every year, and more than 99 percent of cervical cancers are caused by HPV infection.
The major transmission pathway of HPV is sexual transmission, and its main mechanism into the human body is associated with micro-damage of epithelial cells.
At present, HPV of at least two hundred genotypes can be classified into high-risk type and low-risk type according to whether the HPV has carcinogenicity after infection or not, wherein the high-risk type HPV is mainly related to various malignant tumors, and the low-risk type HPV mainly causes benign tumors such as condyloma acuminatum and respiratory system tumors. Although there are some differences in the epidemiological data of HPV in different countries and regions, the high-risk HPV types currently having the most prevalent worldwide are HPV16 and HPV18, which are associated with 70% of cervical cancers, and the other high-risk HPV types having higher prevalence are HPV31, HPV33, HPV45, HPV52, HPV58, etc. The main prevalent types of low risk HPV are HPV6 and HPV 11. Based on the above epidemiological trends, three HPV preventive vaccines are currently on the market, namely, Kurarin Schker
Figure BDA0002289704740000011
(HPV16/18), Merck' s
Figure BDA0002289704740000012
(HPV6/11/16/18) and
Figure BDA0002289704740000013
9(HPV6/11/16/18/31/33/45/52/58), defaultThe two vaccines can simultaneously prevent high-risk and low-risk infections, and greatly reduce the incidence of cervical cancer and condyloma acuminatum caused by corresponding types.
The total length of the HPV double-stranded DNA virus is 7900-8000 bp, and 7 non-structural proteins E1-E7 and 2 capsid proteins L1 and L2 are encoded. Wherein the protein L1 is the main capsid protein, and 360 copies of the protein L1 form 72 pentamers to form virus particles with T-7; the L2 protein is a minor capsid protein and does not assume the function of the structural backbone, but is involved in viral DNA release following receptor binding. The main neutralizing epitope of HPV exists on the L1 protein, and the neutralizing antibody based on the neutralizing epitope can effectively prevent HPV infection.
Studies based on various recombinant expression systems have demonstrated that the expression of the L1 protein alone results in the formation of Virus-like particles (VLPs) with conformation close to that of the native virions, these HPV-VLPs retaining the neutralizing epitopes of the native HPV, while lacking DNA necessary for viral replication, the safety and reliability of which have been conferred by HPV prophylactic vaccines based on these VLPs
Figure BDA0002289704740000021
And
Figure BDA0002289704740000022
9 was well documented.
Figure BDA0002289704740000023
Respectively expressing HPV16L1 and HPV18L1 in a baculovirus system, self-assembling recombinant expression products into VLPs, respectively purifying and preparing the two recombinant expression products, and uniformly mixing to prepare a vaccine preparation;
Figure BDA0002289704740000024
the four recombinant expression products are respectively purified, prepared and then uniformly mixed to form the final product. Second generation HPV prophylactic vaccine by Merck
Figure BDA0002289704740000025
9 is at
Figure BDA0002289704740000026
On the basis, 5 high-risk HPV types, namely HPV31, HPV33, HPV45, HPV52 and HPV58 are added, and the original 4-valent Gardasil is added to form a 9-valent vaccine with wider protection range. But whether it is 2 valent
Figure BDA0002289704740000027
Of value 4
Figure BDA0002289704740000028
And the latest 9 prices
Figure BDA0002289704740000029
Figure BDA0002289704740000029
9, the vaccine needs to prepare a set of process for each valence type separately, and the multivalent vaccine preparation is obtained after monovalent semi-finished preparations are mixed uniformly, so that the process is complex and tedious, and corresponding investment needs to be added when each valence type is added, the investment of equipment and personnel for the processes including preparation of engineering strains, corresponding fermentation, purification, preparation and the like is high in cost and poor in controllability.
Disclosure of Invention
In order to achieve the above and other related objects, the present invention provides a recombinant expression vector system, a recombinant engineering bacterium, and a preparation method and use thereof, which are used for solving the problems of complex and tedious process, high cost and poor controllability of HPV antibody preparation in the prior art.
The first aspect of the present invention provides a recombinant expression vector system, which comprises at least two recombinant expression vectors, each of the recombinant expression vectors comprises one or more nucleic acid expression cassettes, each of the nucleic acid expression cassettes comprises a target gene and a promoter operably linked to the target gene, each of the target genes is independently selected from one of HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV 31L 1, HPV 33L 1, HPV 35L 1, HPV 39L 1, HPV 45L 1, HPV 51L 1, HPV52L1, HPV53L1, HPV 56L 1, HPV 58L 1, HPV 59L 1, HPV 66L 1 or HPV68L1, and all the target genes are different from each other.
The plurality may be two. The plurality may be three. The plurality may be four.
In one embodiment, the HPV6L1 is as set forth in SEQ ID NO:1 is shown.
In one embodiment, the HPV11L1 is as set forth in SEQ ID NO:2, respectively.
In one embodiment, the HPV16L1 is as set forth in SEQ ID NO: 3, respectively.
In one embodiment, the HPV18L1 is as set forth in SEQ ID NO: 4, respectively.
In one embodiment, the sequence of HPV 31L 1 is AEI 60965.1.
In one embodiment, the sequence of HPV 33L 1 is AAA 46964.1.
In one embodiment, the sequence of HPV 35L 1 is ARQ 82609.1.
In one embodiment, the sequence of HPV 39L 1 is AGU 90477.1.
In one embodiment, the sequence of HPV 45L 1 is CAA 52578.1.
In one embodiment, the sequence of HPV 51L 1 is AMK 51256.1.
In one embodiment, the sequence of HPV52L1 is ACX 32362.1.
In one embodiment, the sequence of HPV53L1 is ABU 54083.1.
In one embodiment, the sequence of HPV 56L 1 is ABO 76795.1.
In one embodiment, the sequence of HPV 58L 1 is ACL 12325.1.
In one embodiment, the sequence of HPV 59L 1 is ALT 54908.1.
In one embodiment, the sequence of HPV 66L 1 is ABO 76872.1.
In one embodiment, the sequence of HPV68L1 is AGU 90680.1.
HPV6L1 DNA coding sequence (SEQ ID NO: 1)
atgtggagaccatctgactcgactgtctacgttcctccacctaacccagtctctaaggttgtggctaccgacgcctacgtgactagaaccaacatcttctaccacgcatcgtcctctagattgctggccgttggacatccttacttctccatcaagagagccaacaaaaccgtcgtgccaaaggtttctggctaccagtatagagtcttcaaggtggttctgcctgaccctaacaagtttgccctgccagactcgtccttgttcgatcctacgactcaaagactcgtttgggcctgcactggtcttgaggttggcagaggacagccactcggagtcggcgtttctggtcatcctttcctgaacaagtacgacgatgttgagaactctggttccggaggcaatcctggacaggacaacagagtcaatgttggcatggactacaagcaaactcagctctgtatggtgggatgcgcacctccactgggtgagcattggggaaaaggcaagcagtgcaccaacactccagttcaggcaggtgactgccctccattggagctgattacctcggtcatccaggacggtgatatggttgacaccggattcggtgccatgaacttcgctgacctgcagaccaacaagtccgatgtgccaatcgacatctgcggaacgacctgtaaataccctgactacttgcagatggcagccgatccatacggcgacagactcttcttctttctgagaaaggagcagatgtttgctagacactttttcaacagagccggagaggtcggtgagcctgttccagacaccctgatcattaagggctcgggaaacagaacttctgtcggctcctcgatctacgtgaacaccccttccggttcgttggtgtcgtccgaggctcaactgttcaacaagccatactggctccagaaagcccaaggtcacaacaatggcatctgctggggtaaccagctgttcgttactgtcgtggacaccacgagatcgaccaacatgactctgtgtgcctccgttaccacatcttcgacttacaccaactccgactataaggagtacatgagacacgttgaagagtacgacttgcaattcatctttcagctctgctcgattaccctgtccgcagaagtcatggcctacatccacactatgaatccatcggtgcttgaggactggaactttggcttgtccccacctccaaacggaactctggaagacacctatagatacgtgcagtcgcaagccatcacgtgccagaagccaacccctgagaaggaaaagccagatccttacaagaacctgtccttctgggaggtcaatctcaaggagaagttctcgtccgaactggaccagtacccattgggtagaaagttcctgctccagtctggataccgtggtagatcgtccatcagaaccggagtgaagagaccagcagtttccaaggcttctgccgcacctaagcgtaaaagagccaagaccaagagataatag
HPV11L1 DNA coding sequence (SEQ ID NO: 2)
atgtggagaccatctgactcgaccgtctacgttcctccacctaacccagtctccaaagtggttgctactgatgcctacgtcaagagaaccaacatcttctaccacgcctcttcgtccagattgctggcagttggacatccttactattccatcaagaaagttaacaagaccgtggtccctaaggtttcgggctatcagtacagagtgttcaaggtcgttctgccagaccctaacaagtttgccttgccagattcctcgctgttcgaccctaccactcaaagactggtttgggcttgcacgggtttggaagtgggcagaggacaaccactgggtgtcggagtttccggccatccattgctgaacaagtacgacgatgttgagaactcgggtggatacggtggcaatccaggacaagacaacagagtgaatgtcggcatggactacaagcaaacccagctgtgcatggttggttgtgcaccacctctgggtgaacattggggaaagggcactcagtgctccaacacctctgttcagaatggtgactgtcctccactggagctgatcacctcggttattcaggatggagacatggtggacactggcttcggtgcaatgaactttgccgacttgcagaccaacaagtccgatgttccattggacatttgcggcactgtgtgcaaatatcctgactacctgcagatggctgcagatccatacggtgacagactcttcttctacctgagaaaggagcagatgttcgccagacacttctttaacagagctggcaccgttggtgagcctgttccagatgacctgttggtgaagggtggaaacaatagatcctctgttgcctcgtccatctacgtccacaccccatcgggctctctggtgtcctcggaggcccaactgttcaacaagccttactggctccagaaggcccaaggacacaacaatggcatctgctggggaaaccacctgttcgtcaccgttgtggacacgactagatccaccaacatgactttgtgtgcctctgtttccaagtcggctacctacacgaactcggactataaggagtacatgagacatgttgaagagttcgacctgcagtttatcttccagctctgctccattaccttgtcggcagaagtgatggcctacatccacacgatgaacccatccgtcttggaagactggaacttcggtctgtctccacctccaaatggcaccctggaagacacgtacagatatgtgcagtcgcaagccatcacctgtcagaaaccaactcctgaaaaggagaaacaggacccatacaaggatatgtcgttctgggaggtcaacctgaaggagaagttctcttcggaactggaccagttcccattgggaagaaagttcctgctccagtctggttatagaggaagaacctccgcaagaactggcatcaagagacctgccgtgtcgaagccatccaccgctcctaagagaaagagaacgaagaccaaaaagtaatag
HPV16L1 DNA coding sequence (SEQ ID NO: 3)
atgtctctttggttgccatccgaggctactgtttacttgccacctgtcccagtttctaaggtcgtgtcaactgatgaatacgttgctagaaccaacatctattaccacgctggaacttctagattgcttgctgttggtcatccatacttccctatcaagaaacctaacaataacaagatccttgttccaaaggtctctggattgcagtacagagttttcagaatccacttgcctgacccaaacaagttcggttttcctgacacctccttctacaacccagatactcaaagattggtgtgggcctgtgttggtgtcgaggttggacgtggtcaaccattgggagttggtatttctggacatcctttgcttaacaagttggatgacactgaaaatgcttctgcctacgcagccaacgctggtgttgacaacagagagtgtatctctatggattacaaacagacccaattgtgtcttattggttgcaagccacctattggtgaacactggggtaagggatctccatgtaccaacgttgctgtcaatccaggtgattgtcctccattggagcttatcaacactgttattcaagatggtgacatggttgatactggatttggtgctatggacttcactaccttgcaagctaacaagtccgaggttccattggacatttgtacttctatctgcaagtatccagactacattaagatggtctctgaaccatacggtgactccttgtttttctaccttcgtagagagcaaatgtttgttagacacttgttcaacagagctggtgccgttggtgagaatgtcccagacgatttgtacatcaagggatctggttccactgcaaacttggcctcttccaactacttcccaactccttcaggttctatggttacctccgatgctcaaatcttcaacaagccttactggttgcaaagagcacagggtcacaacaatggaatttgttggggtaaccaacttttcgttactgtcgttgataccacaagatccaccaacatgtccttgtgtgctgccatttctacttcagaaaccacatacaagaatactaacttcaaggagtaccttagacatggtgaggaatacgacttgcagttcatctttcaattgtgcaagattaccttgactgctgacgttatgacctacatccactctatgaactccactatccttgaagattggaacttcggattgcaaccacctccaggtggaaccttggaagatacttacagatttgtcacatcccaagccattgcttgtcagaagcacactcctccagctcctaaggaagatccacttaagaaatacaccttctgggaagtcaacttgaaagagaagttctctgctgaccttgatcagtttcctttgggtagaaagttcttacttcaagctggattgaaggccaaaccaaagttcaccttgggtaaaagaaaggctactccaaccacatcatctacttccacaaccgccaaacgtaagaagagaaaactgtaatag
HPV18L1 DNA coding sequence (SEQ ID NO: 4)
atggctctgtggagaccatctgacaacaccgtctacttgccacctccatctgttgccagagtcgttaacaccgacgattacgtcactagaacatccatcttctaccacgctggttcctctagattgcttactgttggtaatccatacttcagagttccagctggtggaggtaataagcaagacattcctaaggtttctgcttaccaatatagagtctttagagttcagttgcctgacccaaacaagttcggtttgcctgatacttctatctacaacccagagactcaaagattggtttgggcctgtgctggtgtcgagatcggtagaggacaacctttgggagttggtctttctggacacccattctacaacaagttggatgacactgaatcttcacatgctgccacttccaatgtttctgaggatgttcgtgacaacgtctctgttgattacaagcaaactcagttgtgtatccttggatgtgcccctgctattggtgaacactgggctaaaggtactgcctgtaagtccagaccattgtctcaaggtgactgcccacctttggagcttaagaacactgttttggaagatggtgacatggttgatactggatatggtgctatggacttctctaccctgcaggatactaagtgtgaggttccattggacatctgtcaatctatttgtaagtaccctgattatcttcagatgtctgctgacccatacggtgattctatgttcttttgcttgagacgtgagcaacttttcgctagacacttttggaacagagcaggtactatgggtgacaccgttccacaatctttgtatatcaaaggaactggtatgagagcttctcctggatcctgtgtttactcaccatctccttccggttctattgttacctcagactctcaattgtttaacaagccatactggttacacaaggctcagggtcataacaatggtgtttgctggcacaaccaattgttcgttactgtcgttgacaccactagatccaccaatttgacaatttgtgcttctactcagtcccctgttccaggtcaatatgatgctaccaaattcaagcaatactctagacacgttgaggaatatgacttgcagtttatcttccaattgtgtactattaccttgactgccgatgttatgtcttacattcattccatgaactcttcaatcttggaggattggaactttggtgttccaccacctccaacaacttctttggttgacacctacagattcgtccaatctgttgctattacctgtcagaaggatgccgcaccagctgaaaacaaggatccatacgacaagttgaagttttggaatgtcgatttgaaggagaagttctcccttgacttggatcaatacccacttggtagaaagttcttggttcaggctggtttgagacgtaagccaactatcggtccaagaaaacgttctgctccatcagccactacctcttccaaacctgctaagagagttcgtgtcagagctagaaagtaatag
Further, the promoter in each of said nucleic acid expression cassettes is selected from the group consisting of MOX. The sequence of the MOX is shown as SEQ ID NO: 5, respectively.
Further, the termination sequence within each of said nucleic acid expression cassettes is selected from the group consisting of AOXTT. The sequence of the AOXTT is shown in SEQ ID NO: and 6.
MOX DNA sequence (SEQ ID NO: 5)
gtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaa
AOXTT DNA sequence (SEQ ID NO: 6)
gtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatc
Further, each of the recombinant expression vectors further comprises one or more nucleic acid expression cassettes containing a selectable marker gene.
Further, the selectable marker gene is selected from antibiotic resistance genes. The antibiotic resistance gene may be Zeocin resistance gene, G418 resistance gene or other currently commonly used antibiotic resistance genes.
Further, the promoter is selected from yeast expression fragments. The promoter gene may be selected from MOX, GAPDH, DAS or other yeast available promoters.
Further, the recombinant expression vector is obtained by inserting a gene of interest into a plasmid. For example, the plasmid may be pPICZB.
Further, the recombinant expression vector system comprises two recombinant expression vectors, each recombinant expression vector comprises two nucleic acid expression cassettes, target genes of the two nucleic acid expression cassettes are respectively and independently selected from one of HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV 31L 1, HPV 33L 1, HPV 35L 1, HPV 39L 1, HPV 45L 1, HPV 51L 1, HPV52L1, HPV53L1, HPV 56L 1, HPV 58L 1, HPV 59L 1, HPV 66L 1 or HPV68L1, and the selected target genes are different from each other.
In a second aspect, the invention provides a recombinant engineered bacterium comprising a recombinant expression vector system as described above.
Further, the recombinant engineering bacteria are yeasts.
Further, the yeast is selected from any one or more of saccharomyces cerevisiae (saccharomyces cerevisiae), Hansenula polymorpha (Hansenula polymorpha), pichia pastoris (Pichiapastoris), Kluyveromyces fragilis (Kluyveromyces fragilis), Kluyveromyces lactis (Kluyveromyces lactis), or Schizosaccharomyces pombe (Schizosaccharomyces pombe).
The third aspect of the present invention provides a method for preparing the above recombinant expression vector system, wherein the method is obtained by transforming an original plasmid, and specifically comprises the following steps: respectively replacing the promoter and the termination sequence of the two original plasmids with the promoter and the termination sequence of the target gene; then the target genes are respectively inserted between the promoter and the termination sequence.
Further, the method comprises the following steps: the plasmid pPICZB is obtained by transformation, and specifically comprises the following steps:
(1) construction of plasmid pMMZ: respectively replacing a promoter and a termination sequence in the plasmid pPICZB by using a first promoter MOX insert and a first termination sequence AOXTT to obtain a plasmid pMOXZ-1; replacing a promoter and a termination sequence in the plasmid pPICZB with a second promoter MOX insert and a second termination sequence AOXTT respectively to obtain a plasmid pMOXZ-2; connecting the plasmid pMOXZ-1 and the plasmid pMOXZ-2;
(2) replacing the selection marker gene on the plasmid to obtain a plasmid with a different selection marker gene from the plasmid in step (1);
(3) and (3) respectively inserting the target gene into the plasmids constructed in the step (1) and the step (2) to obtain the recombinant expression vector.
Further, the target genes are respectively and independently selected from at least one of HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV 31L 1, HPV 33L 1, HPV 35L 1, HPV 39L 1, HPV 45L 1, HPV 51L 1, HPV52L1, HPV53L1, HPV 58L 1, HPV 59L 1, HPV 66L 1 or HPV68L 1.
Further, the first promoter MOX insert is a promoter sequence represented by SEQ ID NO: 7. SEQ ID NO: 8 is obtained by amplification with a primer and hansenula polymorpha genome DNA as a template.
Further, the first termination sequence AOXTT employs SEQ ID NO: 10. SEQ ID NO: 11 is obtained by amplification with a primer and a pPICZB plasmid as a template.
Further, plasmid pMOXZ-1 was prepared by treating the MOX promoter insert with BglII + NdeI in combination, the AOXTT terminator sequence fragment with NdeI + BamHI, the pPICZB with BglII + BamHI, the pPICZB-BglII + BamHI fragment with calf intestinal alkaline phosphatase, and ligating the three fragments.
Further, the second promoter MOX insert is a promoter sequence represented by SEQ ID NO: 7. SEQ ID NO: 9 is obtained by amplification with Hansenula polymorpha genome DNA as a template.
Further, the second termination sequence AOXTT is a sequence that uses SEQ ID NO: 11. SEQ ID NO: the sequence shown in 12 is a primer, and the pPICZB plasmid is a template and is obtained by amplification.
Further, the plasmid pMOXZ-2 was obtained by treating the MOX promoter insert with BglII + HindIII in combination, the AOXTT terminator fragment with HindIII + BamHI, the pPICZB with BglII + BamHI, the pPICZB-BglII + BamHI fragment with calf intestinal alkaline phosphatase, and ligating the three fragments.
The fourth aspect of the invention provides a preparation method of recombinant engineering bacteria, which is obtained by transforming the recombinant expression vector system into engineering bacteria.
The fifth aspect of the invention provides the use of the recombinant expression vector system or the recombinant engineering bacteria in the preparation of the HPVL1 protein.
A sixth aspect of the present invention provides a method of producing an hpv l1 protein, selected from any one of:
a. transforming the recombinant expression vector system into recombinant engineering bacteria, and expressing the HPVL1 protein by using the recombinant engineering bacteria;
b. the HPVL1 protein is expressed by the recombinant engineering bacteria.
The HPVL1 protein obtained by the above method has at least two subtypes.
In a seventh aspect the invention provides a protein obtainable by a process for the preparation of an hpv l1 protein as described above.
As mentioned above, the polygene recombinant expression fragment, the expression vector system, the recombinant engineering bacteria, the preparation method and the application thereof have the following beneficial effects:
the recombinant engineering bacteria prepared by the invention can be used for preparing HPV vaccines. The recombinant engineering bacteria can simultaneously express a plurality of HPVL1 proteins. Experimental results show that after Quadri-L1 immunization, serum can induce HPV antibodies, the antibody titer levels are similar, and a certain dose-effect relationship is achieved.
Drawings
FIG. 1 shows a schematic diagram of the structure of the yeast dual expression plasmid pMMZ of the present invention.
FIG. 2 shows the schematic structure of the yeast double expression plasmid pMMG of the present invention.
FIG. 3 shows the structural diagram of the HPV6L 1/HPV 11L1 double expression plasmid of the present invention.
FIG. 4 shows the structural diagram of the HPV16L 1/HPV 18L1 double expression plasmid of the invention.
FIG. 5 shows the restriction enzyme identification structure of 6L1-11L1-pMMG, 16L1-18L1-pMMZ of the present invention.
Wherein 1.6L1-11L1-pMMG is not digested; 2.6L1-11L1-pMMG NheI + NotI enzyme digestion identification; 3.6L1-11L1-pMMG BstBI + SbfI enzyme digestion identification; 4.16L1-18L1-pMMZ not digested; 5.16L1-18L1-pMMZ NheI + NotI enzyme digestion identification; 6.16L1-18L1-pMMZ BstBI + SbfI enzyme digestion identification; 7.250bp DNAmarker (Takara).
FIG. 6 shows the type-specific neutralizing monoclonal antibody of the present invention detecting the broken supernatant of HPV16L 1/HPV 18L1 double expression strain.
A1-A6, B1-B2: h16.V5 detects 16L1-18L1-CBS 4732-1-8 #; b3: positive control for v5 detection (HPV 16L1 standard); C1-C6, D1-D2: h18.J4 detects 16L1-18L1-CBS 4732-1-8 #; d3: j4 positive control (HPV 18L1 standard) was detected.
FIG. 7 shows the type-specific neutralizing monoclonal antibody of the present invention for detecting the broken supernatant of four expression strains HPV6L 1/HPV 11L 1/HPV 16L 1/HPV 18L 1.
A1-A10, B7: h6.M48 detects 6L1-11L1-16L1-18L1-CBS 4732-1-11 #; b1, B8: h6.m48 detection positive control (HPV 6L1 standard);
C1-C10, D7: h11.H3 detection 6L1-11L1-16L1-18L1-CBS 4732-1-11 #; d1, D8: h11.h3 detection positive control (HPV 11L1 standard);
E1-E10, F7: h16.V5 detects 6L1-11L1-16L1-18L1-CBS 4732-1-11 #; f1, F8: positive control for v5 detection (HPV 16L1 standard);
A1-A10, H7: h18.J4 detects 6L1-11L1-16L1-18L1-CBS 4732-1-11 #; h1, H8: j4 positive control (HPV 18L1 standard);
FIG. 8 shows a transmission electron microscope image of the Quadri-L1-VLP of the present invention.
FIG. 9 shows the mean geometric antibody titer (GMT) of Quadri-L1 mouse serum according to the present invention.
Detailed Description
The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and generally refer to a polymer of any length consisting essentially of nucleotides, such as deoxyribonucleotides and/or ribonucleotides. Nucleic acids may comprise purine and/or pyrimidine bases, and/or other natural, chemically or biochemically modified (e.g., methylated), non-natural, or derivatized nucleotide bases. The backbone of the nucleic acid may comprise a sugar and a phosphate group, as may typically be found in RNA or DNA, and/or one or more modified or substituted (e.g., 2' -O-alkylated, e.g., 2' -O-methylated or 2' -O-ethylated; or 2' -O,4' -C-alkynylated, e.g., 2' -O,4' -C-ethylated) sugars or one or more modified or substituted phosphate groups.
The term "nucleic acid expression cassette" refers to a nucleic acid molecule comprising one or more transcriptional control elements (such as, but not limited to, promoters, enhancers, polyadenylation sequences, and introns) that direct the expression of a (trans) gene to which they are operably linked.
