WO1998032849A2

WO1998032849A2 - Polynucleotide associated with the bo or bor syndrome, its corresponding polypeptide and diagnostic and therapeutic applications

Info

Publication number: WO1998032849A2
Application number: PCT/EP1998/000433
Authority: WO
Inventors: Christine Petit; Sonia Abdelhak; Kalatzis Vasiliki; Sylvie Compain; Christophe Vincent; Dominique Weil
Original assignee: Institut Pasteur
Priority date: 1997-01-29
Filing date: 1998-01-28
Publication date: 1998-07-30
Also published as: WO1998032849A3; AU6293798A

Abstract

This invention concerns a polynucleotide which is selected from the group consisting of: (a) a polynucleotide comprising at least 8 consecutive nucleotides of SEQ ID No. 1; (b) a polynucleotide differing from the polynucleotide defined in (a) by mutation, insertion, deletion of substitution of one or more bases; (c) a polynucleotide, the sequence of which is complementary to the sequence of anyone of polynucleotides defined in (a) and (b); (d) a polynucleotide which hybridizes specifically with anyone of the polynucleotides defined in (a), (b) or (c).

Description

POLYNUCLEOTIDE ASSOCIATED WITH THE BO OR BOR SYNDROME, ITS CORRESPONDING POLYPEPΗDE AND DIAGNOSTIC AND THERAPEUTIC APPLICATIONS

The subject of the present invention pertains to a polynucleotide, the alteration of which is associated with Branchio-Oto-Renal syndrome (BOR) and which may be also involved in some carcinogenetics processes, especially in kidney tumorogenesis. The present invention is also directed to new polynucleotides which share a strong homology with the polynucleotide associated with the BOR syndrome, thus defining the BOR-syndrome-linked polynucleotide as a member of a new human gene family. The present invention also concerns a polypeptide encoded by anyone of the above cited polynucleotides. The invention is also directed to the use of said polypeptides or said polynucleotides, or fragments thereof, for example as tools useful for the in vitro detection of their presence in a biological sample, as well as for the detection of any kind of alteration of a gene belonging to the new gene family to which the polynucleotide associated to the BOR syndrome belongs. Finally, the present invention concerns the use of the polynucleotide or polypeptide for the preparation of therapeutic compositions useful for the treatment of genetic diseases or for the control of tumor proliferation, especially of cancers, particularly kidney tumors.

The association of branchial arch anomalies and hearing impairment has been recognized since the nineteenth century. In 1976 Melnick et al, observing the additional association of renal aberrations, used the term Branchio-Oto-Renal (BOR) syndrome to describe individuals presenting with such branchial, otic and renal anomalies^'1. BOR syndrome is an autosomal dominant disorder with incomplete penetrance and variable expressivity. Several reports have described members within the same family who show different combinations of symptoms with varying degrees of severity, especially with regards to renal anomalies which range from undetected (BO syndrome) to bilateral aplasia²-³.

The branchial anomalies of BOR syndrome consist of latero-cervical fistulas or cysts⁴. The otic anomalies involve the outer, middle and inner ear, affecting both the cochlea and the vestibular apparatus. The outer ear anomalies most frequently include preauricular pits and tags, malformed auricle and, less commonly, malpositioned ears, microtia (undersized auricle), and atresia to stenosis of the external auditory canal^.5. Anomalies of the middle ear mainly comprise hypoplasia or absence of the three ossicles (malleus, incus and stapes) and malformation of the middle ear cavity. Inner ear anomalies include an absent cochlea, an underdeveloped cochlea showing only 1 ¹/2 turns instead of the normal 2 ¹/2 turns, as well as absent or abnormal semicircular canals of the vestibular apparatus⁶. Hearing loss is the most commonly (93% of affected individuals) observed feature of BOR syndrome. Hearing impairment can range from mild to profound? and can be either conductive (i.e. due to external and/or middle ear anomalies), sensorineural (i.e. due to inner ear anomalies) or mixed, as seen in half of the cases⁵. BOR syndrome accounts for approximately 2% of profoundly deaf children⁴. Renal anomalies include unilateral or bilateral hypoplasia, dysplasia and aplasia⁶. In addition, anomalies of the collecting system, such as duplication or absence of the ureter, megaureter, blunted or distorted calyces and extra or bifid pelvis have been observed²-⁸ The incidence of BOR syndrome is difficult to estimate Branchial anomalies and minor renal aberrations may be overlooked, and due to the lethal consequence of the most severe kidney anomalies BOR syndrome may often remain undetected