The term "operably linked" refers to the arrangement of various nucleic acid molecule elements relative to each such that the elements are functionally linked and capable of interacting with each other in the context of gene expression. Such elements may include, but are not limited to, promoters, enhancers, polyadenylation sequences, one or more introns, and coding sequences of the gene of interest to be expressed (e.g., the gene of interest). When properly oriented or operably linked, the nucleic acid sequence elements function together to ensure or regulate expression of the coding sequence. Modulation refers to increasing, decreasing, or maintaining the level of activity of a particular element. The position of each element relative to other elements can be expressed in terms of the 5 'end and 3' end of each element, and the distance between any particular element can be expressed in terms of the number of intervening nucleotides or base pairs between the elements.
The term "gene of interest" refers to a specific nucleic acid sequence encoding a polypeptide or a portion of a polypeptide to be expressed in a host cell into which the nucleic acid sequence is introduced. It is not essential how the nucleic acid sequence is introduced into the host cell, for example it may be integrated into the genome or as an episomal plasmid.
The term "promoter" refers to a nucleic acid sequence capable of binding RNA polymerase and initiating transcription of one or more nucleic acid coding sequences (e.g., a gene of interest) to which it is operably linked. Promoters are typically located near the transcription start site of a gene on the same strand and upstream (5' in the sense strand) of the nucleotide coding sequence. Promoters may function individually to regulate transcription or may be further regulated by one or more regulatory sequences (e.g., enhancers or silencers).
The term "transcription termination sequence" includes control sequences at the end of a transcriptional unit that indicate the 3' processing and termination of transcription.
The term "selectable marker gene" includes any gene that confers a phenotype on a host cell in which it is expressed to facilitate identification and/or selection of host cells transfected or transformed with the transgene.
The term "vector" refers to a polynucleotide molecule, preferably a DNA molecule derived from, for example, a plasmid, phage, or plant virus, into which a polynucleotide can be inserted or cloned. The vector preferably contains one or more unique restriction endonuclease sites and may be capable of autonomous replication in a defined host cell or may integrate into the genome of a defined host such that the cloned sequence is replicable. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
The term "recombinantly engineered bacteria" refers to those cells used for transformation, i.e., cells used to express a gene of interest. The recombinant engineered bacteria may be isolated cells or cell lines cultured in culture, or cells present in living tissue or organisms. In the context of the present invention, a host cell is preferably a cell capable of growing in culture.
The term "transformation" refers to the introduction of an exogenous nucleic acid into an organism such that the nucleic acid can be replicated as an extrachromosomal element or by chromosomal integration.
Expressing the fragment:
the expression fragments provided herein comprise at least a first nucleic acid expression cassette and a second nucleic acid expression cassette, wherein the first nucleic acid expression cassette and the second nucleic acid expression cassette are not copies of each other, and wherein each expression cassette comprises a promoter operably linked to one or more genes of interest. The promoters linked to each gene of interest may be the same or different for each expression cassette. In a preferred embodiment, the same promoter is used.
Other sequences may be incorporated into the nucleic acid expression cassettes according to the invention. More specifically, the nucleic acid expression cassette includes sequences (e.g., introns and/or transcription termination sequences) that further increase or stabilize expression of the transgene product.
Preferably, the expression fragments encompassed herein comprise a selectable marker gene. The selectable marker gene is preferably not contained in the nucleic acid expression cassette of the expression fragment. Thus, in embodiments, an expression fragment encompassed herein further comprises one or more nucleic acid expression cassettes comprising a selectable marker gene. Expression of the selectable marker gene can indicate that the nucleic acid expression cassette of the host cell has been transformed, thus allowing for selection of transformed host cells. The selectable marker gene cassette also typically includes a promoter and a transcription terminator sequence, which are operably linked to the selectable marker gene.
Suitable markers may be selected from markers that confer antibiotic resistance, visual markers, or complement host cell auxotrophy. For example, a selectable marker may confer resistance to an antibiotic such as hygromycin B (e.g., the hph gene), zeocin/phleomycin (e.g., the ble gene), kanamycin or G418 (e.g., the nptII or aphVIII gene), spectinomycin (e.g., the aadA gene), neomycin (e.g., the aphVIII gene), blasticidin (e.g., the bsd gene), nourseothricin (e.g., the natR gene), puromycin (e.g., the pac gene), and paromomycin (e.g., the aphVIII gene). Visual markers may also be used and include, for example, β -Glucuronidase (GUS), luciferase, and fluorescent proteins, such as Green Fluorescent Protein (GFP), yellow fluorescent protein, and the like. Two prominent examples of auxotrophs are amino acid leucine deficiency (e.g., the LEU2 gene) or uracil deficiency (e.g., the URA3 gene). Cells that are orotidine-5' -phosphate decarboxylase negative (ura3-) cannot grow on medium lacking uracil. Thus, a functional URA3 gene can be used as a selection marker on host cells that are uracil deficient, and successful transformants can be selected on medium lacking uracil. Cells transformed with only the functional URA3 gene are able to synthesize uracil and grow on such media. If the wild-type strain does not have a uracil deficiency, it is necessary to prepare auxotrophic mutants having the deficiency in order to use URA3 as a selection marker for the strain. Methods of achieving this are well known in the art.
Carrier system：
The expression cassettes encompassed herein may be used as such or, in general, they may be part of (i.e., introduced into) a nucleic acid vector. The nucleic acid expression cassettes for the expression fragments disclosed herein can be located on the same vector or on different vectors. The invention specifically encompasses vector systems comprising at least two vectors, wherein each vector comprises at least two nucleic acid expression cassettes. The vectors of the vector systems encompassed herein may be the same or different.
The vectors taught herein may further contain various types of restriction sites for linearization or fragmentation.
Many vectors are known to those skilled in the art, and any such vector may be used. Selection of an appropriate vector is a matter of choice. The vector may be a non-viral vector or a viral vector. Non-viral vectors include, but are not limited to, plasmids, cationic lipids, liposomes, nanoparticles, PEG, PEI, and the like. Viral vectors are derived from viruses, including but not limited to: retroviruses, lentiviruses, adeno-associated viruses, adenoviruses, herpes viruses, hepatitis viruses, and the like.
The methods used herein for transforming recombinant engineered bacteria are well known to the skilled artisan. For example, electroporation and/or chemical (e.g., calcium chloride or lithium acetate based) transformation methods or Agrobacterium tumefaciens (Agrobacterium tumefaciens) mediated transformation methods as known in the art may be used.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.
Example 1 construction and engineering of Yeast Dual expression plasmids
In order to express HPV6L1, HPV11L1, HPV16L1 and HPV18L1 in yeast at the same time, two expression plasmids capable of expressing two proteins at the same time need to be obtained by modification, each plasmid contains two independent expression cassettes, and the two plasmids contain different screening markers, so that two different double expression plasmids can be introduced into the yeast by a method of two times of transformation and screening. The specific plasmid construction scheme is as follows:
1.1 construction of the Yeast Dual expression plasmid pMMZ
By PCR, using SEQ ID NO: 7-8 as a primer, and hansenula polymorpha whole genome DNA as a template, and amplifying to obtain a MOX promoter insert (SEQ ID NO: 26); using SEQ ID NO: 10, SEQ ID NO: the sequence shown in 12 is a primer, pPICZB plasmid is a template, and a fragment containing AOXTT termination sequence (SEQ ID NO: 27) is obtained by amplification. The MOX promoter insert was treated with BglII + NdeI in combination with digestion, the AOXTT terminator fragment was treated with NdeI + BamHI, pPICZB was treated with BglII + BamHI, and the pPICZB-BglII + BamHI fragment was treated with calf intestinal alkaline phosphatase, and the three fragments were ligated to give plasmid pMOXZ-1(SEQ ID NO: 28).
Primer sequence (SEQ ID NO: 7)
5’-accaaaagatctgtcgacgcggag-3；
Primer sequence (SEQ ID NO: 8)
5’-cttggctacatatgatcgatcgtgtacacgtttcgaatttgtttttgtacttt-3’；
Primer sequence (SEQ ID NO: 10)
5’-gcccgatcatatgcctgcaggcagctttctagaacaa-3’
Primer sequence (SEQ ID NO: 12)
5’-tgggggatccgcacaaacgaa-3’
PCR sequence (SEQ ID NO: 26)
accaaaagatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgtgtacacgatcgatcatatgtagccaag
PCR sequence (SEQ ID NO: 27)
gcccgatcatatgcctgcaggcagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatccccca
pMOXZ-1 sequence (SEQ ID NO: 28)
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgtgtacacgatcgatcatatgcctgcaggcagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
By PCR, using SEQ ID NO: 7,9 is used as a primer, hansenula polymorpha genome DNA is used as a template, and a MOX promoter insert (SEQ ID NO: 29) is obtained by amplification; using SEQ ID NO: 11-12 as primer, pPICZB plasmid as template, and amplifying to obtain the segment containing AOXTT termination sequence (SEQ ID NO: 30). The MOX promoter insert was treated with BglII + HindIII in combination with digestion, the AOXTT terminator fragment was treated with HindIII + BamHI, pPICZB was treated with BglII + BamHI, the pPICZB-BglII + BamHI fragment was treated with calf intestinal alkaline phosphatase, and the three fragments were ligated to give plasmid pMOXZ-2(SEQ ID NO: 31).
Primer sequence (SEQ ID NO: 9)
5’-tcaagtcaagcttcacgtggctagctttgtttttgtacttt-3’；
Primer sequence (SEQ ID NO: 11)
5’-aaacgtgaagcttgaattcgcggccgccagctttct-3’；
PCR sequence (SEQ ID NO: 29)
accaaaagatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaagctagccacgtgaagcttgacttga
PCR sequence (SEQ ID NO: 30)
aaacgtgaagcttgaattcgcggccgccagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatccccca
pMOXZ-2 sequence (SEQ ID NO: 31)
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaagctagccacgtgaagcttgaattcgcggccgccagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
The pMOXZ-2 plasmid expression cassette was inserted into the BglII-treated pMOXZ-1 plasmid by means of BglII and BamHI ligation (see in vitro multicopy plasmid construction protocol mentioned in the Invitrogen pPICZB operating manual) to give the Zeocin-resistant Hansenula yeast dual expression plasmid pMMZ (SEQ ID NO: 32) (FIG. 1).
pMMZ sequence (SEQ ID NO: 32)
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaagctagccacgtgaagcttgaattcgcggccgccagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgtgtacacgatcgatcatatgcctgcaggcagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
1.2 construction of Yeast Dual expression plasmid pMMG
Amplifying a TEF promoter + EM7 promoter fragment (SEQ ID NO: 33) by adopting a sequence shown in SEQ ID NO:13-14 as a primer and a pPICZB plasmid as a template in a PCR (polymerase chain reaction) mode; the sequence shown in SEQ ID NO. 15-16 is used as a primer, pPIC9K plasmid is used as a template, and a G418 resistance gene segment (SEQ ID NO. 34) is obtained by amplification; the combined enzyme digestion treatment of BglII + AflII on a TEF promoter + EM7 promoter fragment, the combined enzyme digestion treatment of AflII + SacII on a G418 resistant gene fragment, the combined enzyme digestion treatment of BglII + SacII on a pMOXZ-1 plasmid, and the connection of the three fragments to obtain a pMOXZG plasmid (SEQ ID NO: 35);
primer sequence (SEQ ID NO: 13)
5’-cccttagatctcccacacaccatagct-3’
Primer sequence (SEQ ID NO: 14)
5’-tgcggtcttaaggtttagttcctcacc-3’
Primer sequence (SEQ ID NO: 15)
5’-tattggcttaagatgagccatattcaac-3’
Primer sequence (SEQ ID NO: 16)
5’-gggtatcgtcaccgcggccggctacagcacttgaagtcggacag-3’
PCR sequence (SEQ ID NO: 33)
cccttagatctcccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccttaagaccgca
PCR sequence (SEQ ID NO: 34)
gggtatcgtcaccgcggccggctacagcacttgaagtcggacagtgagtgtagtcttgagaaattctgaagccgtatttttattatcagtgagtcagtcatcaggagatcctctacgccggacgcatcgtggccgacctgcagggggggggggggcgctgaggtctgcctcgtgaagaaggtgttgctgactcataccaggcctgaatcgccccatcatccagccagaaagtgagggagccacggttgatgagagctttgttgtaggtggaccagttggtgattttgaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaaagttcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttgaatatggctcatcttaagccaata
pMOXZG sequence (SEQ ID NO: 35)
agatctcccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccttaagatgagccatattcaacgggaaacgtcttgctcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatcagaattggttaattggttgtaacactggcagagcattacgctgacttgacgggacggcggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcacgcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacctacaacaaagctctcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctggtatgagtcagcaacaccttcttcacgaggcagacctcagcgccccccccccccctgcaggtcggccacgatgcgtccggcgtagaggatctcctgatgactgactcactgataataaaaatacggcttcagaatttctcaagactacactcactgtccgacttcaagtgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgtgtacacgatcgatcatatgcctgcaggcagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
Through a PCR mode, a sequence shown in SEQ ID NO 17-18 is used as a primer, pMOXZG is used as a template, and a pUC replicon + TEF1 promoter + EM7 promoter + G418 resistance gene fragment (SEQ ID NO 36) is obtained through amplification; BamHI digestion treatment of pUC replicon + TEF promoter + EM7 promoter + G418 resistance gene fragment, BamHI + BglII combined digestion treatment of pMMZ plasmid (elimination of Zeocin resistance expression cassette and pUC replicon of original pMMZ plasmid), two fragments were ligated to obtain the G418 resistance-carrying Hansenula yeast double expression plasmid pMMG (SEQ ID NO: 37) (FIG. 2).
Primer sequence (SEQ ID NO: 17)
5’-gttggatcctcgatcacatgtgagc-3’
Primer sequence (SEQ ID NO: 18)
5’-aaaggatcctgaggtctgcctcgt-3’
PCR sequence (SEQ ID NO: 36)
gttggatcctcgatcacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatcagatctcccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccttaagatgagccatattcaacgggaaacgtcttgctcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatcagaattggttaattggttgtaacactggcagagcattacgctgacttgacgggacggcggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcacgcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacctacaacaaagctctcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctggtatgagtcagcaacaccttcttcacgaggcagacctcaggatccttt
pMMG sequence (SEQ ID NO: 37)
ggatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaagctagccacgtgaagcttgaattcgcggccgccagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgtgtacacgatcgatcatatgcctgcaggcagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatcctcgatcacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatcagatctcccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccttaagatgagccatattcaacgggaaacgtcttgctcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatcagaattggttaattggttgtaacactggcagagcattacgctgacttgacgggacggcggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcacgcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacctacaacaaagctctcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctggtatgagtcagcaacaccttcttcacgaggcagacctca
EXAMPLE 2 construction of 6L1-11L1-pMMG dual expression plasmid and 16L1-18L1-pMMZ dual expression plasmid
2.1HPVL1 Dual expression plasmid construction
The DNA sequences of the codes HPV6L1, HPV11L1, HPV16L1 and HPV18L1 are shown in SEQ ID NO: 1-4, synthesized by Jinzhi corporation, Suzhou, with codon usage frequency referenced to yeast preferred codons, linked to the universal cloning plasmid pUC57 supplied by Jinzhi corporation, to obtain HPV6L1-pUC57, HPV11L1-pUC57, HPV16L1-pUC57, and HPV18L1-pUC57, respectively. The HPV6L1 and HPV11L1 are respectively inserted into pMMG plasmid, and the HPV16L1 and HPV18L1 genes are inserted into pMMZ plasmid.
2.2 construction of the HPV6L 1/HPV 11L1 Dual expression plasmid
Amplifying an HPV11L1 fragment (SEQ ID NO: 38) by a PCR mode by using a sequence shown in SEQ ID NO:19-20 as a primer and an HPV11L1-pUC57 plasmid as a template; the HPV11L1 fragment and the pMMG plasmid were treated with BstBI + SbfI in combination with enzyme digestion, and the two fragments were ligated to obtain 11L1-pMMG (SEQ ID NO: 39).
By a PCR mode, a sequence shown by SEQ ID NO:21-22 is used as a primer, HPV6L1-pUC57 plasmid is used as a template, an HPV6L1 fragment (SEQ ID NO: 40) is obtained through amplification, an HPV6L1 fragment and 11L1-pMMG plasmid are treated through NheI + NotI combined enzyme digestion, and the two fragments are connected to obtain 6L1-11L1-pMMG (SEQ ID NO: 41) (figure 3).
2.3 HPV16L 1/HPV 18L1 double expression plasmid construction
Amplifying an HPV16L1 fragment (SEQ ID NO: 42) by a PCR mode by using sequences shown in SEQ ID NO:19 and 23 as primers and an HPV16L1-pUC57 plasmid as a template; the HPV16L1 fragment and the pMMZ plasmid were treated with BstBI + SbfI in combination with enzymatic cleavage, and the two fragments were ligated to yield 16L1-pMMZ (SEQ ID NO: 43).
By a PCR mode, a sequence shown by SEQ ID NO:24-25 is used as a primer, HPV18L1-pUC57 plasmid is used as a template, an HPV18L1 fragment (SEQ ID NO: 44) is obtained through amplification, an HPV18L1 fragment and a 16L1-pMMZ plasmid are treated by NheI + NotI combined enzyme digestion, and the two fragments are connected to obtain 16L1-18L1-pMMZ (SEQ ID NO: 45) (figure 4).
Primer sequence (SEQ ID NO: 19)
5’-cggacaaattcgaaacgatgt-3’
Primer sequence (SEQ ID NO: 20)
5’-acagaaacctgcaggctattactttttggtc-3’
Primer sequence (SEQ ID NO: 21)
5’-aatcgctagcatgtggagaccatctg-3’
Primer sequence (SEQ ID NO: 22)
5’-ttggtaatgcggccgcctattatctcttggtc-3’
Primer sequence (SEQ ID NO: 23)
5’-tgcttcctgcaggctattacagttttctc-3’
Primer sequence (SEQ ID NO: 24)
5’-attcgctagcatggctctgtggagac-3’
Primer sequence (SEQ ID NO: 25)
5’-ttccttatgcggccgctattactttctagct-3’
PCR sequence (SEQ ID NO: 38)
cggacaaattcgaaacgatgtggagaccatctgactcgaccgtctacgttcctccacctaacccagtctccaaagtggttgctactgatgcctacgtcaagagaaccaacatcttctaccacgcctcttcgtccagattgctggcagttggacatccttactattccatcaagaaagttaacaagaccgtggtccctaaggtttcgggctatcagtacagagtgttcaaggtcgttctgccagaccctaacaagtttgccttgccagattcctcgctgttcgaccctaccactcaaagactggtttgggcttgcacgggtttggaagtgggcagaggacaaccactgggtgtcggagtttccggccatccattgctgaacaagtacgacgatgttgagaactcgggtggatacggtggcaatccaggacaagacaacagagtgaatgtcggcatggactacaagcaaacccagctgtgcatggttggttgtgcaccacctctgggtgaacattggggaaagggcactcagtgctccaacacctctgttcagaatggtgactgtcctccactggagctgatcacctcggttattcaggatggagacatggtggacactggcttcggtgcaatgaactttgccgacttgcagaccaacaagtccgatgttccattggacatttgcggcactgtgtgcaaatatcctgactacctgcagatggctgcagatccatacggtgacagactcttcttctacctgagaaaggagcagatgttcgccagacacttctttaacagagctggcaccgttggtgagcctgttccagatgacctgttggtgaagggtggaaacaatagatcctctgttgcctcgtccatctacgtccacaccccatcgggctctctggtgtcctcggaggcccaactgttcaacaagccttactggctccagaaggcccaaggacacaacaatggcatctgctggggaaaccacctgttcgtcaccgttgtggacacgactagatccaccaacatgactttgtgtgcctctgtttccaagtcggctacctacacgaactcggactataaggagtacatgagacatgttgaagagttcgacctgcagtttatcttccagctctgctccattaccttgtcggcagaagtgatggcctacatccacacgatgaacccatccgtcttggaagactggaacttcggtctgtctccacctccaaatggcaccctggaagacacgtacagatatgtgcagtcgcaagccatcacctgtcagaaaccaactcctgaaaaggagaaacaggacccatacaaggatatgtcgttctgggaggtcaacctgaaggagaagttctcttcggaactggaccagttcccattgggaagaaagttcctgctccagtctggttatagaggaagaacctccgcaagaactggcatcaagagacctgccgtgtcgaagccatccaccgctcctaagagaaagagaacgaagaccaaaaagtaatagcctgcaggtttctgt
11L1-pMMG sequence (SEQ ID NO: 39)
ggatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaagctagccacgtgaagcttgaattcgcggccgccagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatgtggagaccatctgactcgaccgtctacgttcctccacctaacccagtctccaaagtggttgctactgatgcctacgtcaagagaaccaacatcttctaccacgcctcttcgtccagattgctggcagttggacatccttactattccatcaagaaagttaacaagaccgtggtccctaaggtttcgggctatcagtacagagtgttcaaggtcgttctgccagaccctaacaagtttgccttgccagattcctcgctgttcgaccctaccactcaaagactggtttgggcttgcacgggtttggaagtgggcagaggacaaccactgggtgtcggagtttccggccatccattgctgaacaagtacgacgatgttgagaactcgggtggatacggtggcaatccaggacaagacaacagagtgaatgtcggcatggactacaagcaaacccagctgtgcatggttggttgtgcaccacctctgggtgaacattggggaaagggcactcagtgctccaacacctctgttcagaatggtgactgtcctccactggagctgatcacctcggttattcaggatggagacatggtggacactggcttcggtgcaatgaactttgccgacttgcagaccaacaagtccgatgttccattggacatttgcggcactgtgtgcaaatatcctgactacctgcagatggctgcagatccatacggtgacagactcttcttctacctgagaaaggagcagatgttcgccagacacttctttaacagagctggcaccgttggtgagcctgttccagatgacctgttggtgaagggtggaaacaatagatcctctgttgcctcgtccatctacgtccacaccccatcgggctctctggtgtcctcggaggcccaactgttcaacaagccttactggctccagaaggcccaaggacacaacaatggcatctgctggggaaaccacctgttcgtcaccgttgtggacacgactagatccaccaacatgactttgtgtgcctctgtttccaagtcggctacctacacgaactcggactataaggagtacatgagacatgttgaagagttcgacctgcagtttatcttccagctctgctccattaccttgtcggcagaagtgatggcctacatccacacgatgaacccatccgtcttggaagactggaacttcggtctgtctccacctccaaatggcaccctggaagacacgtacagatatgtgcagtcgcaagccatcacctgtcagaaaccaactcctgaaaaggagaaacaggacccatacaaggatatgtcgttctgggaggtcaacctgaaggagaagttctcttcggaactggaccagttcccattgggaagaaagttcctgctccagtctggttatagaggaagaacctccgcaagaactggcatcaagagacctgccgtgtcgaagccatccaccgctcctaagagaaagagaacgaagaccaaaaagtaatagcctgcaggcagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatcctcgatcacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatcagatctcccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccttaagatgagccatattcaacgggaaacgtcttgctcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatcagaattggttaattggttgtaacactggcagagcattacgctgacttgacgggacggcggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcacgcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacctacaacaaagctctcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctggtatgagtcagcaacaccttcttcacgaggcagacctca
PCR sequence (SEQ ID NO: 40)
aatcgctagcatgtggagaccatctgactcgactgtctacgttcctccacctaacccagtctctaaggttgtggctaccgacgcctacgtgactagaaccaacatcttctaccacgcatcgtcctctagattgctggccgttggacatccttacttctccatcaagagagccaacaaaaccgtcgtgccaaaggtttctggctaccagtatagagtcttcaaggtggttctgcctgaccctaacaagtttgccctgccagactcgtccttgttcgatcctacgactcaaagactcgtttgggcctgcactggtcttgaggttggcagaggacagccactcggagtcggcgtttctggtcatcctttcctgaacaagtacgacgatgttgagaactctggttccggaggcaatcctggacaggacaacagagtcaatgttggcatggactacaagcaaactcagctctgtatggtgggatgcgcacctccactgggtgagcattggggaaaaggcaagcagtgcaccaacactccagttcaggcaggtgactgccctccattggagctgattacctcggtcatccaggacggtgatatggttgacaccggattcggtgccatgaacttcgctgacctgcagaccaacaagtccgatgtgccaatcgacatctgcggaacgacctgtaaataccctgactacttgcagatggcagccgatccatacggcgacagactcttcttctttctgagaaaggagcagatgtttgctagacactttttcaacagagccggagaggtcggtgagcctgttccagacaccctgatcattaagggctcgggaaacagaacttctgtcggctcctcgatctacgtgaacaccccttccggttcgttggtgtcgtccgaggctcaactgttcaacaagccatactggctccagaaagcccaaggtcacaacaatggcatctgctggggtaaccagctgttcgttactgtcgtggacaccacgagatcgaccaacatgactctgtgtgcctccgttaccacatcttcgacttacaccaactccgactataaggagtacatgagacacgttgaagagtacgacttgcaattcatctttcagctctgctcgattaccctgtccgcagaagtcatggcctacatccacactatgaatccatcggtgcttgaggactggaactttggcttgtccccacctccaaacggaactctggaagacacctatagatacgtgcagtcgcaagccatcacgtgccagaagccaacccctgagaaggaaaagccagatccttacaagaacctgtccttctgggaggtcaatctcaaggagaagttctcgtccgaactggaccagtacccattgggtagaaagttcctgctccagtctggataccgtggtagatcgtccatcagaaccggagtgaagagaccagcagtttccaaggcttctgccgcacctaagcgtaaaagagccaagaccaagagataataggcggccgcattaccaa
6L1-11L1-pMMG sequence (SEQ ID NO: 41)
ggatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaagctagcatgtggagaccatctgactcgactgtctacgttcctccacctaacccagtctctaaggttgtggctaccgacgcctacgtgactagaaccaacatcttctaccacgcatcgtcctctagattgctggccgttggacatccttacttctccatcaagagagccaacaaaaccgtcgtgccaaaggtttctggctaccagtatagagtcttcaaggtggttctgcctgaccctaacaagtttgccctgccagactcgtccttgttcgatcctacgactcaaagactcgtttgggcctgcactggtcttgaggttggcagaggacagccactcggagtcggcgtttctggtcatcctttcctgaacaagtacgacgatgttgagaactctggttccggaggcaatcctggacaggacaacagagtcaatgttggcatggactacaagcaaactcagctctgtatggtgggatgcgcacctccactgggtgagcattggggaaaaggcaagcagtgcaccaacactccagttcaggcaggtgactgccctccattggagctgattacctcggtcatccaggacggtgatatggttgacaccggattcggtgccatgaacttcgctgacctgcagaccaacaagtccgatgtgccaatcgacatctgcggaacgacctgtaaataccctgactacttgcagatggcagccgatccatacggcgacagactcttcttctttctgagaaaggagcagatgtttgctagacactttttcaacagagccggagaggtcggtgagcctgttccagacaccctgatcattaagggctcgggaaacagaacttctgtcggctcctcgatctacgtgaacaccccttccggttcgttggtgtcgtccgaggctcaactgttcaacaagccatactggctccagaaagcccaaggtcacaacaatggcatctgctggggtaaccagctgttcgttactgtcgtggacaccacgagatcgaccaacatgactctgtgtgcctccgttaccacatcttcgacttacaccaactccgactataaggagtacatgagacacgttgaagagtacgacttgcaattcatctttcagctctgctcgattaccctgtccgcagaagtcatggcctacatccacactatgaatccatcggtgcttgaggactggaactttggcttgtccccacctccaaacggaactctggaagacacctatagatacgtgcagtcgcaagccatcacgtgccagaagccaacccctgagaaggaaaagccagatccttacaagaacctgtccttctgggaggtcaatctcaaggagaagttctcgtccgaactggaccagtacccattgggtagaaagttcctgctccagtctggataccgtggtagatcgtccatcagaaccggagtgaagagaccagcagtttccaaggcttctgccgcacctaagcgtaaaagagccaagaccaagagataataggcggccgccagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatgtggagaccatctgactcgaccgtctacgttcctccacctaacccagtctccaaagtggttgctactgatgcctacgtcaagagaaccaacatcttctaccacgcctcttcgtccagattgctggcagttggacatccttactattccatcaagaaagttaacaagaccgtggtccctaaggtttcgggctatcagtacagagtgttcaaggtcgttctgccagaccctaacaagtttgccttgccagattcctcgctgttcgaccctaccactcaaagactggtttgggcttgcacgggtttggaagtgggcagaggacaaccactgggtgtcggagtttccggccatccattgctgaacaagtacgacgatgttgagaactcgggtggatacggtggcaatccaggacaagacaacagagtgaatgtcggcatggactacaagcaaacccagctgtgcatggttggttgtgcaccacctctgggtgaacattggggaaagggcactcagtgctccaacacctctgttcagaatggtgactgtcctccactggagctgatcacctcggttattcaggatggagacatggtggacactggcttcggtgcaatgaactttgccgacttgcagaccaacaagtccgatgttccattggacatttgcggcactgtgtgcaaatatcctgactacctgcagatggctgcagatccatacggtgacagactcttcttctacctgagaaaggagcagatgttcgccagacacttctttaacagagctggcaccgttggtgagcctgttccagatgacctgttggtgaagggtggaaacaatagatcctctgttgcctcgtccatctacgtccacaccccatcgggctctctggtgtcctcggaggcccaactgttcaacaagccttactggctccagaaggcccaaggacacaacaatggcatctgctggggaaaccacctgttcgtcaccgttgtggacacgactagatccaccaacatgactttgtgtgcctctgtttccaagtcggctacctacacgaactcggactataaggagtacatgagacatgttgaagagttcgacctgcagtttatcttccagctctgctccattaccttgtcggcagaagtgatggcctacatccacacgatgaacccatccgtcttggaagactggaacttcggtctgtctccacctccaaatggcaccctggaagacacgtacagatatgtgcagtcgcaagccatcacctgtcagaaaccaactcctgaaaaggagaaacaggacccatacaaggatatgtcgttctgggaggtcaacctgaaggagaagttctcttcggaactggaccagttcccattgggaagaaagttcctgctccagtctggttatagaggaagaacctccgcaagaactggcatcaagagacctgccgtgtcgaagccatccaccgctcctaagagaaagagaacgaagaccaaaaagtaatagcctgcaggcagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatcctcgatcacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatcagatctcccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccttaagatgagccatattcaacgggaaacgtcttgctcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatcagaattggttaattggttgtaacactggcagagcattacgctgacttgacgggacggcggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcacgcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacctacaacaaagctctcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctggtatgagtcagcaacaccttcttcacgaggcagacctca
PCR sequence (SEQ ID NO: 42)
cggacaaattcgaaacgatgtctctttggttgccatccgaggctactgtttacttgccacctgtcccagtttctaaggtcgtgtcaactgatgaatacgttgctagaaccaacatctattaccacgctggaacttctagattgcttgctgttggtcatccatacttccctatcaagaaacctaacaataacaagatccttgttccaaaggtctctggattgcagtacagagttttcagaatccacttgcctgacccaaacaagttcggttttcctgacacctccttctacaacccagatactcaaagattggtgtgggcctgtgttggtgtcgaggttggacgtggtcaaccattgggagttggtatttctggacatcctttgcttaacaagttggatgacactgaaaatgcttctgcctacgcagccaacgctggtgttgacaacagagagtgtatctctatggattacaaacagacccaattgtgtcttattggttgcaagccacctattggtgaacactggggtaagggatctccatgtaccaacgttgctgtcaatccaggtgattgtcctccattggagcttatcaacactgttattcaagatggtgacatggttgatactggatttggtgctatggacttcactaccttgcaagctaacaagtccgaggttccattggacatttgtacttctatctgcaagtatccagactacattaagatggtctctgaaccatacggtgactccttgtttttctaccttcgtagagagcaaatgtttgttagacacttgttcaacagagctggtgccgttggtgagaatgtcccagacgatttgtacatcaagggatctggttccactgcaaacttggcctcttccaactacttcccaactccttcaggttctatggttacctccgatgctcaaatcttcaacaagccttactggttgcaaagagcacagggtcacaacaatggaatttgttggggtaaccaacttttcgttactgtcgttgataccacaagatccaccaacatgtccttgtgtgctgccatttctacttcagaaaccacatacaagaatactaacttcaaggagtaccttagacatggtgaggaatacgacttgcagttcatctttcaattgtgcaagattaccttgactgctgacgttatgacctacatccactctatgaactccactatccttgaagattggaacttcggattgcaaccacctccaggtggaaccttggaagatacttacagatttgtcacatcccaagccattgcttgtcagaagcacactcctccagctcctaaggaagatccacttaagaaatacaccttctgggaagtcaacttgaaagagaagttctctgctgaccttgatcagtttcctttgggtagaaagttcttacttcaagctggattgaaggccaaaccaaagttcaccttgggtaaaagaaaggctactccaaccacatcatctacttccacaaccgccaaacgtaagaagagaaaactgtaatagcctgcaggaagca
16L1-pMMZ sequence (SEQ ID NO: 43)
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaagctagccacgtgaagcttgaattcgcggccgccagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatgtctctttggttgccatccgaggctactgtttacttgccacctgtcccagtttctaaggtcgtgtcaactgatgaatacgttgctagaaccaacatctattaccacgctggaacttctagattgcttgctgttggtcatccatacttccctatcaagaaacctaacaataacaagatccttgttccaaaggtctctggattgcagtacagagttttcagaatccacttgcctgacccaaacaagttcggttttcctgacacctccttctacaacccagatactcaaagattggtgtgggcctgtgttggtgtcgaggttggacgtggtcaaccattgggagttggtatttctggacatcctttgcttaacaagttggatgacactgaaaatgcttctgcctacgcagccaacgctggtgttgacaacagagagtgtatctctatggattacaaacagacccaattgtgtcttattggttgcaagccacctattggtgaacactggggtaagggatctccatgtaccaacgttgctgtcaatccaggtgattgtcctccattggagcttatcaacactgttattcaagatggtgacatggttgatactggatttggtgctatggacttcactaccttgcaagctaacaagtccgaggttccattggacatttgtacttctatctgcaagtatccagactacattaagatggtctctgaaccatacggtgactccttgtttttctaccttcgtagagagcaaatgtttgttagacacttgttcaacagagctggtgccgttggtgagaatgtcccagacgatttgtacatcaagggatctggttccactgcaaacttggcctcttccaactacttcccaactccttcaggttctatggttacctccgatgctcaaatcttcaacaagccttactggttgcaaagagcacagggtcacaacaatggaatttgttggggtaaccaacttttcgttactgtcgttgataccacaagatccaccaacatgtccttgtgtgctgccatttctacttcagaaaccacatacaagaatactaacttcaaggagtaccttagacatggtgaggaatacgacttgcagttcatctttcaattgtgcaagattaccttgactgctgacgttatgacctacatccactctatgaactccactatccttgaagattggaacttcggattgcaaccacctccaggtggaaccttggaagatacttacagatttgtcacatcccaagccattgcttgtcagaagcacactcctccagctcctaaggaagatccacttaagaaatacaccttctgggaagtcaacttgaaagagaagttctctgctgaccttgatcagtttcctttgggtagaaagttcttacttcaagctggattgaaggccaaaccaaagttcaccttgggtaaaagaaaggctactccaaccacatcatctacttccacaaccgccaaacgtaagaagagaaaactgtaatagcctgcaggcagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
PCR sequence (SEQ ID NO: 44)
attcgctagcatggctctgtggagaccatctgacaacaccgtctacttgccacctccatctgttgccagagtcgttaacaccgacgattacgtcactagaacatccatcttctaccacgctggttcctctagattgcttactgttggtaatccatacttcagagttccagctggtggaggtaataagcaagacattcctaaggtttctgcttaccaatatagagtctttagagttcagttgcctgacccaaacaagttcggtttgcctgatacttctatctacaacccagagactcaaagattggtttgggcctgtgctggtgtcgagatcggtagaggacaacctttgggagttggtctttctggacacccattctacaacaagttggatgacactgaatcttcacatgctgccacttccaatgtttctgaggatgttcgtgacaacgtctctgttgattacaagcaaactcagttgtgtatccttggatgtgcccctgctattggtgaacactgggctaaaggtactgcctgtaagtccagaccattgtctcaaggtgactgcccacctttggagcttaagaacactgttttggaagatggtgacatggttgatactggatatggtgctatggacttctctaccctgcaggatactaagtgtgaggttccattggacatctgtcaatctatttgtaagtaccctgattatcttcagatgtctgctgacccatacggtgattctatgttcttttgcttgagacgtgagcaacttttcgctagacacttttggaacagagcaggtactatgggtgacaccgttccacaatctttgtatatcaaaggaactggtatgagagcttctcctggatcctgtgtttactcaccatctccttccggttctattgttacctcagactctcaattgtttaacaagccatactggttacacaaggctcagggtcataacaatggtgtttgctggcacaaccaattgttcgttactgtcgttgacaccactagatccaccaatttgacaatttgtgcttctactcagtcccctgttccaggtcaatatgatgctaccaaattcaagcaatactctagacacgttgaggaatatgacttgcagtttatcttccaattgtgtactattaccttgactgccgatgttatgtcttacattcattccatgaactcttcaatcttggaggattggaactttggtgttccaccacctccaacaacttctttggttgacacctacagattcgtccaatctgttgctattacctgtcagaaggatgccgcaccagctgaaaacaaggatccatacgacaagttgaagttttggaatgtcgatttgaaggagaagttctcccttgacttggatcaatacccacttggtagaaagttcttggttcaggctggtttgagacgtaagccaactatcggtccaagaaaacgttctgctccatcagccactacctcttccaaacctgctaagagagttcgtgtcagagctagaaagtaatagcggccgcataaggaa
16L1-18L1-pMMZ sequence (SEQ ID NO: 45)
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaagctagcatggctctgtggagaccatctgacaacaccgtctacttgccacctccatctgttgccagagtcgttaacaccgacgattacgtcactagaacatccatcttctaccacgctggttcctctagattgcttactgttggtaatccatacttcagagttccagctggtggaggtaataagcaagacattcctaaggtttctgcttaccaatatagagtctttagagttcagttgcctgacccaaacaagttcggtttgcctgatacttctatctacaacccagagactcaaagattggtttgggcctgtgctggtgtcgagatcggtagaggacaacctttgggagttggtctttctggacacccattctacaacaagttggatgacactgaatcttcacatgctgccacttccaatgtttctgaggatgttcgtgacaacgtctctgttgattacaagcaaactcagttgtgtatccttggatgtgcccctgctattggtgaacactgggctaaaggtactgcctgtaagtccagaccattgtctcaaggtgactgcccacctttggagcttaagaacactgttttggaagatggtgacatggttgatactggatatggtgctatggacttctctaccctgcaggatactaagtgtgaggttccattggacatctgtcaatctatttgtaagtaccctgattatcttcagatgtctgctgacccatacggtgattctatgttcttttgcttgagacgtgagcaacttttcgctagacacttttggaacagagcaggtactatgggtgacaccgttccacaatctttgtatatcaaaggaactggtatgagagcttctcctggatcctgtgtttactcaccatctccttccggttctattgttacctcagactctcaattgtttaacaagccatactggttacacaaggctcagggtcataacaatggtgtttgctggcacaaccaattgttcgttactgtcgttgacaccactagatccaccaatttgacaatttgtgcttctactcagtcccctgttccaggtcaatatgatgctaccaaattcaagcaatactctagacacgttgaggaatatgacttgcagtttatcttccaattgtgtactattaccttgactgccgatgttatgtcttacattcattccatgaactcttcaatcttggaggattggaactttggtgttccaccacctccaacaacttctttggttgacacctacagattcgtccaatctgttgctattacctgtcagaaggatgccgcaccagctgaaaacaaggatccatacgacaagttgaagttttggaatgtcgatttgaaggagaagttctcccttgacttggatcaatacccacttggtagaaagttcttggttcaggctggtttgagacgtaagccaactatcggtccaagaaaacgttctgctccatcagccactacctcttccaaacctgctaagagagttcgtgtcagagctagaaagtaatagcggccgccagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatgtctctttggttgccatccgaggctactgtttacttgccacctgtcccagtttctaaggtcgtgtcaactgatgaatacgttgctagaaccaacatctattaccacgctggaacttctagattgcttgctgttggtcatccatacttccctatcaagaaacctaacaataacaagatccttgttccaaaggtctctggattgcagtacagagttttcagaatccacttgcctgacccaaacaagttcggttttcctgacacctccttctacaacccagatactcaaagattggtgtgggcctgtgttggtgtcgaggttggacgtggtcaaccattgggagttggtatttctggacatcctttgcttaacaagttggatgacactgaaaatgcttctgcctacgcagccaacgctggtgttgacaacagagagtgtatctctatggattacaaacagacccaattgtgtcttattggttgcaagccacctattggtgaacactggggtaagggatctccatgtaccaacgttgctgtcaatccaggtgattgtcctccattggagcttatcaacactgttattcaagatggtgacatggttgatactggatttggtgctatggacttcactaccttgcaagctaacaagtccgaggttccattggacatttgtacttctatctgcaagtatccagactacattaagatggtctctgaaccatacggtgactccttgtttttctaccttcgtagagagcaaatgtttgttagacacttgttcaacagagctggtgccgttggtgagaatgtcccagacgatttgtacatcaagggatctggttccactgcaaacttggcctcttccaactacttcccaactccttcaggttctatggttacctccgatgctcaaatcttcaacaagccttactggttgcaaagagcacagggtcacaacaatggaatttgttggggtaaccaacttttcgttactgtcgttgataccacaagatccaccaacatgtccttgtgtgctgccatttctacttcagaaaccacatacaagaatactaacttcaaggagtaccttagacatggtgaggaatacgacttgcagttcatctttcaattgtgcaagattaccttgactgctgacgttatgacctacatccactctatgaactccactatccttgaagattggaacttcggattgcaaccacctccaggtggaaccttggaagatacttacagatttgtcacatcccaagccattgcttgtcagaagcacactcctccagctcctaaggaagatccacttaagaaatacaccttctgggaagtcaacttgaaagagaagttctctgctgaccttgatcagtttcctttgggtagaaagttcttacttcaagctggattgaaggccaaaccaaagttcaccttgggtaaaagaaaggctactccaaccacatcatctacttccacaaccgccaaacgtaagaagagaaaactgtaatagcctgcaggcagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
The enzyme digestion identification results of 6L1-11L1-pMMG and 16L1-18L1-pMMZ are shown in figure 5, and the results show that 6L1-11L1-pMMG can obtain a 7719bp +1513bp fragment after NheI + NotI enzyme digestion treatment, and can obtain a 7710bp +1522bp fragment after BstBI + SbfI enzyme digestion treatment; the 16L1-18L1-pMMZ is digested by NheI and NotI to obtain a fragment of about 7322bp +1533bp, and is digested by BstBI and SbfI to obtain a fragment of about 4496+2825+1534 bp. The cleavage result is consistent with the theoretical band.
Example 3 construction and screening of four expression strains
Based on the 6L1-11L1-pMMG double expression plasmid and the 16L1-18L1-pMMZ double expression plasmid constructed in the example 2, the two double expression plasmids are integrated into the Hansenula polymorpha host strain CBS4732(ATCC 34438) by a method of twice transformation and twice screening (two screening markers), so as to obtain a recombinant engineering yeast strain which simultaneously expresses HPV6L1, HPV11L1, HPV16L1 and HPV18L1, and the specific steps are as follows:
3.1 construction and screening of HPV16L 1/HPV 18L1 double expression strain
The recombinant 16L1-18L1-pMMZ plasmid was linearized by BglII enzyme, electrotransferred to Hansenula yeast CBS4732 under 1500V, 120. omega. and 50. mu.F. After electroporation, YPD plates (200. mu.g/ml Zeocin) were plated with the inoculum and cultured overnight in an inverted manner at 37 ℃. A portion of the clones were streaked onto YPD plates (1500. mu.g/ml Zeocin), and cultured in an inverted state at 37 ℃ for 2 days. Several strains with good growth conditions were selected and inoculated in YPD liquid medium, cultured with shaking at 37 ℃ for one day, centrifuged to collect the cells, discarded in YPD medium, added with BMMY medium (0.5% methanol), and cultured with shaking at 37 ℃ for 48 hours. Collecting thallus, collecting supernatant after breaking glass beads by vigorous shaking, detecting the expression of HPV16L1 and HPV18L1 by dot blot hybridization (FIG. 6), and detecting the neutralizing monoclonal antibodies of HPV16L1 type specificity H16.V5 and HPV18L1 type specificity H18.J4 (supplied by Neil Christensen laboratory of Pennsylvania State university). One strain with ideal expression conditions of HPV16L1 and HPV18L1 is picked, stored and used for further construction, and the strain is named as Dual-L1-HP.
3.2 construction and screening of HPV6L 1/HPV 11L 1/HPV 16L 1/HPV 18L1 four-expression strain
The recombinant 6L1-11L1-pMMG plasmid was linearized by BglII enzyme and electrotransferred to Dual-L1-HP, which was constructed in example 3.1, under conditions of 1500V, 120. omega. and 50. mu.F. After electroporation, YPD plates (200. mu.g/ml G418) were plated with the resulting suspension and cultured overnight in an inverted manner at 37 ℃. A portion of the clones was streaked onto YPD plates (1200. mu.g/ml G418) and cultured in an inverted state at 37 ℃ for 2 days. Several strains with good growth conditions were selected and inoculated in YPD liquid medium, cultured with shaking at 37 ℃ for one day, centrifuged to collect the cells, discarded in YPD medium, added with BMMY medium (0.5% methanol), and cultured with shaking at 37 ℃ for 48 hours. Collecting thalli, collecting a germ-breaking supernatant after violent shaking and breaking of glass beads, detecting the expression conditions of HPV6L1, HPV11L1, HPV16L1 and HPV18L1 by a dot blot hybridization method (figure 7), wherein the antibodies for detection are an HPV6L1 type specific neutralizing monoclonal antibody H6.M48, an HPV11L1 type specific neutralizing monoclonal antibody H11.H3 neutralizing monoclonal antibody, an HPV16L1 type specific neutralizing monoclonal antibody H16.V5 neutralizing monoclonal antibody and an HPV18L1 type specific neutralizing monoclonal antibody H18.J4 neutralizing monoclonal antibody respectively. One strain with ideal expression conditions of HPV6L1, HPV11L1, HPV16L1 and HPV18L1 is picked and stored, and the strain is named as Quadri-L1-HP.
Example 4 preparation of HPV6L 1/HPV 11L 1/HPV 16L 1/HPV 18L1 antigen
Quadri-L1-HP was inoculated into 500ml YPD medium, and the mixture was subjected to shaking culture at 37 ℃ for 16-20 hours, and the cultured bacterial solution was used as a fermentation seed bacterial solution in the following ratio of 1: 15 in proportion was inoculated in a BIOTECH-7JG-7000A fermenter (Boehringer Bio) with BSM as the basic salt. Setting the initial fermentation temperature at 37 ℃, setting the initial pH value to 5-6, rotating speed at 300rpm, ventilation volume at 0.5vvm and DO value at 100%, and adding PTM1 trace salt. Culturing for about 20-24 hours in the initial proliferation stage, and maintaining the dissolved oxygen value at 20-40% by adjusting the stirring speed, the air flow, the tank pressure and supplementing pure oxygen. When the wet weight of the thalli reaches 60-90 g/L, 50% glycerol or glucose solution is supplemented. And maintaining the dissolved oxygen value at 20-40%, and supplementing for 4-8 hours. And stopping feeding when the wet weight of the thalli is detected to be increased to 100-200 g/L. Setting the temperature at 30-37 ℃, controlling the pH value to be 6-7, and starting to add methanol for induction, wherein the supplement speed of the methanol is 50-100 mL/min. Maintaining the dissolved oxygen value to be more than 20%, setting the temperature to be 30-37 ℃, controlling the pH value to be 6-7, and finishing the fermentation after 40 hours of induction. Centrifuging at 8000rpm for 10 min at 4 deg.C, and freezing at-20 deg.C. According to the following steps: 3 adding bacteria breaking buffer solution, 1200bar, homogenizing under high pressure and breaking for 4 times. After the disruption, the suspension was centrifuged at 8000rpm at 4 ℃ for 30min to collect the supernatant. And (3) carrying out gradient super-separation on the broken bacteria supernatant by using 20-50% of sucrose at 4 ℃, carrying out super-separation for 4 hours at 40000rpm, collecting a 40% gradient sample, and carrying out ultrafiltration to remove the sucrose to obtain a purified Quadri-L1 sample.
Example 5 detection of antigen content of HPV6L 1/HPV 11L 1/HPV 16L 1/HPV 18L1
Detecting the contents of HPV6L1, HPV11L1, HPV16L1 and HPV18L1 in Quadri-L1 by sandwich ELISA, wherein a coating antibody is self-made rabbit polyclonal antibody, and a detection antibody is type specificity neutralizing monoclonal antibody: quantifying the content of HPV6L1 by using HPV6L1 rabbit polyclonal antibody and H6.M48 neutralizing monoclonal antibody; the content of HPV11L1 is quantified by using HPV11L1 rabbit polyclonal antibody (and H11.H3 neutralizing monoclonal antibody; the content of HPV16L1 is quantified by using HPV16L1 rabbit polyclonal antibody and H16.V5 neutralizing monoclonal antibody; and the content of HPV18L1 is quantified by using HPV18L1 rabbit polyclonal antibody and H18.J4 neutralizing monoclonal antibody.
The operation steps are briefly described as follows: adding different types of homemade rabbit polyclonal antibodies into a 96-hole enzyme label plate respectively, and coating overnight at 4 ℃; discarding the coating solution, washing, and sealing at 37 deg.C for 2 hr; adding a Quadri-L1 purified sample and corresponding HPV6L1, HPV11L1, HPV16L1 and HPV18L1 standard substances (6-12 gradients of double dilution), and standing at 37 ℃ for 1 hour; adding different type specificity neutralizing monoclonal antibodies corresponding to rabbit polyclonal antibody types respectively, and standing at 37 ℃ for 1 hour; adding goat anti-mouse IgG labeled with HRP, and keeping the temperature at 37 ℃ for 1 hour; adding TMB color development liquid, and standing for 5-20 minutes at 37 ℃; and (4) adding a stop solution to stop the color reaction, and measuring the absorption value of 450nm by using an enzyme-labeled instrument. Standard curves were prepared from the readings of HPV6L1, HPV11L1, HPV16L1 and HPV18L1 standards to determine the corresponding antigen content in the Quadri-L1 purified sample (table 1).
TABLE 1Quadri-L1 detection of antigen content of HPV6L1, HPV11L1, HPV16L1 and HPV18L1 in purified samples (Sandwich ELISA)
Quadri-L1 antigen content Antigen content (μ g/ml)
HPV6 L1 6.984
HPV11L1 15.201
HPV16L1 7.112
HPV18L1 5.018
Example 6 confirmation of particle Structure of HPV6L 1/HPV 11L 1/HPV 16L 1/HPV 18L1
The Transmission Electron Microscope (TEM) observes the Quadri-L1 purified sample, and the Quadri-L1 purified sample forms VLP with the particle diameter of about 55-70 nm as shown in FIG. 8.
Example 7 preparation of HPV6L 1/HPV 11L 1/HPV 16L 1/HPV 18L1 vaccine
Adsorbing an aluminum phosphate adjuvant on a purified sample of the Quadri-L1 to prepare the Quadri-L1 vaccine.
Example 8 HPV6L 1/HPV 11L 1/HPV 16L 1/HPV 18L1 vaccine immunogenicity assay
6-8 weeks old BALB/c mice (SCXK (Jing) 2016-. Immunization procedure: three times of immunization (subcutaneous injection) are carried out for 0 day, 14 days and 28 days, the mice are killed in 42 days, blood is taken from eyeballs, centrifugation is carried out for 10 minutes at 4000g, and serum is taken and frozen at the temperature of 20 ℃ below zero.
ELISA is used for detecting the positive conversion rate of the mouse serum, and the method is briefly described as follows: respectively adding HPV6L1, HPV11L1, HPV16L1 and HPV18L1 standard substances into a 96-hole enzyme label plate, and coating overnight at 4 ℃; discarding the coating solution, washing, and sealing at 37 deg.C for 2 hr; adding diluted rat serum, and standing at 37 deg.C for 1 hr; discarding mouse serum, washing, adding goat anti-mouse IgG labeled by HRP, and keeping the temperature at 37 ℃ for 1 hour; adding TMB color development liquid, and standing for 5-20 minutes at 37 ℃; the chromogenic reaction was stopped by adding stop solution, and the absorbance at 450nm was measured by a microplate reader, and the absorbance at 450nm was set as the Cutoff value by adding 3-fold Standard Deviation (SD) to the average of the absorbance at 450nm of 8 negative control sera (mouse sera adjuvanted with immunoaluminum). And (4) determining that the OD450 value of the diluted serum to be detected is greater than the Cutoff value as positive, and determining that the diluted serum is negative otherwise. The highest positive dilution factor of all the sera to be tested was taken as the titer value of the serum antibody, and the Geometric Mean Titer (GMT) of the antibody of each group was calculated, respectively, and the results are shown in FIG. 9.
The result shows that serum after Quadri-L1 immunization can induce HPV6L1, HPV11L1, HPV16L1 and HPV18L1 antibodies, the antibody titer levels are similar, and a certain dose-effect relationship is achieved.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Sequence listing
<110> Chongqing Bowei Baitai biopharmaceutical Co., Ltd
SHANGHAI BOWEI BIOTECHNOLOGY Co.,Ltd.
<120> recombinant expression vector system, recombinant engineering bacteria, preparation method and application thereof
<160> 45
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1506
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgtggagac catctgactc gactgtctac gttcctccac ctaacccagt ctctaaggtt 60
gtggctaccg acgcctacgt gactagaacc aacatcttct accacgcatc gtcctctaga 120
ttgctggccg ttggacatcc ttacttctcc atcaagagag ccaacaaaac cgtcgtgcca 180
aaggtttctg gctaccagta tagagtcttc aaggtggttc tgcctgaccc taacaagttt 240
gccctgccag actcgtcctt gttcgatcct acgactcaaa gactcgtttg ggcctgcact 300
ggtcttgagg ttggcagagg acagccactc ggagtcggcg tttctggtca tcctttcctg 360
aacaagtacg acgatgttga gaactctggt tccggaggca atcctggaca ggacaacaga 420
gtcaatgttg gcatggacta caagcaaact cagctctgta tggtgggatg cgcacctcca 480
ctgggtgagc attggggaaa aggcaagcag tgcaccaaca ctccagttca ggcaggtgac 540
tgccctccat tggagctgat tacctcggtc atccaggacg gtgatatggt tgacaccgga 600
ttcggtgcca tgaacttcgc tgacctgcag accaacaagt ccgatgtgcc aatcgacatc 660
tgcggaacga cctgtaaata ccctgactac ttgcagatgg cagccgatcc atacggcgac 720
agactcttct tctttctgag aaaggagcag atgtttgcta gacacttttt caacagagcc 780
ggagaggtcg gtgagcctgt tccagacacc ctgatcatta agggctcggg aaacagaact 840
tctgtcggct cctcgatcta cgtgaacacc ccttccggtt cgttggtgtc gtccgaggct 900
caactgttca acaagccata ctggctccag aaagcccaag gtcacaacaa tggcatctgc 960
tggggtaacc agctgttcgt tactgtcgtg gacaccacga gatcgaccaa catgactctg 1020
tgtgcctccg ttaccacatc ttcgacttac accaactccg actataagga gtacatgaga 1080
cacgttgaag agtacgactt gcaattcatc tttcagctct gctcgattac cctgtccgca 1140
gaagtcatgg cctacatcca cactatgaat ccatcggtgc ttgaggactg gaactttggc 1200
ttgtccccac ctccaaacgg aactctggaa gacacctata gatacgtgca gtcgcaagcc 1260
atcacgtgcc agaagccaac ccctgagaag gaaaagccag atccttacaa gaacctgtcc 1320
ttctgggagg tcaatctcaa ggagaagttc tcgtccgaac tggaccagta cccattgggt 1380
agaaagttcc tgctccagtc tggataccgt ggtagatcgt ccatcagaac cggagtgaag 1440
agaccagcag tttccaaggc ttctgccgca cctaagcgta aaagagccaa gaccaagaga 1500
taatag 1506
<210> 2
<211> 1509
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgtggagac catctgactc gaccgtctac gttcctccac ctaacccagt ctccaaagtg 60
gttgctactg atgcctacgt caagagaacc aacatcttct accacgcctc ttcgtccaga 120
ttgctggcag ttggacatcc ttactattcc atcaagaaag ttaacaagac cgtggtccct 180
aaggtttcgg gctatcagta cagagtgttc aaggtcgttc tgccagaccc taacaagttt 240
gccttgccag attcctcgct gttcgaccct accactcaaa gactggtttg ggcttgcacg 300
ggtttggaag tgggcagagg acaaccactg ggtgtcggag tttccggcca tccattgctg 360
aacaagtacg acgatgttga gaactcgggt ggatacggtg gcaatccagg acaagacaac 420
agagtgaatg tcggcatgga ctacaagcaa acccagctgt gcatggttgg ttgtgcacca 480
cctctgggtg aacattgggg aaagggcact cagtgctcca acacctctgt tcagaatggt 540
gactgtcctc cactggagct gatcacctcg gttattcagg atggagacat ggtggacact 600
ggcttcggtg caatgaactt tgccgacttg cagaccaaca agtccgatgt tccattggac 660
atttgcggca ctgtgtgcaa atatcctgac tacctgcaga tggctgcaga tccatacggt 720
gacagactct tcttctacct gagaaaggag cagatgttcg ccagacactt ctttaacaga 780
gctggcaccg ttggtgagcc tgttccagat gacctgttgg tgaagggtgg aaacaataga 840
tcctctgttg cctcgtccat ctacgtccac accccatcgg gctctctggt gtcctcggag 900
gcccaactgt tcaacaagcc ttactggctc cagaaggccc aaggacacaa caatggcatc 960
tgctggggaa accacctgtt cgtcaccgtt gtggacacga ctagatccac caacatgact 1020
ttgtgtgcct ctgtttccaa gtcggctacc tacacgaact cggactataa ggagtacatg 1080
agacatgttg aagagttcga cctgcagttt atcttccagc tctgctccat taccttgtcg 1140
gcagaagtga tggcctacat ccacacgatg aacccatccg tcttggaaga ctggaacttc 1200
ggtctgtctc cacctccaaa tggcaccctg gaagacacgt acagatatgt gcagtcgcaa 1260
gccatcacct gtcagaaacc aactcctgaa aaggagaaac aggacccata caaggatatg 1320
tcgttctggg aggtcaacct gaaggagaag ttctcttcgg aactggacca gttcccattg 1380
ggaagaaagt tcctgctcca gtctggttat agaggaagaa cctccgcaag aactggcatc 1440
aagagacctg ccgtgtcgaa gccatccacc gctcctaaga gaaagagaac gaagaccaaa 1500
aagtaatag 1509
<210> 3
<211> 1521
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgtctcttt ggttgccatc cgaggctact gtttacttgc cacctgtccc agtttctaag 60
gtcgtgtcaa ctgatgaata cgttgctaga accaacatct attaccacgc tggaacttct 120
agattgcttg ctgttggtca tccatacttc cctatcaaga aacctaacaa taacaagatc 180
cttgttccaa aggtctctgg attgcagtac agagttttca gaatccactt gcctgaccca 240
aacaagttcg gttttcctga cacctccttc tacaacccag atactcaaag attggtgtgg 300
gcctgtgttg gtgtcgaggt tggacgtggt caaccattgg gagttggtat ttctggacat 360
cctttgctta acaagttgga tgacactgaa aatgcttctg cctacgcagc caacgctggt 420
gttgacaaca gagagtgtat ctctatggat tacaaacaga cccaattgtg tcttattggt 480
tgcaagccac ctattggtga acactggggt aagggatctc catgtaccaa cgttgctgtc 540
aatccaggtg attgtcctcc attggagctt atcaacactg ttattcaaga tggtgacatg 600
gttgatactg gatttggtgc tatggacttc actaccttgc aagctaacaa gtccgaggtt 660
ccattggaca tttgtacttc tatctgcaag tatccagact acattaagat ggtctctgaa 720
ccatacggtg actccttgtt tttctacctt cgtagagagc aaatgtttgt tagacacttg 780
ttcaacagag ctggtgccgt tggtgagaat gtcccagacg atttgtacat caagggatct 840
ggttccactg caaacttggc ctcttccaac tacttcccaa ctccttcagg ttctatggtt 900
acctccgatg ctcaaatctt caacaagcct tactggttgc aaagagcaca gggtcacaac 960
aatggaattt gttggggtaa ccaacttttc gttactgtcg ttgataccac aagatccacc 1020
aacatgtcct tgtgtgctgc catttctact tcagaaacca catacaagaa tactaacttc 1080
aaggagtacc ttagacatgg tgaggaatac gacttgcagt tcatctttca attgtgcaag 1140
attaccttga ctgctgacgt tatgacctac atccactcta tgaactccac tatccttgaa 1200
gattggaact tcggattgca accacctcca ggtggaacct tggaagatac ttacagattt 1260
gtcacatccc aagccattgc ttgtcagaag cacactcctc cagctcctaa ggaagatcca 1320
cttaagaaat acaccttctg ggaagtcaac ttgaaagaga agttctctgc tgaccttgat 1380
cagtttcctt tgggtagaaa gttcttactt caagctggat tgaaggccaa accaaagttc 1440
accttgggta aaagaaaggc tactccaacc acatcatcta cttccacaac cgccaaacgt 1500
aagaagagaa aactgtaata g 1521
<210> 4
<211> 1527
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atggctctgt ggagaccatc tgacaacacc gtctacttgc cacctccatc tgttgccaga 60
gtcgttaaca ccgacgatta cgtcactaga acatccatct tctaccacgc tggttcctct 120
agattgctta ctgttggtaa tccatacttc agagttccag ctggtggagg taataagcaa 180
gacattccta aggtttctgc ttaccaatat agagtcttta gagttcagtt gcctgaccca 240
aacaagttcg gtttgcctga tacttctatc tacaacccag agactcaaag attggtttgg 300
gcctgtgctg gtgtcgagat cggtagagga caacctttgg gagttggtct ttctggacac 360
ccattctaca acaagttgga tgacactgaa tcttcacatg ctgccacttc caatgtttct 420
gaggatgttc gtgacaacgt ctctgttgat tacaagcaaa ctcagttgtg tatccttgga 480
tgtgcccctg ctattggtga acactgggct aaaggtactg cctgtaagtc cagaccattg 540
tctcaaggtg actgcccacc tttggagctt aagaacactg ttttggaaga tggtgacatg 600
gttgatactg gatatggtgc tatggacttc tctaccctgc aggatactaa gtgtgaggtt 660
ccattggaca tctgtcaatc tatttgtaag taccctgatt atcttcagat gtctgctgac 720
ccatacggtg attctatgtt cttttgcttg agacgtgagc aacttttcgc tagacacttt 780
tggaacagag caggtactat gggtgacacc gttccacaat ctttgtatat caaaggaact 840
ggtatgagag cttctcctgg atcctgtgtt tactcaccat ctccttccgg ttctattgtt 900
acctcagact ctcaattgtt taacaagcca tactggttac acaaggctca gggtcataac 960
aatggtgttt gctggcacaa ccaattgttc gttactgtcg ttgacaccac tagatccacc 1020
aatttgacaa tttgtgcttc tactcagtcc cctgttccag gtcaatatga tgctaccaaa 1080
ttcaagcaat actctagaca cgttgaggaa tatgacttgc agtttatctt ccaattgtgt 1140
actattacct tgactgccga tgttatgtct tacattcatt ccatgaactc ttcaatcttg 1200
gaggattgga actttggtgt tccaccacct ccaacaactt ctttggttga cacctacaga 1260
ttcgtccaat ctgttgctat tacctgtcag aaggatgccg caccagctga aaacaaggat 1320
ccatacgaca agttgaagtt ttggaatgtc gatttgaagg agaagttctc ccttgacttg 1380
gatcaatacc cacttggtag aaagttcttg gttcaggctg gtttgagacg taagccaact 1440
atcggtccaa gaaaacgttc tgctccatca gccactacct cttccaaacc tgctaagaga 1500
gttcgtgtca gagctagaaa gtaatag 1527
<210> 5
<211> 1512
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gtcgacgcgg agaacgatct cctcgagctg ctcgcggatc agcttgtggc ccggtaatgg 60
aaccaggccg acggcacgct ccttgcggac cacggtggct ggcgagccca gtttgtgaac 120
gaggtcgttt agaacgtcct gcgcaaagtc cagtgtcaga tgaatgtcct cctcggacca 180
attcagcatg ttctcgagca gccatctgtc tttggagtag aagcgtaatc tctgctcctc 240
gttactgtac cggaagaggt agtttgcctc gccgcccata atgaacaggt tctctttctg 300
gtggcctgtg agcagcgggg acgtctggac ggcgtcgatg aggcccttga ggcgctcgta 360
gtacttgttc gcgtcgctgt agccggccgc ggtgacgata cccacataga ggtccttggc 420
cattagtttg atgaggtggg gcaggatggg cgactcggca tcgaaatttt tgccgtcgtc 480
gtacagtgtg atgtcaccat cgaatgtaat gagctgcagc ttgcgatctc ggatggtttt 540
ggaatggaag aaccgcgaca tctccaacag ctgggccgtg ttgagaatga gccggacgtc 600
gttgaacgag ggggccacaa gccggcgttt gctgatggcg cggcgctcgt cctcgatgta 660
gaaggccttt tccagaggca gtctcgtgaa gaagctgcca acgctcggaa ccagctgcac 720
gagccgagac aattcggggg tgccggcttt ggtcatttca atgttgtcgt cgatgaggag 780
ttcgaggtcg tggaagattt ccgcgtagcg gcgttttgcc tcagagttta ccatgaggtc 840
gtccactgca gagatgccgt tgctcttcac cgcgtacagg acgaacggcg tggccagcag 900
gcccttgatc cattctatga ggccatctcg acggtgttcc ttgagtgcgt actccactct 960
gtagcgactg gacatctcga gactgggctt gctgtgctgg atgcaccaat taattgttgc 1020
cgcatgcatc cttgcaccgc aagtttttaa aacccactcg ctttagccgt cgcgtaaaac 1080
ttgtgaatct ggcaactgag ggggttctgc agccgcaacc gaacttttcg cttcgaggac 1140
gcagctggat ggtgtcatgt gaggctctgt ttgctggcgt agcctacaac gtgaccttgc 1200
ctaaccggac ggcgctaccc actgctgtct gtgcctgcta ccagaaaatc accagagcag 1260
cagagggccg atgtggcaac tggtggggtg tcggacaggc tgtttctcca cagtgcaaat 1320
gcgggtgaac cggccagaaa gtaaattctt atgctaccgt gcagtgactc cgacatcccc 1380
agtttttgcc ctacttgatc acagatgggg tcagcgctgc cgctaagtgt acccaaccgt 1440
ccccacacgg tccatctata aatactgctg ccagtgcacg gtggtgacat caatctaaag 1500
tacaaaaaca aa 1512
<210> 6
<211> 341
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gtttgtagcc ttagacatga ctgttcctca gttcaagttg ggcacttacg agaagaccgg 60
tcttgctaga ttctaatcaa gaggatgtca gaatgccatt tgcctgagag atgcaggctt 120
catttttgat acttttttat ttgtaaccta tatagtatag gatttttttt gtcattttgt 180
ttcttctcgt acgagcttgc tcctgatcag cctatctcgc agctgatgaa tatcttgtgg 240
taggggtttg ggaaaatcat tcgagtttga tgtttttctt ggtatttccc actcctcttc 300
agagtacaga agattaagtg agaccttcgt ttgtgcggat c 341
<210> 7
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
accaaaagat ctgtcgacgc ggag 24
<210> 8
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cttggctaca tatgatcgat cgtgtacacg tttcgaattt gtttttgtac ttt 53
<210> 9
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
tcaagtcaag cttcacgtgg ctagctttgt ttttgtactt t 41
<210> 10
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gcccgatcat atgcctgcag gcagctttct agaacaa 37
<210> 11
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
aaacgtgaag cttgaattcg cggccgccag ctttct 36
<210> 12
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tgggggatcc gcacaaacga a 21
<210> 13
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
cccttagatc tcccacacac catagct 27
<210> 14
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
tgcggtctta aggtttagtt cctcacc 27
<210> 15
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
tattggctta agatgagcca tattcaac 28
<210> 16
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gggtatcgtc accgcggccg gctacagcac ttgaagtcgg acag 44
<210> 17
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gttggatcct cgatcacatg tgagc 25
<210> 18
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
aaaggatcct gaggtctgcc tcgt 24
<210> 19
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
cggacaaatt cgaaacgatg t 21
<210> 20
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
acagaaacct gcaggctatt actttttggt c 31
<210> 21
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
aatcgctagc atgtggagac catctg 26
<210> 22
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ttggtaatgc ggccgcctat tatctcttgg tc 32
<210> 23
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
tgcttcctgc aggctattac agttttctc 29
<210> 24
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
attcgctagc atggctctgt ggagac 26
<210> 25
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
ttccttatgc ggccgctatt actttctagc t 31
<210> 26
<211> 1561
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
accaaaagat ctgtcgacgc ggagaacgat ctcctcgagc tgctcgcgga tcagcttgtg 60
gcccggtaat ggaaccaggc cgacggcacg ctccttgcgg accacggtgg ctggcgagcc 120
cagtttgtga acgaggtcgt ttagaacgtc ctgcgcaaag tccagtgtca gatgaatgtc 180
ctcctcggac caattcagca tgttctcgag cagccatctg tctttggagt agaagcgtaa 240
tctctgctcc tcgttactgt accggaagag gtagtttgcc tcgccgccca taatgaacag 300
gttctctttc tggtggcctg tgagcagcgg ggacgtctgg acggcgtcga tgaggccctt 360
gaggcgctcg tagtacttgt tcgcgtcgct gtagccggcc gcggtgacga tacccacata 420
gaggtccttg gccattagtt tgatgaggtg gggcaggatg ggcgactcgg catcgaaatt 480
tttgccgtcg tcgtacagtg tgatgtcacc atcgaatgta atgagctgca gcttgcgatc 540
tcggatggtt ttggaatgga agaaccgcga catctccaac agctgggccg tgttgagaat 600
gagccggacg tcgttgaacg agggggccac aagccggcgt ttgctgatgg cgcggcgctc 660
gtcctcgatg tagaaggcct tttccagagg cagtctcgtg aagaagctgc caacgctcgg 720
aaccagctgc acgagccgag acaattcggg ggtgccggct ttggtcattt caatgttgtc 780
gtcgatgagg agttcgaggt cgtggaagat ttccgcgtag cggcgttttg cctcagagtt 840
taccatgagg tcgtccactg cagagatgcc gttgctcttc accgcgtaca ggacgaacgg 900
cgtggccagc aggcccttga tccattctat gaggccatct cgacggtgtt ccttgagtgc 960
gtactccact ctgtagcgac tggacatctc gagactgggc ttgctgtgct ggatgcacca 1020
attaattgtt gccgcatgca tccttgcacc gcaagttttt aaaacccact cgctttagcc 1080
gtcgcgtaaa acttgtgaat ctggcaactg agggggttct gcagccgcaa ccgaactttt 1140
cgcttcgagg acgcagctgg atggtgtcat gtgaggctct gtttgctggc gtagcctaca 1200
acgtgacctt gcctaaccgg acggcgctac ccactgctgt ctgtgcctgc taccagaaaa 1260
tcaccagagc agcagagggc cgatgtggca actggtgggg tgtcggacag gctgtttctc 1320
cacagtgcaa atgcgggtga accggccaga aagtaaattc ttatgctacc gtgcagtgac 1380
tccgacatcc ccagtttttg ccctacttga tcacagatgg ggtcagcgct gccgctaagt 1440
gtacccaacc gtccccacac ggtccatcta taaatactgc tgccagtgca cggtggtgac 1500
atcaatctaa agtacaaaaa caaattcgaa acgtgtacac gatcgatcat atgtagccaa 1560
g 1561
<210> 27
<211> 443
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gcccgatcat atgcctgcag gcagctttct agaacaaaaa ctcatctcag aagaggatct 60
gaatagcgcc gtcgaccatc atcatcatca tcattgagtt tgtagcctta gacatgactg 120
ttcctcagtt caagttgggc acttacgaga agaccggtct tgctagattc taatcaagag 180
gatgtcagaa tgccatttgc ctgagagatg caggcttcat ttttgatact tttttatttg 240
taacctatat agtataggat tttttttgtc attttgtttc ttctcgtacg agcttgctcc 300
tgatcagcct atctcgcagc tgatgaatat cttgtggtag gggtttggga aaatcattcg 360
agtttgatgt ttttcttggt atttcccact cctcttcaga gtacagaaga ttaagtgaga 420
ccttcgtttg tgcggatccc cca 443
<210> 28
<211> 3884
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgg cacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctgcgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttcgcgt cgctgtagcc ggccgcggtg acgataccca catagaggtc 420
cttggccatt agtttgatga ggtggggcag gatgggcgac tcggcatcga aatttttgcc 480
gtcgtcgtac agtgtgatgt caccatcgaa tgtaatgagc tgcagcttgc gatctcggat 540
ggttttggaa tggaagaacc gcgacatctc caacagctgg gccgtgttga gaatgagccg 600
gacgtcgttg aacgaggggg ccacaagccg gcgtttgctg atggcgcggc gctcgtcctc 660
gatgtagaag gccttttcca gaggcagtct cgtgaagaag ctgccaacgc tcggaaccag 720
ctgcacgagc cgagacaatt cgggggtgcc ggctttggtc atttcaatgt tgtcgtcgat 780
gaggagttcg aggtcgtgga agatttccgc gtagcggcgt tttgcctcag agtttaccat 840
gaggtcgtcc actgcagaga tgccgttgct cttcaccgcg tacaggacga acggcgtggc 900
cagcaggccc ttgatccatt ctatgaggcc atctcgacgg tgttccttga gtgcgtactc 960
cactctgtag cgactggaca tctcgagact gggcttgctg tgctggatgc accaattaat 1020
tgttgccgca tgcatccttg caccgcaagt ttttaaaacc cactcgcttt agccgtcgcg 1080
taaaacttgt gaatctggca actgaggggg ttctgcagcc gcaaccgaac ttttcgcttc 1140
gaggacgcag ctggatggtg tcatgtgagg ctctgtttgc tggcgtagcc tacaacgtga 1200
ccttgcctaa ccggacggcg ctacccactg ctgtctgtgc ctgctaccag aaaatcacca 1260
gagcagcaga gggccgatgt ggcaactggt ggggtgtcgg acaggctgtt tctccacagt 1320
gcaaatgcgg gtgaaccggc cagaaagtaa attcttatgc taccgtgcag tgactccgac 1380
atccccagtt tttgccctac ttgatcacag atggggtcag cgctgccgct aagtgtaccc 1440
aaccgtcccc acacggtcca tctataaata ctgctgccag tgcacggtgg tgacatcaat 1500
ctaaagtaca aaaacaaatt cgaaacgtgt acacgatcga tcatatgcct gcaggcagct 1560
ttctagaaca aaaactcatc tcagaagagg atctgaatag cgccgtcgac catcatcatc 1620
atcatcattg agtttgtagc cttagacatg actgttcctc agttcaagtt gggcacttac 1680
gagaagaccg gtcttgctag attctaatca agaggatgtc agaatgccat ttgcctgaga 1740
gatgcaggct tcatttttga tactttttta tttgtaacct atatagtata ggattttttt 1800
tgtcattttg tttcttctcg tacgagcttg ctcctgatca gcctatctcg cagctgatga 1860
atatcttgtg gtaggggttt gggaaaatca ttcgagtttg atgtttttct tggtatttcc 1920
cactcctctt cagagtacag aagattaagt gagaccttcg tttgtgcgga tcccccacac 1980
accatagctt caaaatgttt ctactccttt tttactcttc cagattttct cggactccgc 2040
gcatcgccgt accacttcaa aacacccaag cacagcatac taaattttcc ctctttcttc 2100
ctctagggtg tcgttaatta cccgtactaa aggtttggaa aagaaaaaag agaccgcctc 2160
gtttcttttt cttcgtcgaa aaaggcaata aaaattttta tcacgtttct ttttcttgaa 2220
attttttttt ttagtttttt tctctttcag tgacctccat tgatatttaa gttaataaac 2280
ggtcttcaat ttctcaagtt tcagtttcat ttttcttgtt ctattacaac tttttttact 2340
tcttgttcat tagaaagaaa gcatagcaat ctaatctaag gggcggtgtt gacaattaat 2400
catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca 2460
agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct 2520
ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc 2580
gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc 2640
tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt 2700
ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg 2760
ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc 2820
aggactgaca cgtccgacgg cggcccacgg gtcccaggcc tcggagatcc gtcccccttt 2880
tcctttgtcg atatcatgta attagttatg tcacgcttac attcacgccc tccccccaca 2940
tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt 3000
tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg 3060
tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga 3120
cgctcgaagg ctttaatttg caagctggag accaacatgt gagcaaaagg ccagcaaaag 3180
gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 3240
gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 3300
taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 3360
accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc 3420
tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 3480
cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 3540
agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 3600
gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 3660
gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 3720
tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 3780
acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 3840
cagtggaacg aaaactcacg ttaagggatt ttggtcatga gatc 3884
<210> 29
<211> 1549
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
accaaaagat ctgtcgacgc ggagaacgat ctcctcgagc tgctcgcgga tcagcttgtg 60
gcccggtaat ggaaccaggc cgacggcacg ctccttgcgg accacggtgg ctggcgagcc 120
cagtttgtga acgaggtcgt ttagaacgtc ctgcgcaaag tccagtgtca gatgaatgtc 180
ctcctcggac caattcagca tgttctcgag cagccatctg tctttggagt agaagcgtaa 240
tctctgctcc tcgttactgt accggaagag gtagtttgcc tcgccgccca taatgaacag 300
gttctctttc tggtggcctg tgagcagcgg ggacgtctgg acggcgtcga tgaggccctt 360
gaggcgctcg tagtacttgt tcgcgtcgct gtagccggcc gcggtgacga tacccacata 420
gaggtccttg gccattagtt tgatgaggtg gggcaggatg ggcgactcgg catcgaaatt 480
tttgccgtcg tcgtacagtg tgatgtcacc atcgaatgta atgagctgca gcttgcgatc 540
tcggatggtt ttggaatgga agaaccgcga catctccaac agctgggccg tgttgagaat 600
gagccggacg tcgttgaacg agggggccac aagccggcgt ttgctgatgg cgcggcgctc 660
gtcctcgatg tagaaggcct tttccagagg cagtctcgtg aagaagctgc caacgctcgg 720
aaccagctgc acgagccgag acaattcggg ggtgccggct ttggtcattt caatgttgtc 780
gtcgatgagg agttcgaggt cgtggaagat ttccgcgtag cggcgttttg cctcagagtt 840
taccatgagg tcgtccactg cagagatgcc gttgctcttc accgcgtaca ggacgaacgg 900
cgtggccagc aggcccttga tccattctat gaggccatct cgacggtgtt ccttgagtgc 960
gtactccact ctgtagcgac tggacatctc gagactgggc ttgctgtgct ggatgcacca 1020
attaattgtt gccgcatgca tccttgcacc gcaagttttt aaaacccact cgctttagcc 1080
gtcgcgtaaa acttgtgaat ctggcaactg agggggttct gcagccgcaa ccgaactttt 1140
cgcttcgagg acgcagctgg atggtgtcat gtgaggctct gtttgctggc gtagcctaca 1200
acgtgacctt gcctaaccgg acggcgctac ccactgctgt ctgtgcctgc taccagaaaa 1260
tcaccagagc agcagagggc cgatgtggca actggtgggg tgtcggacag gctgtttctc 1320
cacagtgcaa atgcgggtga accggccaga aagtaaattc ttatgctacc gtgcagtgac 1380
tccgacatcc ccagtttttg ccctacttga tcacagatgg ggtcagcgct gccgctaagt 1440
gtacccaacc gtccccacac ggtccatcta taaatactgc tgccagtgca cggtggtgac 1500
atcaatctaa agtacaaaaa caaagctagc cacgtgaagc ttgacttga 1549
<210> 30
<211> 449
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
aaacgtgaag cttgaattcg cggccgccag ctttctagaa caaaaactca tctcagaaga 60
ggatctgaat agcgccgtcg accatcatca tcatcatcat tgagtttgta gccttagaca 120
tgactgttcc tcagttcaag ttgggcactt acgagaagac cggtcttgct agattctaat 180
caagaggatg tcagaatgcc atttgcctga gagatgcagg cttcattttt gatacttttt 240
tatttgtaac ctatatagta taggattttt tttgtcattt tgtttcttct cgtacgagct 300
tgctcctgat cagcctatct cgcagctgat gaatatcttg tggtaggggt ttgggaaaat 360
cattcgagtt tgatgttttt cttggtattt cccactcctc ttcagagtac agaagattaa 420
gtgagacctt cgtttgtgcg gatccccca 449
<210> 31
<211> 3879
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgg cacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctgcgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttcgcgt cgctgtagcc ggccgcggtg acgataccca catagaggtc 420
cttggccatt agtttgatga ggtggggcag gatgggcgac tcggcatcga aatttttgcc 480
gtcgtcgtac agtgtgatgt caccatcgaa tgtaatgagc tgcagcttgc gatctcggat 540
ggttttggaa tggaagaacc gcgacatctc caacagctgg gccgtgttga gaatgagccg 600
gacgtcgttg aacgaggggg ccacaagccg gcgtttgctg atggcgcggc gctcgtcctc 660
gatgtagaag gccttttcca gaggcagtct cgtgaagaag ctgccaacgc tcggaaccag 720
ctgcacgagc cgagacaatt cgggggtgcc ggctttggtc atttcaatgt tgtcgtcgat 780
gaggagttcg aggtcgtgga agatttccgc gtagcggcgt tttgcctcag agtttaccat 840
gaggtcgtcc actgcagaga tgccgttgct cttcaccgcg tacaggacga acggcgtggc 900
cagcaggccc ttgatccatt ctatgaggcc atctcgacgg tgttccttga gtgcgtactc 960
cactctgtag cgactggaca tctcgagact gggcttgctg tgctggatgc accaattaat 1020
tgttgccgca tgcatccttg caccgcaagt ttttaaaacc cactcgcttt agccgtcgcg 1080
taaaacttgt gaatctggca actgaggggg ttctgcagcc gcaaccgaac ttttcgcttc 1140
gaggacgcag ctggatggtg tcatgtgagg ctctgtttgc tggcgtagcc tacaacgtga 1200
ccttgcctaa ccggacggcg ctacccactg ctgtctgtgc ctgctaccag aaaatcacca 1260
gagcagcaga gggccgatgt ggcaactggt ggggtgtcgg acaggctgtt tctccacagt 1320
gcaaatgcgg gtgaaccggc cagaaagtaa attcttatgc taccgtgcag tgactccgac 1380
atccccagtt tttgccctac ttgatcacag atggggtcag cgctgccgct aagtgtaccc 1440
aaccgtcccc acacggtcca tctataaata ctgctgccag tgcacggtgg tgacatcaat 1500
ctaaagtaca aaaacaaagc tagccacgtg aagcttgaat tcgcggccgc cagctttcta 1560
gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca tcatcatcat 1620
cattgagttt gtagccttag acatgactgt tcctcagttc aagttgggca cttacgagaa 1680
gaccggtctt gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc 1740
aggcttcatt tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca 1800
ttttgtttct tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc 1860
ttgtggtagg ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc 1920
ctcttcagag tacagaagat taagtgagac cttcgtttgt gcggatcccc cacacaccat 1980
agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac tccgcgcatc 2040
gccgtaccac ttcaaaacac ccaagcacag catactaaat tttccctctt tcttcctcta 2100
gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc 2160
tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc ttgaaatttt 2220
tttttttagt ttttttctct ttcagtgacc tccattgata tttaagttaa taaacggtct 2280
tcaatttctc aagtttcagt ttcatttttc ttgttctatt acaacttttt ttacttcttg 2340
ttcattagaa agaaagcata gcaatctaat ctaaggggcg gtgttgacaa ttaatcatcg 2400
gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat ggccaagttg 2460
accagtgccg ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc 2520
gaccggctcg ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac 2580
gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc 2640
tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg 2700
aacttccggg acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg 2760
gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac 2820
tgacacgtcc gacggcggcc cacgggtccc aggcctcgga gatccgtccc ccttttcctt 2880
tgtcgatatc atgtaattag ttatgtcacg cttacattca cgccctcccc ccacatccgc 2940
tctaaccgaa aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat 3000
agttatgtta gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag 3060
acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc 3120
gaaggcttta atttgcaagc tggagaccaa catgtgagca aaaggccagc aaaaggccag 3180
gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 3240
tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 3300
ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 3360
atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag 3420
gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 3480
tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 3540
cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 3600
cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 3660
tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 3720
cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 3780
cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 3840
gaacgaaaac tcacgttaag ggattttggt catgagatc 3879
<210> 32
<211> 5846
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgg cacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctgcgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttcgcgt cgctgtagcc ggccgcggtg acgataccca catagaggtc 420
cttggccatt agtttgatga ggtggggcag gatgggcgac tcggcatcga aatttttgcc 480
gtcgtcgtac agtgtgatgt caccatcgaa tgtaatgagc tgcagcttgc gatctcggat 540
ggttttggaa tggaagaacc gcgacatctc caacagctgg gccgtgttga gaatgagccg 600
gacgtcgttg aacgaggggg ccacaagccg gcgtttgctg atggcgcggc gctcgtcctc 660
gatgtagaag gccttttcca gaggcagtct cgtgaagaag ctgccaacgc tcggaaccag 720
ctgcacgagc cgagacaatt cgggggtgcc ggctttggtc atttcaatgt tgtcgtcgat 780
gaggagttcg aggtcgtgga agatttccgc gtagcggcgt tttgcctcag agtttaccat 840
gaggtcgtcc actgcagaga tgccgttgct cttcaccgcg tacaggacga acggcgtggc 900
cagcaggccc ttgatccatt ctatgaggcc atctcgacgg tgttccttga gtgcgtactc 960
cactctgtag cgactggaca tctcgagact gggcttgctg tgctggatgc accaattaat 1020
tgttgccgca tgcatccttg caccgcaagt ttttaaaacc cactcgcttt agccgtcgcg 1080
taaaacttgt gaatctggca actgaggggg ttctgcagcc gcaaccgaac ttttcgcttc 1140
gaggacgcag ctggatggtg tcatgtgagg ctctgtttgc tggcgtagcc tacaacgtga 1200
ccttgcctaa ccggacggcg ctacccactg ctgtctgtgc ctgctaccag aaaatcacca 1260
gagcagcaga gggccgatgt ggcaactggt ggggtgtcgg acaggctgtt tctccacagt 1320
gcaaatgcgg gtgaaccggc cagaaagtaa attcttatgc taccgtgcag tgactccgac 1380
atccccagtt tttgccctac ttgatcacag atggggtcag cgctgccgct aagtgtaccc 1440
aaccgtcccc acacggtcca tctataaata ctgctgccag tgcacggtgg tgacatcaat 1500
ctaaagtaca aaaacaaagc tagccacgtg aagcttgaat tcgcggccgc cagctttcta 1560
gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca tcatcatcat 1620
cattgagttt gtagccttag acatgactgt tcctcagttc aagttgggca cttacgagaa 1680
gaccggtctt gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc 1740
aggcttcatt tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca 1800
ttttgtttct tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc 1860
ttgtggtagg ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc 1920
ctcttcagag tacagaagat taagtgagac cttcgtttgt gcggatctgt cgacgcggag 1980
aacgatctcc tcgagctgct cgcggatcag cttgtggccc ggtaatggaa ccaggccgac 2040
ggcacgctcc ttgcggacca cggtggctgg cgagcccagt ttgtgaacga ggtcgtttag 2100
aacgtcctgc gcaaagtcca gtgtcagatg aatgtcctcc tcggaccaat tcagcatgtt 2160
ctcgagcagc catctgtctt tggagtagaa gcgtaatctc tgctcctcgt tactgtaccg 2220
gaagaggtag tttgcctcgc cgcccataat gaacaggttc tctttctggt ggcctgtgag 2280
cagcggggac gtctggacgg cgtcgatgag gcccttgagg cgctcgtagt acttgttcgc 2340
gtcgctgtag ccggccgcgg tgacgatacc cacatagagg tccttggcca ttagtttgat 2400
gaggtggggc aggatgggcg actcggcatc gaaatttttg ccgtcgtcgt acagtgtgat 2460
gtcaccatcg aatgtaatga gctgcagctt gcgatctcgg atggttttgg aatggaagaa 2520
ccgcgacatc tccaacagct gggccgtgtt gagaatgagc cggacgtcgt tgaacgaggg 2580
ggccacaagc cggcgtttgc tgatggcgcg gcgctcgtcc tcgatgtaga aggccttttc 2640
cagaggcagt ctcgtgaaga agctgccaac gctcggaacc agctgcacga gccgagacaa 2700
ttcgggggtg ccggctttgg tcatttcaat gttgtcgtcg atgaggagtt cgaggtcgtg 2760
gaagatttcc gcgtagcggc gttttgcctc agagtttacc atgaggtcgt ccactgcaga 2820
gatgccgttg ctcttcaccg cgtacaggac gaacggcgtg gccagcaggc ccttgatcca 2880
ttctatgagg ccatctcgac ggtgttcctt gagtgcgtac tccactctgt agcgactgga 2940
catctcgaga ctgggcttgc tgtgctggat gcaccaatta attgttgccg catgcatcct 3000
tgcaccgcaa gtttttaaaa cccactcgct ttagccgtcg cgtaaaactt gtgaatctgg 3060
caactgaggg ggttctgcag ccgcaaccga acttttcgct tcgaggacgc agctggatgg 3120
tgtcatgtga ggctctgttt gctggcgtag cctacaacgt gaccttgcct aaccggacgg 3180
cgctacccac tgctgtctgt gcctgctacc agaaaatcac cagagcagca gagggccgat 3240
gtggcaactg gtggggtgtc ggacaggctg tttctccaca gtgcaaatgc gggtgaaccg 3300
gccagaaagt aaattcttat gctaccgtgc agtgactccg acatccccag tttttgccct 3360
acttgatcac agatggggtc agcgctgccg ctaagtgtac ccaaccgtcc ccacacggtc 3420
catctataaa tactgctgcc agtgcacggt ggtgacatca atctaaagta caaaaacaaa 3480
ttcgaaacgt gtacacgatc gatcatatgc ctgcaggcag ctttctagaa caaaaactca 3540
tctcagaaga ggatctgaat agcgccgtcg accatcatca tcatcatcat tgagtttgta 3600
gccttagaca tgactgttcc tcagttcaag ttgggcactt acgagaagac cggtcttgct 3660
agattctaat caagaggatg tcagaatgcc atttgcctga gagatgcagg cttcattttt 3720
gatacttttt tatttgtaac ctatatagta taggattttt tttgtcattt tgtttcttct 3780
cgtacgagct tgctcctgat cagcctatct cgcagctgat gaatatcttg tggtaggggt 3840
ttgggaaaat cattcgagtt tgatgttttt cttggtattt cccactcctc ttcagagtac 3900
agaagattaa gtgagacctt cgtttgtgcg gatcccccac acaccatagc ttcaaaatgt 3960
ttctactcct tttttactct tccagatttt ctcggactcc gcgcatcgcc gtaccacttc 4020
aaaacaccca agcacagcat actaaatttt ccctctttct tcctctaggg tgtcgttaat 4080
tacccgtact aaaggtttgg aaaagaaaaa agagaccgcc tcgtttcttt ttcttcgtcg 4140
aaaaaggcaa taaaaatttt tatcacgttt ctttttcttg aaattttttt ttttagtttt 4200
tttctctttc agtgacctcc attgatattt aagttaataa acggtcttca atttctcaag 4260
tttcagtttc atttttcttg ttctattaca acttttttta cttcttgttc attagaaaga 4320
aagcatagca atctaatcta aggggcggtg ttgacaatta atcatcggca tagtatatcg 4380
gcatagtata atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc 4440
cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt 4500
tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt 4560
tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc 4620
gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg 4680
cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc 4740
gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtccgac 4800
ggcggcccac gggtcccagg cctcggagat ccgtccccct tttcctttgt cgatatcatg 4860
taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag 4920
gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta 4980
ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca 5040
tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt 5100
tgcaagctgg agaccaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 5160
gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 5220
cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 5280
ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 5340
tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg 5400
gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 5460
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 5520
ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 5580
ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 5640
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 5700
accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 5760
tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 5820
cgttaaggga ttttggtcat gagatc 5846
<210> 33
<211> 502
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
cccttagatc tcccacacac catagcttca aaatgtttct actccttttt tactcttcca 60
gattttctcg gactccgcgc atcgccgtac cacttcaaaa cacccaagca cagcatacta 120
aattttccct ctttcttcct ctagggtgtc gttaattacc cgtactaaag gtttggaaaa 180
gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa aggcaataaa aatttttatc 240
acgtttcttt ttcttgaaat tttttttttt agtttttttc tctttcagtg acctccattg 300
atatttaagt taataaacgg tcttcaattt ctcaagtttc agtttcattt ttcttgttct 360
attacaactt tttttacttc ttgttcatta gaaagaaagc atagcaatct aatctaaggg 420
gcggtgttga caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga 480
ggaactaaac cttaagaccg ca 502
<210> 34
<211> 1269
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gggtatcgtc accgcggccg gctacagcac ttgaagtcgg acagtgagtg tagtcttgag 60
aaattctgaa gccgtatttt tattatcagt gagtcagtca tcaggagatc ctctacgccg 120
gacgcatcgt ggccgacctg cagggggggg gggggcgctg aggtctgcct cgtgaagaag 180
gtgttgctga ctcataccag gcctgaatcg ccccatcatc cagccagaaa gtgagggagc 240
cacggttgat gagagctttg ttgtaggtgg accagttggt gattttgaac ttttgctttg 300
ccacggaacg gtctgcgttg tcgggaagat gcgtgatctg atccttcaac tcagcaaaag 360
ttcgatttat tcaacaaagc cgccgtcccg tcaagtcagc gtaatgctct gccagtgtta 420
caaccaatta accaattctg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt 480
attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga 540
aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac 600
tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga 660
gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagcttat gcatttcttt 720
ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa 780
accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg 840
acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat 900
attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc 960
agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg 1020
cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct 1080
acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat 1140
tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc 1200
catgttggaa tttaatcgcg gcctcgagca agacgtttcc cgttgaatat ggctcatctt 1260
aagccaata 1269
<210> 35
<211> 5223
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
agatctccca cacaccatag cttcaaaatg tttctactcc ttttttactc ttccagattt 60
tctcggactc cgcgcatcgc cgtaccactt caaaacaccc aagcacagca tactaaattt 120
tccctctttc ttcctctagg gtgtcgttaa ttacccgtac taaaggtttg gaaaagaaaa 180
aagagaccgc ctcgtttctt tttcttcgtc gaaaaaggca ataaaaattt ttatcacgtt 240
tctttttctt gaaatttttt tttttagttt ttttctcttt cagtgacctc cattgatatt 300
taagttaata aacggtcttc aatttctcaa gtttcagttt catttttctt gttctattac 360
aacttttttt acttcttgtt cattagaaag aaagcatagc aatctaatct aaggggcggt 420
gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac 480
taaaccttaa gatgagccat attcaacggg aaacgtcttg ctcgaggccg cgattaaatt 540
ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag 600
gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg 660
gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg 720
aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac 780
tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag 840
gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt 900
gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga 960
ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac 1020
aagtctggaa agaaatgcat aagcttttgc cattctcacc ggattcagtc gtcactcatg 1080
gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg 1140
ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg 1200
gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg 1260
atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta 1320
attggttgta acactggcag agcattacgc tgacttgacg ggacggcggc tttgttgaat 1380
aaatcgaact tttgctgagt tgaaggatca gatcacgcat cttcccgaca acgcagaccg 1440
ttccgtggca aagcaaaagt tcaaaatcac caactggtcc acctacaaca aagctctcat 1500
caaccgtggc tccctcactt tctggctgga tgatggggcg attcaggcct ggtatgagtc 1560
agcaacacct tcttcacgag gcagacctca gcgccccccc cccccctgca ggtcggccac 1620
gatgcgtccg gcgtagagga tctcctgatg actgactcac tgataataaa aatacggctt 1680
cagaatttct caagactaca ctcactgtcc gacttcaagt gctgtagccg gccgcggtga 1740
cgatacccac atagaggtcc ttggccatta gtttgatgag gtggggcagg atgggcgact 1800
cggcatcgaa atttttgccg tcgtcgtaca gtgtgatgtc accatcgaat gtaatgagct 1860
gcagcttgcg atctcggatg gttttggaat ggaagaaccg cgacatctcc aacagctggg 1920
ccgtgttgag aatgagccgg acgtcgttga acgagggggc cacaagccgg cgtttgctga 1980
tggcgcggcg ctcgtcctcg atgtagaagg ccttttccag aggcagtctc gtgaagaagc 2040
tgccaacgct cggaaccagc tgcacgagcc gagacaattc gggggtgccg gctttggtca 2100
tttcaatgtt gtcgtcgatg aggagttcga ggtcgtggaa gatttccgcg tagcggcgtt 2160
ttgcctcaga gtttaccatg aggtcgtcca ctgcagagat gccgttgctc ttcaccgcgt 2220
acaggacgaa cggcgtggcc agcaggccct tgatccattc tatgaggcca tctcgacggt 2280
gttccttgag tgcgtactcc actctgtagc gactggacat ctcgagactg ggcttgctgt 2340
gctggatgca ccaattaatt gttgccgcat gcatccttgc accgcaagtt tttaaaaccc 2400
actcgcttta gccgtcgcgt aaaacttgtg aatctggcaa ctgagggggt tctgcagccg 2460
caaccgaact tttcgcttcg aggacgcagc tggatggtgt catgtgaggc tctgtttgct 2520
ggcgtagcct acaacgtgac cttgcctaac cggacggcgc tacccactgc tgtctgtgcc 2580
tgctaccaga aaatcaccag agcagcagag ggccgatgtg gcaactggtg gggtgtcgga 2640
caggctgttt ctccacagtg caaatgcggg tgaaccggcc agaaagtaaa ttcttatgct 2700
accgtgcagt gactccgaca tccccagttt ttgccctact tgatcacaga tggggtcagc 2760
gctgccgcta agtgtaccca accgtcccca cacggtccat ctataaatac tgctgccagt 2820
gcacggtggt gacatcaatc taaagtacaa aaacaaattc gaaacgtgta cacgatcgat 2880
catatgcctg caggcagctt tctagaacaa aaactcatct cagaagagga tctgaatagc 2940
gccgtcgacc atcatcatca tcatcattga gtttgtagcc ttagacatga ctgttcctca 3000
gttcaagttg ggcacttacg agaagaccgg tcttgctaga ttctaatcaa gaggatgtca 3060
gaatgccatt tgcctgagag atgcaggctt catttttgat acttttttat ttgtaaccta 3120
tatagtatag gatttttttt gtcattttgt ttcttctcgt acgagcttgc tcctgatcag 3180
cctatctcgc agctgatgaa tatcttgtgg taggggtttg ggaaaatcat tcgagtttga 3240
tgtttttctt ggtatttccc actcctcttc agagtacaga agattaagtg agaccttcgt 3300
ttgtgcggat cccccacaca ccatagcttc aaaatgtttc tactcctttt ttactcttcc 3360
agattttctc ggactccgcg catcgccgta ccacttcaaa acacccaagc acagcatact 3420
aaattttccc tctttcttcc tctagggtgt cgttaattac ccgtactaaa ggtttggaaa 3480
agaaaaaaga gaccgcctcg tttctttttc ttcgtcgaaa aaggcaataa aaatttttat 3540
cacgtttctt tttcttgaaa tttttttttt tagttttttt ctctttcagt gacctccatt 3600
gatatttaag ttaataaacg gtcttcaatt tctcaagttt cagtttcatt tttcttgttc 3660
tattacaact ttttttactt cttgttcatt agaaagaaag catagcaatc taatctaagg 3720
ggcggtgttg acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg 3780
aggaactaaa ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc 3840
gccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac 3900
gacttcgccg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag 3960
gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc 4020
gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag 4080
atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg 4140
cacttcgtgg ccgaggagca ggactgacac gtccgacggc ggcccacggg tcccaggcct 4200
cggagatccg tccccctttt cctttgtcga tatcatgtaa ttagttatgt cacgcttaca 4260
ttcacgccct ccccccacat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt 4320
ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat ttatatttca 4380
aatttttctt ttttttctgt acagacgcgt gtacgcatgt aacattatac tgaaaacctt 4440
gcttgagaag gttttgggac gctcgaaggc tttaatttgc aagctggaga ccaacatgtg 4500
agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 4560
taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 4620
cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 4680
tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 4740
gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 4800
gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 4860
tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 4920
gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 4980
cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 5040
aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 5100
tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 5160
ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 5220
atc 5223
<210> 36
<211> 2344
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
gttggatcct cgatcacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 60
ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 120
gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 180
gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 240
ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg 300
tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 360
gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 420
tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 480
tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 540
tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 600
ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 660
ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 720
gttaagggat tttggtcatg agatcagatc tcccacacac catagcttca aaatgtttct 780
actccttttt tactcttcca gattttctcg gactccgcgc atcgccgtac cacttcaaaa 840
cacccaagca cagcatacta aattttccct ctttcttcct ctagggtgtc gttaattacc 900
cgtactaaag gtttggaaaa gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa 960
aggcaataaa aatttttatc acgtttcttt ttcttgaaat tttttttttt agtttttttc 1020
tctttcagtg acctccattg atatttaagt taataaacgg tcttcaattt ctcaagtttc 1080
agtttcattt ttcttgttct attacaactt tttttacttc ttgttcatta gaaagaaagc 1140
atagcaatct aatctaaggg gcggtgttga caattaatca tcggcatagt atatcggcat 1200
agtataatac gacaaggtga ggaactaaac cttaagatga gccatattca acgggaaacg 1260
tcttgctcga ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg 1320
gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat 1380
gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag 1440
atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc 1500
cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac agcattccag 1560
gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg 1620
cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt 1680
ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac 1740
gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataagct tttgccattc 1800
tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag 1860
gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat 1920
cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt 1980
caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat 2040
gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcagagcat tacgctgact 2100
tgacgggacg gcggctttgt tgaataaatc gaacttttgc tgagttgaag gatcagatca 2160
cgcatcttcc cgacaacgca gaccgttccg tggcaaagca aaagttcaaa atcaccaact 2220
ggtccaccta caacaaagct ctcatcaacc gtggctccct cactttctgg ctggatgatg 2280
gggcgattca ggcctggtat gagtcagcaa caccttcttc acgaggcaga cctcaggatc 2340
cttt 2344
<210> 37
<211> 6261
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
ggatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgg cacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctgcgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttcgcgt cgctgtagcc ggccgcggtg acgataccca catagaggtc 420
cttggccatt agtttgatga ggtggggcag gatgggcgac tcggcatcga aatttttgcc 480
gtcgtcgtac agtgtgatgt caccatcgaa tgtaatgagc tgcagcttgc gatctcggat 540
ggttttggaa tggaagaacc gcgacatctc caacagctgg gccgtgttga gaatgagccg 600
gacgtcgttg aacgaggggg ccacaagccg gcgtttgctg atggcgcggc gctcgtcctc 660
gatgtagaag gccttttcca gaggcagtct cgtgaagaag ctgccaacgc tcggaaccag 720
ctgcacgagc cgagacaatt cgggggtgcc ggctttggtc atttcaatgt tgtcgtcgat 780
gaggagttcg aggtcgtgga agatttccgc gtagcggcgt tttgcctcag agtttaccat 840
gaggtcgtcc actgcagaga tgccgttgct cttcaccgcg tacaggacga acggcgtggc 900
cagcaggccc ttgatccatt ctatgaggcc atctcgacgg tgttccttga gtgcgtactc 960
cactctgtag cgactggaca tctcgagact gggcttgctg tgctggatgc accaattaat 1020
tgttgccgca tgcatccttg caccgcaagt ttttaaaacc cactcgcttt agccgtcgcg 1080
taaaacttgt gaatctggca actgaggggg ttctgcagcc gcaaccgaac ttttcgcttc 1140
gaggacgcag ctggatggtg tcatgtgagg ctctgtttgc tggcgtagcc tacaacgtga 1200
ccttgcctaa ccggacggcg ctacccactg ctgtctgtgc ctgctaccag aaaatcacca 1260
gagcagcaga gggccgatgt ggcaactggt ggggtgtcgg acaggctgtt tctccacagt 1320
gcaaatgcgg gtgaaccggc cagaaagtaa attcttatgc taccgtgcag tgactccgac 1380
atccccagtt tttgccctac ttgatcacag atggggtcag cgctgccgct aagtgtaccc 1440
aaccgtcccc acacggtcca tctataaata ctgctgccag tgcacggtgg tgacatcaat 1500
ctaaagtaca aaaacaaagc tagccacgtg aagcttgaat tcgcggccgc cagctttcta 1560
gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca tcatcatcat 1620
cattgagttt gtagccttag acatgactgt tcctcagttc aagttgggca cttacgagaa 1680
gaccggtctt gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc 1740
aggcttcatt tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca 1800
ttttgtttct tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc 1860
ttgtggtagg ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc 1920
ctcttcagag tacagaagat taagtgagac cttcgtttgt gcggatctgt cgacgcggag 1980
aacgatctcc tcgagctgct cgcggatcag cttgtggccc ggtaatggaa ccaggccgac 2040
ggcacgctcc ttgcggacca cggtggctgg cgagcccagt ttgtgaacga ggtcgtttag 2100
aacgtcctgc gcaaagtcca gtgtcagatg aatgtcctcc tcggaccaat tcagcatgtt 2160
ctcgagcagc catctgtctt tggagtagaa gcgtaatctc tgctcctcgt tactgtaccg 2220
gaagaggtag tttgcctcgc cgcccataat gaacaggttc tctttctggt ggcctgtgag 2280
cagcggggac gtctggacgg cgtcgatgag gcccttgagg cgctcgtagt acttgttcgc 2340
gtcgctgtag ccggccgcgg tgacgatacc cacatagagg tccttggcca ttagtttgat 2400
gaggtggggc aggatgggcg actcggcatc gaaatttttg ccgtcgtcgt acagtgtgat 2460
gtcaccatcg aatgtaatga gctgcagctt gcgatctcgg atggttttgg aatggaagaa 2520
ccgcgacatc tccaacagct gggccgtgtt gagaatgagc cggacgtcgt tgaacgaggg 2580
ggccacaagc cggcgtttgc tgatggcgcg gcgctcgtcc tcgatgtaga aggccttttc 2640
cagaggcagt ctcgtgaaga agctgccaac gctcggaacc agctgcacga gccgagacaa 2700
ttcgggggtg ccggctttgg tcatttcaat gttgtcgtcg atgaggagtt cgaggtcgtg 2760
gaagatttcc gcgtagcggc gttttgcctc agagtttacc atgaggtcgt ccactgcaga 2820
gatgccgttg ctcttcaccg cgtacaggac gaacggcgtg gccagcaggc ccttgatcca 2880
ttctatgagg ccatctcgac ggtgttcctt gagtgcgtac tccactctgt agcgactgga 2940
catctcgaga ctgggcttgc tgtgctggat gcaccaatta attgttgccg catgcatcct 3000
tgcaccgcaa gtttttaaaa cccactcgct ttagccgtcg cgtaaaactt gtgaatctgg 3060
caactgaggg ggttctgcag ccgcaaccga acttttcgct tcgaggacgc agctggatgg 3120
tgtcatgtga ggctctgttt gctggcgtag cctacaacgt gaccttgcct aaccggacgg 3180
cgctacccac tgctgtctgt gcctgctacc agaaaatcac cagagcagca gagggccgat 3240
gtggcaactg gtggggtgtc ggacaggctg tttctccaca gtgcaaatgc gggtgaaccg 3300
gccagaaagt aaattcttat gctaccgtgc agtgactccg acatccccag tttttgccct 3360
acttgatcac agatggggtc agcgctgccg ctaagtgtac ccaaccgtcc ccacacggtc 3420
catctataaa tactgctgcc agtgcacggt ggtgacatca atctaaagta caaaaacaaa 3480
ttcgaaacgt gtacacgatc gatcatatgc ctgcaggcag ctttctagaa caaaaactca 3540
tctcagaaga ggatctgaat agcgccgtcg accatcatca tcatcatcat tgagtttgta 3600
gccttagaca tgactgttcc tcagttcaag ttgggcactt acgagaagac cggtcttgct 3660
agattctaat caagaggatg tcagaatgcc atttgcctga gagatgcagg cttcattttt 3720
gatacttttt tatttgtaac ctatatagta taggattttt tttgtcattt tgtttcttct 3780
cgtacgagct tgctcctgat cagcctatct cgcagctgat gaatatcttg tggtaggggt 3840
ttgggaaaat cattcgagtt tgatgttttt cttggtattt cccactcctc ttcagagtac 3900
agaagattaa gtgagacctt cgtttgtgcg gatcctcgat cacatgtgag caaaaggcca 3960
gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 4020
ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 4080
ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 4140
gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg 4200
ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 4260
cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 4320
cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 4380
gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 4440
aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 4500
tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 4560
gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 4620
tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat cagatctccc 4680
acacaccata gcttcaaaat gtttctactc cttttttact cttccagatt ttctcggact 4740
ccgcgcatcg ccgtaccact tcaaaacacc caagcacagc atactaaatt ttccctcttt 4800
cttcctctag ggtgtcgtta attacccgta ctaaaggttt ggaaaagaaa aaagagaccg 4860
cctcgtttct ttttcttcgt cgaaaaaggc aataaaaatt tttatcacgt ttctttttct 4920
tgaaattttt ttttttagtt tttttctctt tcagtgacct ccattgatat ttaagttaat 4980
aaacggtctt caatttctca agtttcagtt tcatttttct tgttctatta caactttttt 5040
tacttcttgt tcattagaaa gaaagcatag caatctaatc taaggggcgg tgttgacaat 5100
taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa ctaaacctta 5160
agatgagcca tattcaacgg gaaacgtctt gctcgaggcc gcgattaaat tccaacatgg 5220
atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa 5280
tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta 5340
gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc 5400
ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg 5460
cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata 5520
ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc 5580
cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt 5640
tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga 5700
aagaaatgca taagcttttg ccattctcac cggattcagt cgtcactcat ggtgatttct 5760
cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag 5820
tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt 5880
ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata 5940
aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt 6000
aacactggca gagcattacg ctgacttgac gggacggcgg ctttgttgaa taaatcgaac 6060
ttttgctgag ttgaaggatc agatcacgca tcttcccgac aacgcagacc gttccgtggc 6120
aaagcaaaag ttcaaaatca ccaactggtc cacctacaac aaagctctca tcaaccgtgg 6180
ctccctcact ttctggctgg atgatggggc gattcaggcc tggtatgagt cagcaacacc 6240
ttcttcacga ggcagacctc a 6261
<210> 38
<211> 1541
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
cggacaaatt cgaaacgatg tggagaccat ctgactcgac cgtctacgtt cctccaccta 60
acccagtctc caaagtggtt gctactgatg cctacgtcaa gagaaccaac atcttctacc 120
acgcctcttc gtccagattg ctggcagttg gacatcctta ctattccatc aagaaagtta 180
acaagaccgt ggtccctaag gtttcgggct atcagtacag agtgttcaag gtcgttctgc 240
cagaccctaa caagtttgcc ttgccagatt cctcgctgtt cgaccctacc actcaaagac 300
tggtttgggc ttgcacgggt ttggaagtgg gcagaggaca accactgggt gtcggagttt 360
ccggccatcc attgctgaac aagtacgacg atgttgagaa ctcgggtgga tacggtggca 420
atccaggaca agacaacaga gtgaatgtcg gcatggacta caagcaaacc cagctgtgca 480
tggttggttg tgcaccacct ctgggtgaac attggggaaa gggcactcag tgctccaaca 540
cctctgttca gaatggtgac tgtcctccac tggagctgat cacctcggtt attcaggatg 600
gagacatggt ggacactggc ttcggtgcaa tgaactttgc cgacttgcag accaacaagt 660
ccgatgttcc attggacatt tgcggcactg tgtgcaaata tcctgactac ctgcagatgg 720
ctgcagatcc atacggtgac agactcttct tctacctgag aaaggagcag atgttcgcca 780
gacacttctt taacagagct ggcaccgttg gtgagcctgt tccagatgac ctgttggtga 840
agggtggaaa caatagatcc tctgttgcct cgtccatcta cgtccacacc ccatcgggct 900
ctctggtgtc ctcggaggcc caactgttca acaagcctta ctggctccag aaggcccaag 960
gacacaacaa tggcatctgc tggggaaacc acctgttcgt caccgttgtg gacacgacta 1020
gatccaccaa catgactttg tgtgcctctg tttccaagtc ggctacctac acgaactcgg 1080
actataagga gtacatgaga catgttgaag agttcgacct gcagtttatc ttccagctct 1140
gctccattac cttgtcggca gaagtgatgg cctacatcca cacgatgaac ccatccgtct 1200
tggaagactg gaacttcggt ctgtctccac ctccaaatgg caccctggaa gacacgtaca 1260
gatatgtgca gtcgcaagcc atcacctgtc agaaaccaac tcctgaaaag gagaaacagg 1320
acccatacaa ggatatgtcg ttctgggagg tcaacctgaa ggagaagttc tcttcggaac 1380
tggaccagtt cccattggga agaaagttcc tgctccagtc tggttataga ggaagaacct 1440
ccgcaagaac tggcatcaag agacctgccg tgtcgaagcc atccaccgct cctaagagaa 1500
agagaacgaa gaccaaaaag taatagcctg caggtttctg t 1541
<210> 39
<211> 7750
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
ggatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgg cacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctgcgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttcgcgt cgctgtagcc ggccgcggtg acgataccca catagaggtc 420
cttggccatt agtttgatga ggtggggcag gatgggcgac tcggcatcga aatttttgcc 480
gtcgtcgtac agtgtgatgt caccatcgaa tgtaatgagc tgcagcttgc gatctcggat 540
ggttttggaa tggaagaacc gcgacatctc caacagctgg gccgtgttga gaatgagccg 600
gacgtcgttg aacgaggggg ccacaagccg gcgtttgctg atggcgcggc gctcgtcctc 660
gatgtagaag gccttttcca gaggcagtct cgtgaagaag ctgccaacgc tcggaaccag 720
ctgcacgagc cgagacaatt cgggggtgcc ggctttggtc atttcaatgt tgtcgtcgat 780
gaggagttcg aggtcgtgga agatttccgc gtagcggcgt tttgcctcag agtttaccat 840
gaggtcgtcc actgcagaga tgccgttgct cttcaccgcg tacaggacga acggcgtggc 900
cagcaggccc ttgatccatt ctatgaggcc atctcgacgg tgttccttga gtgcgtactc 960
cactctgtag cgactggaca tctcgagact gggcttgctg tgctggatgc accaattaat 1020
tgttgccgca tgcatccttg caccgcaagt ttttaaaacc cactcgcttt agccgtcgcg 1080
taaaacttgt gaatctggca actgaggggg ttctgcagcc gcaaccgaac ttttcgcttc 1140
gaggacgcag ctggatggtg tcatgtgagg ctctgtttgc tggcgtagcc tacaacgtga 1200
ccttgcctaa ccggacggcg ctacccactg ctgtctgtgc ctgctaccag aaaatcacca 1260
gagcagcaga gggccgatgt ggcaactggt ggggtgtcgg acaggctgtt tctccacagt 1320
gcaaatgcgg gtgaaccggc cagaaagtaa attcttatgc taccgtgcag tgactccgac 1380
atccccagtt tttgccctac ttgatcacag atggggtcag cgctgccgct aagtgtaccc 1440
aaccgtcccc acacggtcca tctataaata ctgctgccag tgcacggtgg tgacatcaat 1500
ctaaagtaca aaaacaaagc tagccacgtg aagcttgaat tcgcggccgc cagctttcta 1560
gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca tcatcatcat 1620
cattgagttt gtagccttag acatgactgt tcctcagttc aagttgggca cttacgagaa 1680
gaccggtctt gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc 1740
aggcttcatt tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca 1800
ttttgtttct tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc 1860
ttgtggtagg ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc 1920
ctcttcagag tacagaagat taagtgagac cttcgtttgt gcggatctgt cgacgcggag 1980
aacgatctcc tcgagctgct cgcggatcag cttgtggccc ggtaatggaa ccaggccgac 2040
ggcacgctcc ttgcggacca cggtggctgg cgagcccagt ttgtgaacga ggtcgtttag 2100
aacgtcctgc gcaaagtcca gtgtcagatg aatgtcctcc tcggaccaat tcagcatgtt 2160
ctcgagcagc catctgtctt tggagtagaa gcgtaatctc tgctcctcgt tactgtaccg 2220
gaagaggtag tttgcctcgc cgcccataat gaacaggttc tctttctggt ggcctgtgag 2280
cagcggggac gtctggacgg cgtcgatgag gcccttgagg cgctcgtagt acttgttcgc 2340
gtcgctgtag ccggccgcgg tgacgatacc cacatagagg tccttggcca ttagtttgat 2400
gaggtggggc aggatgggcg actcggcatc gaaatttttg ccgtcgtcgt acagtgtgat 2460
gtcaccatcg aatgtaatga gctgcagctt gcgatctcgg atggttttgg aatggaagaa 2520
ccgcgacatc tccaacagct gggccgtgtt gagaatgagc cggacgtcgt tgaacgaggg 2580
ggccacaagc cggcgtttgc tgatggcgcg gcgctcgtcc tcgatgtaga aggccttttc 2640
cagaggcagt ctcgtgaaga agctgccaac gctcggaacc agctgcacga gccgagacaa 2700
ttcgggggtg ccggctttgg tcatttcaat gttgtcgtcg atgaggagtt cgaggtcgtg 2760
gaagatttcc gcgtagcggc gttttgcctc agagtttacc atgaggtcgt ccactgcaga 2820
gatgccgttg ctcttcaccg cgtacaggac gaacggcgtg gccagcaggc ccttgatcca 2880
ttctatgagg ccatctcgac ggtgttcctt gagtgcgtac tccactctgt agcgactgga 2940
catctcgaga ctgggcttgc tgtgctggat gcaccaatta attgttgccg catgcatcct 3000
tgcaccgcaa gtttttaaaa cccactcgct ttagccgtcg cgtaaaactt gtgaatctgg 3060
caactgaggg ggttctgcag ccgcaaccga acttttcgct tcgaggacgc agctggatgg 3120
tgtcatgtga ggctctgttt gctggcgtag cctacaacgt gaccttgcct aaccggacgg 3180
cgctacccac tgctgtctgt gcctgctacc agaaaatcac cagagcagca gagggccgat 3240
gtggcaactg gtggggtgtc ggacaggctg tttctccaca gtgcaaatgc gggtgaaccg 3300
gccagaaagt aaattcttat gctaccgtgc agtgactccg acatccccag tttttgccct 3360
acttgatcac agatggggtc agcgctgccg ctaagtgtac ccaaccgtcc ccacacggtc 3420
catctataaa tactgctgcc agtgcacggt ggtgacatca atctaaagta caaaaacaaa 3480
ttcgaaacga tgtggagacc atctgactcg accgtctacg ttcctccacc taacccagtc 3540
tccaaagtgg ttgctactga tgcctacgtc aagagaacca acatcttcta ccacgcctct 3600
tcgtccagat tgctggcagt tggacatcct tactattcca tcaagaaagt taacaagacc 3660
gtggtcccta aggtttcggg ctatcagtac agagtgttca aggtcgttct gccagaccct 3720
aacaagtttg ccttgccaga ttcctcgctg ttcgacccta ccactcaaag actggtttgg 3780
gcttgcacgg gtttggaagt gggcagagga caaccactgg gtgtcggagt ttccggccat 3840
ccattgctga acaagtacga cgatgttgag aactcgggtg gatacggtgg caatccagga 3900
caagacaaca gagtgaatgt cggcatggac tacaagcaaa cccagctgtg catggttggt 3960
tgtgcaccac ctctgggtga acattgggga aagggcactc agtgctccaa cacctctgtt 4020
cagaatggtg actgtcctcc actggagctg atcacctcgg ttattcagga tggagacatg 4080
gtggacactg gcttcggtgc aatgaacttt gccgacttgc agaccaacaa gtccgatgtt 4140
ccattggaca tttgcggcac tgtgtgcaaa tatcctgact acctgcagat ggctgcagat 4200
ccatacggtg acagactctt cttctacctg agaaaggagc agatgttcgc cagacacttc 4260
tttaacagag ctggcaccgt tggtgagcct gttccagatg acctgttggt gaagggtgga 4320
aacaatagat cctctgttgc ctcgtccatc tacgtccaca ccccatcggg ctctctggtg 4380
tcctcggagg cccaactgtt caacaagcct tactggctcc agaaggccca aggacacaac 4440
aatggcatct gctggggaaa ccacctgttc gtcaccgttg tggacacgac tagatccacc 4500
aacatgactt tgtgtgcctc tgtttccaag tcggctacct acacgaactc ggactataag 4560
gagtacatga gacatgttga agagttcgac ctgcagttta tcttccagct ctgctccatt 4620
accttgtcgg cagaagtgat ggcctacatc cacacgatga acccatccgt cttggaagac 4680
tggaacttcg gtctgtctcc acctccaaat ggcaccctgg aagacacgta cagatatgtg 4740
cagtcgcaag ccatcacctg tcagaaacca actcctgaaa aggagaaaca ggacccatac 4800
aaggatatgt cgttctggga ggtcaacctg aaggagaagt tctcttcgga actggaccag 4860
ttcccattgg gaagaaagtt cctgctccag tctggttata gaggaagaac ctccgcaaga 4920
actggcatca agagacctgc cgtgtcgaag ccatccaccg ctcctaagag aaagagaacg 4980
aagaccaaaa agtaatagcc tgcaggcagc tttctagaac aaaaactcat ctcagaagag 5040
gatctgaata gcgccgtcga ccatcatcat catcatcatt gagtttgtag ccttagacat 5100
gactgttcct cagttcaagt tgggcactta cgagaagacc ggtcttgcta gattctaatc 5160
aagaggatgt cagaatgcca tttgcctgag agatgcaggc ttcatttttg atactttttt 5220
atttgtaacc tatatagtat aggatttttt ttgtcatttt gtttcttctc gtacgagctt 5280
gctcctgatc agcctatctc gcagctgatg aatatcttgt ggtaggggtt tgggaaaatc 5340
attcgagttt gatgtttttc ttggtatttc ccactcctct tcagagtaca gaagattaag 5400
tgagaccttc gtttgtgcgg atcctcgatc acatgtgagc aaaaggccag caaaaggcca 5460
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 5520
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5580
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5640
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta 5700
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5760
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5820
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5880
gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 5940
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 6000
ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 6060
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 6120
ggaacgaaaa ctcacgttaa gggattttgg tcatgagatc agatctccca cacaccatag 6180
cttcaaaatg tttctactcc ttttttactc ttccagattt tctcggactc cgcgcatcgc 6240
cgtaccactt caaaacaccc aagcacagca tactaaattt tccctctttc ttcctctagg 6300
gtgtcgttaa ttacccgtac taaaggtttg gaaaagaaaa aagagaccgc ctcgtttctt 6360
tttcttcgtc gaaaaaggca ataaaaattt ttatcacgtt tctttttctt gaaatttttt 6420
tttttagttt ttttctcttt cagtgacctc cattgatatt taagttaata aacggtcttc 6480
aatttctcaa gtttcagttt catttttctt gttctattac aacttttttt acttcttgtt 6540
cattagaaag aaagcatagc aatctaatct aaggggcggt gttgacaatt aatcatcggc 6600
atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccttaa gatgagccat 6660
attcaacggg aaacgtcttg ctcgaggccg cgattaaatt ccaacatgga tgctgattta 6720
tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg 6780
tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat 6840
gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc 6900
atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg 6960
aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg 7020
ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc 7080
gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg 7140
agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat 7200
aagcttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac 7260
cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca 7320
gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta 7380
cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt 7440
catttgatgc tcgatgagtt tttctaatca gaattggtta attggttgta acactggcag 7500
agcattacgc tgacttgacg ggacggcggc tttgttgaat aaatcgaact tttgctgagt 7560
tgaaggatca gatcacgcat cttcccgaca acgcagaccg ttccgtggca aagcaaaagt 7620
tcaaaatcac caactggtcc acctacaaca aagctctcat caaccgtggc tccctcactt 7680
tctggctgga tgatggggcg attcaggcct ggtatgagtc agcaacacct tcttcacgag 7740
gcagacctca 7750
<210> 40
<211> 1532
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
aatcgctagc atgtggagac catctgactc gactgtctac gttcctccac ctaacccagt 60
ctctaaggtt gtggctaccg acgcctacgt gactagaacc aacatcttct accacgcatc 120
gtcctctaga ttgctggccg ttggacatcc ttacttctcc atcaagagag ccaacaaaac 180
cgtcgtgcca aaggtttctg gctaccagta tagagtcttc aaggtggttc tgcctgaccc 240
taacaagttt gccctgccag actcgtcctt gttcgatcct acgactcaaa gactcgtttg 300
ggcctgcact ggtcttgagg ttggcagagg acagccactc ggagtcggcg tttctggtca 360
tcctttcctg aacaagtacg acgatgttga gaactctggt tccggaggca atcctggaca 420
ggacaacaga gtcaatgttg gcatggacta caagcaaact cagctctgta tggtgggatg 480
cgcacctcca ctgggtgagc attggggaaa aggcaagcag tgcaccaaca ctccagttca 540
ggcaggtgac tgccctccat tggagctgat tacctcggtc atccaggacg gtgatatggt 600
tgacaccgga ttcggtgcca tgaacttcgc tgacctgcag accaacaagt ccgatgtgcc 660
aatcgacatc tgcggaacga cctgtaaata ccctgactac ttgcagatgg cagccgatcc 720
atacggcgac agactcttct tctttctgag aaaggagcag atgtttgcta gacacttttt 780
caacagagcc ggagaggtcg gtgagcctgt tccagacacc ctgatcatta agggctcggg 840
aaacagaact tctgtcggct cctcgatcta cgtgaacacc ccttccggtt cgttggtgtc 900
gtccgaggct caactgttca acaagccata ctggctccag aaagcccaag gtcacaacaa 960
tggcatctgc tggggtaacc agctgttcgt tactgtcgtg gacaccacga gatcgaccaa 1020
catgactctg tgtgcctccg ttaccacatc ttcgacttac accaactccg actataagga 1080
gtacatgaga cacgttgaag agtacgactt gcaattcatc tttcagctct gctcgattac 1140
cctgtccgca gaagtcatgg cctacatcca cactatgaat ccatcggtgc ttgaggactg 1200
gaactttggc ttgtccccac ctccaaacgg aactctggaa gacacctata gatacgtgca 1260
gtcgcaagcc atcacgtgcc agaagccaac ccctgagaag gaaaagccag atccttacaa 1320
gaacctgtcc ttctgggagg tcaatctcaa ggagaagttc tcgtccgaac tggaccagta 1380
cccattgggt agaaagttcc tgctccagtc tggataccgt ggtagatcgt ccatcagaac 1440
cggagtgaag agaccagcag tttccaaggc ttctgccgca cctaagcgta aaagagccaa 1500
gaccaagaga taataggcgg ccgcattacc aa 1532
<210> 41
<211> 9238
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
ggatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgg cacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctgcgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttcgcgt cgctgtagcc ggccgcggtg acgataccca catagaggtc 420
cttggccatt agtttgatga ggtggggcag gatgggcgac tcggcatcga aatttttgcc 480
gtcgtcgtac agtgtgatgt caccatcgaa tgtaatgagc tgcagcttgc gatctcggat 540
ggttttggaa tggaagaacc gcgacatctc caacagctgg gccgtgttga gaatgagccg 600
gacgtcgttg aacgaggggg ccacaagccg gcgtttgctg atggcgcggc gctcgtcctc 660
gatgtagaag gccttttcca gaggcagtct cgtgaagaag ctgccaacgc tcggaaccag 720
ctgcacgagc cgagacaatt cgggggtgcc ggctttggtc atttcaatgt tgtcgtcgat 780
gaggagttcg aggtcgtgga agatttccgc gtagcggcgt tttgcctcag agtttaccat 840
gaggtcgtcc actgcagaga tgccgttgct cttcaccgcg tacaggacga acggcgtggc 900
cagcaggccc ttgatccatt ctatgaggcc atctcgacgg tgttccttga gtgcgtactc 960
cactctgtag cgactggaca tctcgagact gggcttgctg tgctggatgc accaattaat 1020
tgttgccgca tgcatccttg caccgcaagt ttttaaaacc cactcgcttt agccgtcgcg 1080
taaaacttgt gaatctggca actgaggggg ttctgcagcc gcaaccgaac ttttcgcttc 1140
gaggacgcag ctggatggtg tcatgtgagg ctctgtttgc tggcgtagcc tacaacgtga 1200
ccttgcctaa ccggacggcg ctacccactg ctgtctgtgc ctgctaccag aaaatcacca 1260
gagcagcaga gggccgatgt ggcaactggt ggggtgtcgg acaggctgtt tctccacagt 1320
gcaaatgcgg gtgaaccggc cagaaagtaa attcttatgc taccgtgcag tgactccgac 1380
atccccagtt tttgccctac ttgatcacag atggggtcag cgctgccgct aagtgtaccc 1440
aaccgtcccc acacggtcca tctataaata ctgctgccag tgcacggtgg tgacatcaat 1500
ctaaagtaca aaaacaaagc tagcatgtgg agaccatctg actcgactgt ctacgttcct 1560
ccacctaacc cagtctctaa ggttgtggct accgacgcct acgtgactag aaccaacatc 1620
ttctaccacg catcgtcctc tagattgctg gccgttggac atccttactt ctccatcaag 1680
agagccaaca aaaccgtcgt gccaaaggtt tctggctacc agtatagagt cttcaaggtg 1740
gttctgcctg accctaacaa gtttgccctg ccagactcgt ccttgttcga tcctacgact 1800
caaagactcg tttgggcctg cactggtctt gaggttggca gaggacagcc actcggagtc 1860
ggcgtttctg gtcatccttt cctgaacaag tacgacgatg ttgagaactc tggttccgga 1920
ggcaatcctg gacaggacaa cagagtcaat gttggcatgg actacaagca aactcagctc 1980
tgtatggtgg gatgcgcacc tccactgggt gagcattggg gaaaaggcaa gcagtgcacc 2040
aacactccag ttcaggcagg tgactgccct ccattggagc tgattacctc ggtcatccag 2100
gacggtgata tggttgacac cggattcggt gccatgaact tcgctgacct gcagaccaac 2160
aagtccgatg tgccaatcga catctgcgga acgacctgta aataccctga ctacttgcag 2220
atggcagccg atccatacgg cgacagactc ttcttctttc tgagaaagga gcagatgttt 2280
gctagacact ttttcaacag agccggagag gtcggtgagc ctgttccaga caccctgatc 2340
attaagggct cgggaaacag aacttctgtc ggctcctcga tctacgtgaa caccccttcc 2400
ggttcgttgg tgtcgtccga ggctcaactg ttcaacaagc catactggct ccagaaagcc 2460
caaggtcaca acaatggcat ctgctggggt aaccagctgt tcgttactgt cgtggacacc 2520
acgagatcga ccaacatgac tctgtgtgcc tccgttacca catcttcgac ttacaccaac 2580
tccgactata aggagtacat gagacacgtt gaagagtacg acttgcaatt catctttcag 2640
ctctgctcga ttaccctgtc cgcagaagtc atggcctaca tccacactat gaatccatcg 2700
gtgcttgagg actggaactt tggcttgtcc ccacctccaa acggaactct ggaagacacc 2760
tatagatacg tgcagtcgca agccatcacg tgccagaagc caacccctga gaaggaaaag 2820
ccagatcctt acaagaacct gtccttctgg gaggtcaatc tcaaggagaa gttctcgtcc 2880
gaactggacc agtacccatt gggtagaaag ttcctgctcc agtctggata ccgtggtaga 2940
tcgtccatca gaaccggagt gaagagacca gcagtttcca aggcttctgc cgcacctaag 3000
cgtaaaagag ccaagaccaa gagataatag gcggccgcca gctttctaga acaaaaactc 3060
atctcagaag aggatctgaa tagcgccgtc gaccatcatc atcatcatca ttgagtttgt 3120
agccttagac atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc 3180
tagattctaa tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt 3240
tgatactttt ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc 3300
tcgtacgagc ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg 3360
tttgggaaaa tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta 3420
cagaagatta agtgagacct tcgtttgtgc ggatctgtcg acgcggagaa cgatctcctc 3480
gagctgctcg cggatcagct tgtggcccgg taatggaacc aggccgacgg cacgctcctt 3540
gcggaccacg gtggctggcg agcccagttt gtgaacgagg tcgtttagaa cgtcctgcgc 3600
aaagtccagt gtcagatgaa tgtcctcctc ggaccaattc agcatgttct cgagcagcca 3660
tctgtctttg gagtagaagc gtaatctctg ctcctcgtta ctgtaccgga agaggtagtt 3720
tgcctcgccg cccataatga acaggttctc tttctggtgg cctgtgagca gcggggacgt 3780
ctggacggcg tcgatgaggc ccttgaggcg ctcgtagtac ttgttcgcgt cgctgtagcc 3840
ggccgcggtg acgataccca catagaggtc cttggccatt agtttgatga ggtggggcag 3900
gatgggcgac tcggcatcga aatttttgcc gtcgtcgtac agtgtgatgt caccatcgaa 3960
tgtaatgagc tgcagcttgc gatctcggat ggttttggaa tggaagaacc gcgacatctc 4020
caacagctgg gccgtgttga gaatgagccg gacgtcgttg aacgaggggg ccacaagccg 4080
gcgtttgctg atggcgcggc gctcgtcctc gatgtagaag gccttttcca gaggcagtct 4140
cgtgaagaag ctgccaacgc tcggaaccag ctgcacgagc cgagacaatt cgggggtgcc 4200
ggctttggtc atttcaatgt tgtcgtcgat gaggagttcg aggtcgtgga agatttccgc 4260
gtagcggcgt tttgcctcag agtttaccat gaggtcgtcc actgcagaga tgccgttgct 4320
cttcaccgcg tacaggacga acggcgtggc cagcaggccc ttgatccatt ctatgaggcc 4380
atctcgacgg tgttccttga gtgcgtactc cactctgtag cgactggaca tctcgagact 4440
gggcttgctg tgctggatgc accaattaat tgttgccgca tgcatccttg caccgcaagt 4500
ttttaaaacc cactcgcttt agccgtcgcg taaaacttgt gaatctggca actgaggggg 4560
ttctgcagcc gcaaccgaac ttttcgcttc gaggacgcag ctggatggtg tcatgtgagg 4620
ctctgtttgc tggcgtagcc tacaacgtga ccttgcctaa ccggacggcg ctacccactg 4680
ctgtctgtgc ctgctaccag aaaatcacca gagcagcaga gggccgatgt ggcaactggt 4740
ggggtgtcgg acaggctgtt tctccacagt gcaaatgcgg gtgaaccggc cagaaagtaa 4800
attcttatgc taccgtgcag tgactccgac atccccagtt tttgccctac ttgatcacag 4860
atggggtcag cgctgccgct aagtgtaccc aaccgtcccc acacggtcca tctataaata 4920
ctgctgccag tgcacggtgg tgacatcaat ctaaagtaca aaaacaaatt cgaaacgatg 4980
tggagaccat ctgactcgac cgtctacgtt cctccaccta acccagtctc caaagtggtt 5040
gctactgatg cctacgtcaa gagaaccaac atcttctacc acgcctcttc gtccagattg 5100
ctggcagttg gacatcctta ctattccatc aagaaagtta acaagaccgt ggtccctaag 5160
gtttcgggct atcagtacag agtgttcaag gtcgttctgc cagaccctaa caagtttgcc 5220
ttgccagatt cctcgctgtt cgaccctacc actcaaagac tggtttgggc ttgcacgggt 5280
ttggaagtgg gcagaggaca accactgggt gtcggagttt ccggccatcc attgctgaac 5340
aagtacgacg atgttgagaa ctcgggtgga tacggtggca atccaggaca agacaacaga 5400
gtgaatgtcg gcatggacta caagcaaacc cagctgtgca tggttggttg tgcaccacct 5460
ctgggtgaac attggggaaa gggcactcag tgctccaaca cctctgttca gaatggtgac 5520
tgtcctccac tggagctgat cacctcggtt attcaggatg gagacatggt ggacactggc 5580
ttcggtgcaa tgaactttgc cgacttgcag accaacaagt ccgatgttcc attggacatt 5640
tgcggcactg tgtgcaaata tcctgactac ctgcagatgg ctgcagatcc atacggtgac 5700
agactcttct tctacctgag aaaggagcag atgttcgcca gacacttctt taacagagct 5760
ggcaccgttg gtgagcctgt tccagatgac ctgttggtga agggtggaaa caatagatcc 5820
tctgttgcct cgtccatcta cgtccacacc ccatcgggct ctctggtgtc ctcggaggcc 5880
caactgttca acaagcctta ctggctccag aaggcccaag gacacaacaa tggcatctgc 5940
tggggaaacc acctgttcgt caccgttgtg gacacgacta gatccaccaa catgactttg 6000
tgtgcctctg tttccaagtc ggctacctac acgaactcgg actataagga gtacatgaga 6060
catgttgaag agttcgacct gcagtttatc ttccagctct gctccattac cttgtcggca 6120
gaagtgatgg cctacatcca cacgatgaac ccatccgtct tggaagactg gaacttcggt 6180
ctgtctccac ctccaaatgg caccctggaa gacacgtaca gatatgtgca gtcgcaagcc 6240
atcacctgtc agaaaccaac tcctgaaaag gagaaacagg acccatacaa ggatatgtcg 6300
ttctgggagg tcaacctgaa ggagaagttc tcttcggaac tggaccagtt cccattggga 6360
agaaagttcc tgctccagtc tggttataga ggaagaacct ccgcaagaac tggcatcaag 6420
agacctgccg tgtcgaagcc atccaccgct cctaagagaa agagaacgaa gaccaaaaag 6480
taatagcctg caggcagctt tctagaacaa aaactcatct cagaagagga tctgaatagc 6540
gccgtcgacc atcatcatca tcatcattga gtttgtagcc ttagacatga ctgttcctca 6600
gttcaagttg ggcacttacg agaagaccgg tcttgctaga ttctaatcaa gaggatgtca 6660
gaatgccatt tgcctgagag atgcaggctt catttttgat acttttttat ttgtaaccta 6720
tatagtatag gatttttttt gtcattttgt ttcttctcgt acgagcttgc tcctgatcag 6780
cctatctcgc agctgatgaa tatcttgtgg taggggtttg ggaaaatcat tcgagtttga 6840
tgtttttctt ggtatttccc actcctcttc agagtacaga agattaagtg agaccttcgt 6900
ttgtgcggat cctcgatcac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 6960
aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 7020
gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 7080
ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 7140
cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt 7200
cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 7260
gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 7320
cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 7380
agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg 7440
ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 7500
ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 7560
gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 7620
cacgttaagg gattttggtc atgagatcag atctcccaca caccatagct tcaaaatgtt 7680
tctactcctt ttttactctt ccagattttc tcggactccg cgcatcgccg taccacttca 7740
aaacacccaa gcacagcata ctaaattttc cctctttctt cctctagggt gtcgttaatt 7800
acccgtacta aaggtttgga aaagaaaaaa gagaccgcct cgtttctttt tcttcgtcga 7860
aaaaggcaat aaaaattttt atcacgtttc tttttcttga aatttttttt tttagttttt 7920
ttctctttca gtgacctcca ttgatattta agttaataaa cggtcttcaa tttctcaagt 7980
ttcagtttca tttttcttgt tctattacaa ctttttttac ttcttgttca ttagaaagaa 8040
agcatagcaa tctaatctaa ggggcggtgt tgacaattaa tcatcggcat agtatatcgg 8100
catagtataa tacgacaagg tgaggaacta aaccttaaga tgagccatat tcaacgggaa 8160
acgtcttgct cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa 8220
tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc 8280
gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat 8340
gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt 8400
atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc 8460
caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc 8520
ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt 8580
cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat 8640
gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa gcttttgcca 8700
ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac 8760
gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag 8820
gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt 8880
tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc 8940
gatgagtttt tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg 9000
acttgacggg acggcggctt tgttgaataa atcgaacttt tgctgagttg aaggatcaga 9060
tcacgcatct tcccgacaac gcagaccgtt ccgtggcaaa gcaaaagttc aaaatcacca 9120
actggtccac ctacaacaaa gctctcatca accgtggctc cctcactttc tggctggatg 9180
atggggcgat tcaggcctgg tatgagtcag caacaccttc ttcacgaggc agacctca 9238
<210> 42
<211> 1551
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
cggacaaatt cgaaacgatg tctctttggt tgccatccga ggctactgtt tacttgccac 60
ctgtcccagt ttctaaggtc gtgtcaactg atgaatacgt tgctagaacc aacatctatt 120
accacgctgg aacttctaga ttgcttgctg ttggtcatcc atacttccct atcaagaaac 180
ctaacaataa caagatcctt gttccaaagg tctctggatt gcagtacaga gttttcagaa 240
tccacttgcc tgacccaaac aagttcggtt ttcctgacac ctccttctac aacccagata 300
ctcaaagatt ggtgtgggcc tgtgttggtg tcgaggttgg acgtggtcaa ccattgggag 360
ttggtatttc tggacatcct ttgcttaaca agttggatga cactgaaaat gcttctgcct 420
acgcagccaa cgctggtgtt gacaacagag agtgtatctc tatggattac aaacagaccc 480
aattgtgtct tattggttgc aagccaccta ttggtgaaca ctggggtaag ggatctccat 540
gtaccaacgt tgctgtcaat ccaggtgatt gtcctccatt ggagcttatc aacactgtta 600
ttcaagatgg tgacatggtt gatactggat ttggtgctat ggacttcact accttgcaag 660
ctaacaagtc cgaggttcca ttggacattt gtacttctat ctgcaagtat ccagactaca 720
ttaagatggt ctctgaacca tacggtgact ccttgttttt ctaccttcgt agagagcaaa 780
tgtttgttag acacttgttc aacagagctg gtgccgttgg tgagaatgtc ccagacgatt 840
tgtacatcaa gggatctggt tccactgcaa acttggcctc ttccaactac ttcccaactc 900
cttcaggttc tatggttacc tccgatgctc aaatcttcaa caagccttac tggttgcaaa 960
gagcacaggg tcacaacaat ggaatttgtt ggggtaacca acttttcgtt actgtcgttg 1020
ataccacaag atccaccaac atgtccttgt gtgctgccat ttctacttca gaaaccacat 1080
acaagaatac taacttcaag gagtacctta gacatggtga ggaatacgac ttgcagttca 1140
tctttcaatt gtgcaagatt accttgactg ctgacgttat gacctacatc cactctatga 1200
actccactat ccttgaagat tggaacttcg gattgcaacc acctccaggt ggaaccttgg 1260
aagatactta cagatttgtc acatcccaag ccattgcttg tcagaagcac actcctccag 1320
ctcctaagga agatccactt aagaaataca ccttctggga agtcaacttg aaagagaagt 1380
tctctgctga ccttgatcag tttcctttgg gtagaaagtt cttacttcaa gctggattga 1440
aggccaaacc aaagttcacc ttgggtaaaa gaaaggctac tccaaccaca tcatctactt 1500
ccacaaccgc caaacgtaag aagagaaaac tgtaatagcc tgcaggaagc a 1551
<210> 43
<211> 7347
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgg cacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctgcgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttcgcgt cgctgtagcc ggccgcggtg acgataccca catagaggtc 420
cttggccatt agtttgatga ggtggggcag gatgggcgac tcggcatcga aatttttgcc 480
gtcgtcgtac agtgtgatgt caccatcgaa tgtaatgagc tgcagcttgc gatctcggat 540
ggttttggaa tggaagaacc gcgacatctc caacagctgg gccgtgttga gaatgagccg 600
gacgtcgttg aacgaggggg ccacaagccg gcgtttgctg atggcgcggc gctcgtcctc 660
gatgtagaag gccttttcca gaggcagtct cgtgaagaag ctgccaacgc tcggaaccag 720
ctgcacgagc cgagacaatt cgggggtgcc ggctttggtc atttcaatgt tgtcgtcgat 780
gaggagttcg aggtcgtgga agatttccgc gtagcggcgt tttgcctcag agtttaccat 840
gaggtcgtcc actgcagaga tgccgttgct cttcaccgcg tacaggacga acggcgtggc 900
cagcaggccc ttgatccatt ctatgaggcc atctcgacgg tgttccttga gtgcgtactc 960
cactctgtag cgactggaca tctcgagact gggcttgctg tgctggatgc accaattaat 1020
tgttgccgca tgcatccttg caccgcaagt ttttaaaacc cactcgcttt agccgtcgcg 1080
taaaacttgt gaatctggca actgaggggg ttctgcagcc gcaaccgaac ttttcgcttc 1140
gaggacgcag ctggatggtg tcatgtgagg ctctgtttgc tggcgtagcc tacaacgtga 1200
ccttgcctaa ccggacggcg ctacccactg ctgtctgtgc ctgctaccag aaaatcacca 1260
gagcagcaga gggccgatgt ggcaactggt ggggtgtcgg acaggctgtt tctccacagt 1320
gcaaatgcgg gtgaaccggc cagaaagtaa attcttatgc taccgtgcag tgactccgac 1380
atccccagtt tttgccctac ttgatcacag atggggtcag cgctgccgct aagtgtaccc 1440
aaccgtcccc acacggtcca tctataaata ctgctgccag tgcacggtgg tgacatcaat 1500
ctaaagtaca aaaacaaagc tagccacgtg aagcttgaat tcgcggccgc cagctttcta 1560
gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca tcatcatcat 1620
cattgagttt gtagccttag acatgactgt tcctcagttc aagttgggca cttacgagaa 1680
gaccggtctt gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc 1740
aggcttcatt tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca 1800
ttttgtttct tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc 1860
ttgtggtagg ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc 1920
ctcttcagag tacagaagat taagtgagac cttcgtttgt gcggatctgt cgacgcggag 1980
aacgatctcc tcgagctgct cgcggatcag cttgtggccc ggtaatggaa ccaggccgac 2040
ggcacgctcc ttgcggacca cggtggctgg cgagcccagt ttgtgaacga ggtcgtttag 2100
aacgtcctgc gcaaagtcca gtgtcagatg aatgtcctcc tcggaccaat tcagcatgtt 2160
ctcgagcagc catctgtctt tggagtagaa gcgtaatctc tgctcctcgt tactgtaccg 2220
gaagaggtag tttgcctcgc cgcccataat gaacaggttc tctttctggt ggcctgtgag 2280
cagcggggac gtctggacgg cgtcgatgag gcccttgagg cgctcgtagt acttgttcgc 2340
gtcgctgtag ccggccgcgg tgacgatacc cacatagagg tccttggcca ttagtttgat 2400
gaggtggggc aggatgggcg actcggcatc gaaatttttg ccgtcgtcgt acagtgtgat 2460
gtcaccatcg aatgtaatga gctgcagctt gcgatctcgg atggttttgg aatggaagaa 2520
ccgcgacatc tccaacagct gggccgtgtt gagaatgagc cggacgtcgt tgaacgaggg 2580
ggccacaagc cggcgtttgc tgatggcgcg gcgctcgtcc tcgatgtaga aggccttttc 2640
cagaggcagt ctcgtgaaga agctgccaac gctcggaacc agctgcacga gccgagacaa 2700
ttcgggggtg ccggctttgg tcatttcaat gttgtcgtcg atgaggagtt cgaggtcgtg 2760
gaagatttcc gcgtagcggc gttttgcctc agagtttacc atgaggtcgt ccactgcaga 2820
gatgccgttg ctcttcaccg cgtacaggac gaacggcgtg gccagcaggc ccttgatcca 2880
ttctatgagg ccatctcgac ggtgttcctt gagtgcgtac tccactctgt agcgactgga 2940
catctcgaga ctgggcttgc tgtgctggat gcaccaatta attgttgccg catgcatcct 3000
tgcaccgcaa gtttttaaaa cccactcgct ttagccgtcg cgtaaaactt gtgaatctgg 3060
caactgaggg ggttctgcag ccgcaaccga acttttcgct tcgaggacgc agctggatgg 3120
tgtcatgtga ggctctgttt gctggcgtag cctacaacgt gaccttgcct aaccggacgg 3180
cgctacccac tgctgtctgt gcctgctacc agaaaatcac cagagcagca gagggccgat 3240
gtggcaactg gtggggtgtc ggacaggctg tttctccaca gtgcaaatgc gggtgaaccg 3300
gccagaaagt aaattcttat gctaccgtgc agtgactccg acatccccag tttttgccct 3360
acttgatcac agatggggtc agcgctgccg ctaagtgtac ccaaccgtcc ccacacggtc 3420
catctataaa tactgctgcc agtgcacggt ggtgacatca atctaaagta caaaaacaaa 3480
ttcgaaacga tgtctctttg gttgccatcc gaggctactg tttacttgcc acctgtccca 3540
gtttctaagg tcgtgtcaac tgatgaatac gttgctagaa ccaacatcta ttaccacgct 3600
ggaacttcta gattgcttgc tgttggtcat ccatacttcc ctatcaagaa acctaacaat 3660
aacaagatcc ttgttccaaa ggtctctgga ttgcagtaca gagttttcag aatccacttg 3720
cctgacccaa acaagttcgg ttttcctgac acctccttct acaacccaga tactcaaaga 3780
ttggtgtggg cctgtgttgg tgtcgaggtt ggacgtggtc aaccattggg agttggtatt 3840
tctggacatc ctttgcttaa caagttggat gacactgaaa atgcttctgc ctacgcagcc 3900
aacgctggtg ttgacaacag agagtgtatc tctatggatt acaaacagac ccaattgtgt 3960
cttattggtt gcaagccacc tattggtgaa cactggggta agggatctcc atgtaccaac 4020
gttgctgtca atccaggtga ttgtcctcca ttggagctta tcaacactgt tattcaagat 4080
ggtgacatgg ttgatactgg atttggtgct atggacttca ctaccttgca agctaacaag 4140
tccgaggttc cattggacat ttgtacttct atctgcaagt atccagacta cattaagatg 4200
gtctctgaac catacggtga ctccttgttt ttctaccttc gtagagagca aatgtttgtt 4260
agacacttgt tcaacagagc tggtgccgtt ggtgagaatg tcccagacga tttgtacatc 4320
aagggatctg gttccactgc aaacttggcc tcttccaact acttcccaac tccttcaggt 4380
tctatggtta cctccgatgc tcaaatcttc aacaagcctt actggttgca aagagcacag 4440
ggtcacaaca atggaatttg ttggggtaac caacttttcg ttactgtcgt tgataccaca 4500
agatccacca acatgtcctt gtgtgctgcc atttctactt cagaaaccac atacaagaat 4560
actaacttca aggagtacct tagacatggt gaggaatacg acttgcagtt catctttcaa 4620
ttgtgcaaga ttaccttgac tgctgacgtt atgacctaca tccactctat gaactccact 4680
atccttgaag attggaactt cggattgcaa ccacctccag gtggaacctt ggaagatact 4740
tacagatttg tcacatccca agccattgct tgtcagaagc acactcctcc agctcctaag 4800
gaagatccac ttaagaaata caccttctgg gaagtcaact tgaaagagaa gttctctgct 4860
gaccttgatc agtttccttt gggtagaaag ttcttacttc aagctggatt gaaggccaaa 4920
ccaaagttca ccttgggtaa aagaaaggct actccaacca catcatctac ttccacaacc 4980
gccaaacgta agaagagaaa actgtaatag cctgcaggca gctttctaga acaaaaactc 5040
atctcagaag aggatctgaa tagcgccgtc gaccatcatc atcatcatca ttgagtttgt 5100
agccttagac atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc 5160
tagattctaa tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt 5220
tgatactttt ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc 5280
tcgtacgagc ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg 5340
tttgggaaaa tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta 5400
cagaagatta agtgagacct tcgtttgtgc ggatccccca cacaccatag cttcaaaatg 5460
tttctactcc ttttttactc ttccagattt tctcggactc cgcgcatcgc cgtaccactt 5520
caaaacaccc aagcacagca tactaaattt tccctctttc ttcctctagg gtgtcgttaa 5580
ttacccgtac taaaggtttg gaaaagaaaa aagagaccgc ctcgtttctt tttcttcgtc 5640
gaaaaaggca ataaaaattt ttatcacgtt tctttttctt gaaatttttt tttttagttt 5700
ttttctcttt cagtgacctc cattgatatt taagttaata aacggtcttc aatttctcaa 5760
gtttcagttt catttttctt gttctattac aacttttttt acttcttgtt cattagaaag 5820
aaagcatagc aatctaatct aaggggcggt gttgacaatt aatcatcggc atagtatatc 5880
ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt 5940
ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg 6000
ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg 6060
ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg 6120
cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac 6180
gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg 6240
cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtccga 6300
cggcggccca cgggtcccag gcctcggaga tccgtccccc ttttcctttg tcgatatcat 6360
gtaattagtt atgtcacgct tacattcacg ccctcccccc acatccgctc taaccgaaaa 6420
ggaaggagtt agacaacctg aagtctaggt ccctatttat ttttttatag ttatgttagt 6480
attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc 6540
atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga aggctttaat 6600
ttgcaagctg gagaccaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 6660
ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 6720
acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 6780
tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 6840
ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc 6900
ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 6960
ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 7020
actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 7080
gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 7140
tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 7200
caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 7260
atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 7320
acgttaaggg attttggtca tgagatc 7347
<210> 44
<211> 1552
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
attcgctagc atggctctgt ggagaccatc tgacaacacc gtctacttgc cacctccatc 60
tgttgccaga gtcgttaaca ccgacgatta cgtcactaga acatccatct tctaccacgc 120
tggttcctct agattgctta ctgttggtaa tccatacttc agagttccag ctggtggagg 180
taataagcaa gacattccta aggtttctgc ttaccaatat agagtcttta gagttcagtt 240
gcctgaccca aacaagttcg gtttgcctga tacttctatc tacaacccag agactcaaag 300
attggtttgg gcctgtgctg gtgtcgagat cggtagagga caacctttgg gagttggtct 360
ttctggacac ccattctaca acaagttgga tgacactgaa tcttcacatg ctgccacttc 420
caatgtttct gaggatgttc gtgacaacgt ctctgttgat tacaagcaaa ctcagttgtg 480
tatccttgga tgtgcccctg ctattggtga acactgggct aaaggtactg cctgtaagtc 540
cagaccattg tctcaaggtg actgcccacc tttggagctt aagaacactg ttttggaaga 600
tggtgacatg gttgatactg gatatggtgc tatggacttc tctaccctgc aggatactaa 660
gtgtgaggtt ccattggaca tctgtcaatc tatttgtaag taccctgatt atcttcagat 720
gtctgctgac ccatacggtg attctatgtt cttttgcttg agacgtgagc aacttttcgc 780
tagacacttt tggaacagag caggtactat gggtgacacc gttccacaat ctttgtatat 840
caaaggaact ggtatgagag cttctcctgg atcctgtgtt tactcaccat ctccttccgg 900
ttctattgtt acctcagact ctcaattgtt taacaagcca tactggttac acaaggctca 960
gggtcataac aatggtgttt gctggcacaa ccaattgttc gttactgtcg ttgacaccac 1020
tagatccacc aatttgacaa tttgtgcttc tactcagtcc cctgttccag gtcaatatga 1080
tgctaccaaa ttcaagcaat actctagaca cgttgaggaa tatgacttgc agtttatctt 1140
ccaattgtgt actattacct tgactgccga tgttatgtct tacattcatt ccatgaactc 1200
ttcaatcttg gaggattgga actttggtgt tccaccacct ccaacaactt ctttggttga 1260
cacctacaga ttcgtccaat ctgttgctat tacctgtcag aaggatgccg caccagctga 1320
aaacaaggat ccatacgaca agttgaagtt ttggaatgtc gatttgaagg agaagttctc 1380
ccttgacttg gatcaatacc cacttggtag aaagttcttg gttcaggctg gtttgagacg 1440
taagccaact atcggtccaa gaaaacgttc tgctccatca gccactacct cttccaaacc 1500
tgctaagaga gttcgtgtca gagctagaaa gtaatagcgg ccgcataagg aa 1552
<210> 45
<211> 8855
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgg cacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctgcgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttcgcgt cgctgtagcc ggccgcggtg acgataccca catagaggtc 420
cttggccatt agtttgatga ggtggggcag gatgggcgac tcggcatcga aatttttgcc 480
gtcgtcgtac agtgtgatgt caccatcgaa tgtaatgagc tgcagcttgc gatctcggat 540
ggttttggaa tggaagaacc gcgacatctc caacagctgg gccgtgttga gaatgagccg 600
gacgtcgttg aacgaggggg ccacaagccg gcgtttgctg atggcgcggc gctcgtcctc 660
gatgtagaag gccttttcca gaggcagtct cgtgaagaag ctgccaacgc tcggaaccag 720
ctgcacgagc cgagacaatt cgggggtgcc ggctttggtc atttcaatgt tgtcgtcgat 780
gaggagttcg aggtcgtgga agatttccgc gtagcggcgt tttgcctcag agtttaccat 840
gaggtcgtcc actgcagaga tgccgttgct cttcaccgcg tacaggacga acggcgtggc 900
cagcaggccc ttgatccatt ctatgaggcc atctcgacgg tgttccttga gtgcgtactc 960
cactctgtag cgactggaca tctcgagact gggcttgctg tgctggatgc accaattaat 1020
tgttgccgca tgcatccttg caccgcaagt ttttaaaacc cactcgcttt agccgtcgcg 1080
taaaacttgt gaatctggca actgaggggg ttctgcagcc gcaaccgaac ttttcgcttc 1140
gaggacgcag ctggatggtg tcatgtgagg ctctgtttgc tggcgtagcc tacaacgtga 1200
ccttgcctaa ccggacggcg ctacccactg ctgtctgtgc ctgctaccag aaaatcacca 1260
gagcagcaga gggccgatgt ggcaactggt ggggtgtcgg acaggctgtt tctccacagt 1320
gcaaatgcgg gtgaaccggc cagaaagtaa attcttatgc taccgtgcag tgactccgac 1380
atccccagtt tttgccctac ttgatcacag atggggtcag cgctgccgct aagtgtaccc 1440
aaccgtcccc acacggtcca tctataaata ctgctgccag tgcacggtgg tgacatcaat 1500
ctaaagtaca aaaacaaagc tagcatggct ctgtggagac catctgacaa caccgtctac 1560
ttgccacctc catctgttgc cagagtcgtt aacaccgacg attacgtcac tagaacatcc 1620
atcttctacc acgctggttc ctctagattg cttactgttg gtaatccata cttcagagtt 1680
ccagctggtg gaggtaataa gcaagacatt cctaaggttt ctgcttacca atatagagtc 1740
tttagagttc agttgcctga cccaaacaag ttcggtttgc ctgatacttc tatctacaac 1800
ccagagactc aaagattggt ttgggcctgt gctggtgtcg agatcggtag aggacaacct 1860
ttgggagttg gtctttctgg acacccattc tacaacaagt tggatgacac tgaatcttca 1920
catgctgcca cttccaatgt ttctgaggat gttcgtgaca acgtctctgt tgattacaag 1980
caaactcagt tgtgtatcct tggatgtgcc cctgctattg gtgaacactg ggctaaaggt 2040
actgcctgta agtccagacc attgtctcaa ggtgactgcc cacctttgga gcttaagaac 2100
actgttttgg aagatggtga catggttgat actggatatg gtgctatgga cttctctacc 2160
ctgcaggata ctaagtgtga ggttccattg gacatctgtc aatctatttg taagtaccct 2220
gattatcttc agatgtctgc tgacccatac ggtgattcta tgttcttttg cttgagacgt 2280
gagcaacttt tcgctagaca cttttggaac agagcaggta ctatgggtga caccgttcca 2340
caatctttgt atatcaaagg aactggtatg agagcttctc ctggatcctg tgtttactca 2400
ccatctcctt ccggttctat tgttacctca gactctcaat tgtttaacaa gccatactgg 2460
ttacacaagg ctcagggtca taacaatggt gtttgctggc acaaccaatt gttcgttact 2520
gtcgttgaca ccactagatc caccaatttg acaatttgtg cttctactca gtcccctgtt 2580
ccaggtcaat atgatgctac caaattcaag caatactcta gacacgttga ggaatatgac 2640
ttgcagttta tcttccaatt gtgtactatt accttgactg ccgatgttat gtcttacatt 2700
cattccatga actcttcaat cttggaggat tggaactttg gtgttccacc acctccaaca 2760
acttctttgg ttgacaccta cagattcgtc caatctgttg ctattacctg tcagaaggat 2820
gccgcaccag ctgaaaacaa ggatccatac gacaagttga agttttggaa tgtcgatttg 2880
aaggagaagt tctcccttga cttggatcaa tacccacttg gtagaaagtt cttggttcag 2940
gctggtttga gacgtaagcc aactatcggt ccaagaaaac gttctgctcc atcagccact 3000
acctcttcca aacctgctaa gagagttcgt gtcagagcta gaaagtaata gcggccgcca 3060
gctttctaga acaaaaactc atctcagaag aggatctgaa tagcgccgtc gaccatcatc 3120
atcatcatca ttgagtttgt agccttagac atgactgttc ctcagttcaa gttgggcact 3180
tacgagaaga ccggtcttgc tagattctaa tcaagaggat gtcagaatgc catttgcctg 3240
agagatgcag gcttcatttt tgatactttt ttatttgtaa cctatatagt ataggatttt 3300
ttttgtcatt ttgtttcttc tcgtacgagc ttgctcctga tcagcctatc tcgcagctga 3360
tgaatatctt gtggtagggg tttgggaaaa tcattcgagt ttgatgtttt tcttggtatt 3420
tcccactcct cttcagagta cagaagatta agtgagacct tcgtttgtgc ggatctgtcg 3480
acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg taatggaacc 3540
aggccgacgg cacgctcctt gcggaccacg gtggctggcg agcccagttt gtgaacgagg 3600
tcgtttagaa cgtcctgcgc aaagtccagt gtcagatgaa tgtcctcctc ggaccaattc 3660
agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg ctcctcgtta 3720
ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc tttctggtgg 3780
cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg ctcgtagtac 3840
ttgttcgcgt cgctgtagcc ggccgcggtg acgataccca catagaggtc cttggccatt 3900
agtttgatga ggtggggcag gatgggcgac tcggcatcga aatttttgcc gtcgtcgtac 3960
agtgtgatgt caccatcgaa tgtaatgagc tgcagcttgc gatctcggat ggttttggaa 4020
tggaagaacc gcgacatctc caacagctgg gccgtgttga gaatgagccg gacgtcgttg 4080
aacgaggggg ccacaagccg gcgtttgctg atggcgcggc gctcgtcctc gatgtagaag 4140
gccttttcca gaggcagtct cgtgaagaag ctgccaacgc tcggaaccag ctgcacgagc 4200
cgagacaatt cgggggtgcc ggctttggtc atttcaatgt tgtcgtcgat gaggagttcg 4260
aggtcgtgga agatttccgc gtagcggcgt tttgcctcag agtttaccat gaggtcgtcc 4320
actgcagaga tgccgttgct cttcaccgcg tacaggacga acggcgtggc cagcaggccc 4380
ttgatccatt ctatgaggcc atctcgacgg tgttccttga gtgcgtactc cactctgtag 4440
cgactggaca tctcgagact gggcttgctg tgctggatgc accaattaat tgttgccgca 4500
tgcatccttg caccgcaagt ttttaaaacc cactcgcttt agccgtcgcg taaaacttgt 4560
gaatctggca actgaggggg ttctgcagcc gcaaccgaac ttttcgcttc gaggacgcag 4620
ctggatggtg tcatgtgagg ctctgtttgc tggcgtagcc tacaacgtga ccttgcctaa 4680
ccggacggcg ctacccactg ctgtctgtgc ctgctaccag aaaatcacca gagcagcaga 4740
gggccgatgt ggcaactggt ggggtgtcgg acaggctgtt tctccacagt gcaaatgcgg 4800
gtgaaccggc cagaaagtaa attcttatgc taccgtgcag tgactccgac atccccagtt 4860
tttgccctac ttgatcacag atggggtcag cgctgccgct aagtgtaccc aaccgtcccc 4920
acacggtcca tctataaata ctgctgccag tgcacggtgg tgacatcaat ctaaagtaca 4980
aaaacaaatt cgaaacgatg tctctttggt tgccatccga ggctactgtt tacttgccac 5040
ctgtcccagt ttctaaggtc gtgtcaactg atgaatacgt tgctagaacc aacatctatt 5100
accacgctgg aacttctaga ttgcttgctg ttggtcatcc atacttccct atcaagaaac 5160
ctaacaataa caagatcctt gttccaaagg tctctggatt gcagtacaga gttttcagaa 5220
tccacttgcc tgacccaaac aagttcggtt ttcctgacac ctccttctac aacccagata 5280
ctcaaagatt ggtgtgggcc tgtgttggtg tcgaggttgg acgtggtcaa ccattgggag 5340
ttggtatttc tggacatcct ttgcttaaca agttggatga cactgaaaat gcttctgcct 5400
acgcagccaa cgctggtgtt gacaacagag agtgtatctc tatggattac aaacagaccc 5460
aattgtgtct tattggttgc aagccaccta ttggtgaaca ctggggtaag ggatctccat 5520
gtaccaacgt tgctgtcaat ccaggtgatt gtcctccatt ggagcttatc aacactgtta 5580
ttcaagatgg tgacatggtt gatactggat ttggtgctat ggacttcact accttgcaag 5640
ctaacaagtc cgaggttcca ttggacattt gtacttctat ctgcaagtat ccagactaca 5700
ttaagatggt ctctgaacca tacggtgact ccttgttttt ctaccttcgt agagagcaaa 5760
tgtttgttag acacttgttc aacagagctg gtgccgttgg tgagaatgtc ccagacgatt 5820
tgtacatcaa gggatctggt tccactgcaa acttggcctc ttccaactac ttcccaactc 5880
cttcaggttc tatggttacc tccgatgctc aaatcttcaa caagccttac tggttgcaaa 5940
gagcacaggg tcacaacaat ggaatttgtt ggggtaacca acttttcgtt actgtcgttg 6000
ataccacaag atccaccaac atgtccttgt gtgctgccat ttctacttca gaaaccacat 6060
acaagaatac taacttcaag gagtacctta gacatggtga ggaatacgac ttgcagttca 6120
tctttcaatt gtgcaagatt accttgactg ctgacgttat gacctacatc cactctatga 6180
actccactat ccttgaagat tggaacttcg gattgcaacc acctccaggt ggaaccttgg 6240
aagatactta cagatttgtc acatcccaag ccattgcttg tcagaagcac actcctccag 6300
ctcctaagga agatccactt aagaaataca ccttctggga agtcaacttg aaagagaagt 6360
tctctgctga ccttgatcag tttcctttgg gtagaaagtt cttacttcaa gctggattga 6420
aggccaaacc aaagttcacc ttgggtaaaa gaaaggctac tccaaccaca tcatctactt 6480
ccacaaccgc caaacgtaag aagagaaaac tgtaatagcc tgcaggcagc tttctagaac 6540
aaaaactcat ctcagaagag gatctgaata gcgccgtcga ccatcatcat catcatcatt 6600
gagtttgtag ccttagacat gactgttcct cagttcaagt tgggcactta cgagaagacc 6660
ggtcttgcta gattctaatc aagaggatgt cagaatgcca tttgcctgag agatgcaggc 6720
ttcatttttg atactttttt atttgtaacc tatatagtat aggatttttt ttgtcatttt 6780
gtttcttctc gtacgagctt gctcctgatc agcctatctc gcagctgatg aatatcttgt 6840
ggtaggggtt tgggaaaatc attcgagttt gatgtttttc ttggtatttc ccactcctct 6900
tcagagtaca gaagattaag tgagaccttc gtttgtgcgg atcccccaca caccatagct 6960
tcaaaatgtt tctactcctt ttttactctt ccagattttc tcggactccg cgcatcgccg 7020
taccacttca aaacacccaa gcacagcata ctaaattttc cctctttctt cctctagggt 7080
gtcgttaatt acccgtacta aaggtttgga aaagaaaaaa gagaccgcct cgtttctttt 7140
tcttcgtcga aaaaggcaat aaaaattttt atcacgtttc tttttcttga aatttttttt 7200
tttagttttt ttctctttca gtgacctcca ttgatattta agttaataaa cggtcttcaa 7260
tttctcaagt ttcagtttca tttttcttgt tctattacaa ctttttttac ttcttgttca 7320
ttagaaagaa agcatagcaa tctaatctaa ggggcggtgt tgacaattaa tcatcggcat 7380
agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatggcc aagttgacca 7440
gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc 7500
ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg 7560
tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg 7620
tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact 7680
tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt 7740
tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgac 7800
acgtccgacg gcggcccacg ggtcccaggc ctcggagatc cgtccccctt ttcctttgtc 7860
gatatcatgt aattagttat gtcacgctta cattcacgcc ctccccccac atccgctcta 7920
accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt ttttatagtt 7980
atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct gtacagacgc 8040
gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg acgctcgaag 8100
gctttaattt gcaagctgga gaccaacatg tgagcaaaag gccagcaaaa ggccaggaac 8160
cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 8220
aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 8280
tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 8340
ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat 8400
ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 8460
cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 8520
ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 8580
gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 8640
atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 8700
aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 8760
aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 8820
gaaaactcac gttaagggat tttggtcatg agatc 8855

Claims (11)

1. A recombinant expression vector system, comprising: the recombinant expression vector system at least comprises two recombinant expression vectors, each recombinant expression vector contains one or more nucleic acid expression cassettes, each nucleic acid expression cassette contains a target gene and a promoter operably connected with the target gene, each target gene is respectively and independently selected from one of HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV 31L 1, HPV 33L 1, HPV 35L 1, HPV 39L 1, HPV 45L 1, HPV 51L 1, HPV52L1, HPV53L1, HPV 56L 1, HPV 58L 1, HPV 59L 1, HPV 66L 1 or HPV68L1, and all the target genes are different from one another.
2. The recombinant expression vector system of claim 1, wherein: each of the recombinant expression vectors further comprises one or more nucleic acid expression cassettes containing a selectable marker gene.
3. A recombinant engineering bacterium, which is characterized in that: the recombinant engineering bacteria comprise the recombinant expression vector system of claim 1.
4. The recombinant engineered bacterium of claim 3, wherein: the recombinant engineering bacteria are yeasts, and preferably the yeasts are selected from any one or more of saccharomyces cerevisiae, hansenula polymorpha, pichia pastoris, kluyveromyces fragilis, kluyveromyces lactis and schizosaccharomyces pombe.
5. A method for preparing a recombinant expression vector system according to claim 1 or 2, obtained by engineering an original plasmid, comprising in particular the following steps: respectively replacing the promoter and the termination sequence of the two original plasmids with the promoter and the termination sequence of the target gene; then the target genes are respectively inserted between the promoter and the termination sequence.
6. A method of making the recombinant expression vector system of claim 5, the method comprising: the plasmid pPICZB is obtained by transformation, and specifically comprises the following steps:
(1) construction of plasmid pMMZ: respectively replacing a promoter and a termination sequence in the plasmid by using a first promoter MOX insert and a first termination sequence AOXTT to obtain a plasmid pMOXZ-1; replacing a promoter and a termination sequence in the plasmid with a second promoter MOX insert and a second termination sequence AOXTT respectively to obtain a plasmid pMOXZ-2; connecting the plasmid pMOXZ-1 and the plasmid pMOXZ-2;
(2) replacing the selection marker gene on the plasmid to obtain a plasmid with a different selection marker gene from the plasmid in step (1);
(3) and (3) respectively inserting the target gene into the plasmids constructed in the step (1) and the step (2) to obtain the recombinant expression vector.
7. The method for preparing a recombinant expression vector system according to claim 6, wherein: the method further comprises at least one of the following features:
a. the target genes are respectively and independently selected from one of HPV6L1, HPV11L1, HPV16L1, HPV18L1, HPV 31L 1, HPV 33L 1, HPV 35L 1, HPV 39L 1, HPV 45L 1, HPV 51L 1, HPV52L1, HPV53L1, HPV 56L 1, HPV 58L 1, HPV 59L 1, HPV 66L 1 or HPV68L 1;
b. the first promoter MOX insert is a promoter with the sequence shown in SEQ ID NO: 7. SEQ ID NO: 8 is obtained by amplification with a primer and hansenula polymorpha genome DNA as a template;
c. the first termination sequence AOXTT adopts SEQ ID NO: 10. SEQ ID NO: 11 is obtained by amplification with a primer and a pPICZB plasmid as a template;
d. the second promoter MOX insert is a promoter adopting a sequence shown in SEQ ID NO: 7, SEQ ID NO: 9 is obtained by amplification with a primer and hansenula polymorpha genome DNA as a template;
e. the second termination sequence AOXTT adopts SEQ ID NO: 11, SEQ ID NO: the sequence shown in 12 is a primer, and the pPICZB plasmid is a template and is obtained by amplification.
8. The method for preparing recombinant engineering bacteria according to claim 3, which is obtained by transforming the recombinant expression vector system according to claim 1 or 2 into engineering bacteria.
9. Use of the recombinant expression vector system of claim 1 or 2 or the recombinant engineered bacterium of claim 3 or 4 in the preparation of an HPVL1 protein.
10. A method of making an hpv l1 protein, the method selected from any one of:
a. transforming the recombinant expression vector system of claim 1 or 2 into a recombinant engineering bacterium, and expressing the HPVL1 protein by using the recombinant engineering bacterium;
b. the HPVL1 protein is expressed by the recombinant engineering bacteria of claim 3 or 4.
11. A protein produced by the method of claim 10 for producing hpv l1 protein.
CN201911174901.1A 2019-11-26 2019-11-26 Recombinant expression vector system, recombinant engineering bacterium, preparation method and application thereof Pending CN112553240A (en)

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