The clinical features of BOR syndrome are indicative of an early developmental defect taking place between the fourth week and the tenth week of embryonic development The inner ear develops from the otic placode at three and a half weeks⁵* and the cochlea has completed its full two and a half turns at nine weeks The middle ear ossicles, which are visible at week seven, develop from neural crest derived cells of the first and second branchial arches The malleus and incus derive from the first arch and the stapes from the second The external auditory ear canal and the middle ear cavity are derived from the first branchial cleft and pouch respectively, which separate the first and second branchial arch externally (cleft) and internally (pouch) The formation of the auricle requires the correct fusion of six mesenchymal hillocks from the first and second arches that takes place at week Latero-cervical cysts or fistulas are remnants of the second, third and fourth branchial clefts that are normally obliterated at the end of the sixth week Likewise the definitive kidney (metanephros) develops from a reciprocal induction between the metanephπc mesenchyme and the uretenc bud^'O-^{1 '}' that begins during week five and is completed by around week 11

In 1989, Haan et al described a family with an inherited rearrangement of chromosome 8q, dir ins (8)(q24 11 ,q13 3,q21 13), presenting with BO syndrome and Tncho-Rhino-Phalangeal syndrome^''² This report mapped the gene underlying BOR syndrome to chromosome band 8q13 3 or 8q21 13 By linkage analysis the gene was then localised to 8q13 3 between the polymorphic markers D8S543 and D8S286, an interval of approximately 7 cM¹3,14 Subsequent refinement of this interval was then accomplished by the precise mapping of the aforementioned translocation that was shown to be associated with a deletion^{1 5}>^'16 The gene interval was recently redefined to between 470 and 650 kb, flanked by the polymorphic markers D8S1060 and D8S1807¹⁶

In an attempt to clone in eukaryotic genomes the gene underlying BOR syndrome, the inventors constructed a bacteπophage P1 and PAC contig spanning this interval and undertook a positional cloning approach based on a sequencing strategy

For the purpose of the present invention, a Contig is defined as a consensus sequence obtained after alignment of the multiple nucleic sequences of the same clones and of the multiple nucleic sequences of different clones containing common sequences

Computer analysis of the sequences obtained from P1 4405 mapping to the centre of the deletion, demonstrated the presence of putative exons showing homology to the drosophila developmental gene eyes absent (eya) Eya is required prior to the morphogenetic furrow for eye morphogenesis We report here the characterisation of this newly identified gene as underlying BOR syndrome, as well as the identification of two other human homologues of eya. These three newly identified eya related genes are suggestive of a novel gene family that plays a role in development. They do not seem to be linked to the development of eyes in humans, according to the present data.

As it clearly appears from the above detailed teachings of prior art, Branchio- Oto-Renal (BOR) syndrome is a congenital disease characterized by a combination of varying branchial, otic and renal anomalies. The causative gene has been assigned to chromosome band 8q13.3 but it must be underlined that no evidence for genetic heterogeneity has ever been reported in the prior art.

The present inventors have now isolated and sequenced both the genomic and the cDNA corresponding to the gene associated with the BOR syndrome.

Using a positional cloning strategy based on genomic DNA sequencing, the inventors have identified a gene in the candidate region that they have named eyal. A chromosomal deletion and seven mutations predicted to disrupt the eyal gene product have been identified in unrelated BOR-affected individuals, thus establishing that eyal underlies BOR syndrome. The EYA1 polypeptide encoded by the eyal gene was found to share a substantial homology with a drosophila protein that is responsible of the eyes absent phenotype, that is characterized by a reduction or absence of the adult compound eye, thus a phenotype unrelated to the BOR syndrome caused by eyal.

For the purpose of the present invention, the expression « eyal » is used to designate the genomic DNA. As it will be further disclosed, the expressions « eyal- a », « eya1-b » and « eya1-c » are used to designate respectively the three transcripts produced by eyal. The eyal gene was also found to be poorly homologous (43% identity over 72 aminoacids) with an Expressed Sequence Tag (EST) of C. elegans, which is a short transcribed sequence.

Moreover, the inventors have also characterized two other genes, structurally related to eyal, that have been named eya2 and eya3. These two genes are also involved in the development of genetic defects, as it will be detailed further in the specification. For example, eya2 is localized in a chromosome region associated with Fanconi Anemia typel (FA1 ) and epilepsy benign neonatal 1 (EBN1 ) genetic defects.

The inventor's discovery of three members of a new gene family is allowing new means for the detection of genetic defects the causative agent of which was previously remained unknown. The availability of the genes eyal, eya2 and eya3 is also allowing the obtention of their expression products that can be used both to produce diagnostic means to detect a genetic defect in a patient and therapeutic compositions in order to prevent or cure the disease induced by the said genetic defect.

More particularly, eyal is strongly associated with some carcinogenic processes, especially in kidney tumorogenesis. eya2 and eya3 are likely to be responsible for other human developmental diseases and may similarly be linked to the control of the development of various tumors or eukaryotic cell differenciation process. The type of DNA modifications detected in BOR affected patients supports that the disease results from reduced gene dosage, implying that the amount of the protein encoded by eyal is critical for the normal development of the branchial arches, ear and kidney. Consequently, the polynucleotide or polypeptide (including antibodies raised against the polypeptide) detection means made available by the inventors allow not only the determination of the presence or absence of the expression of eyal, eya2 or eya3 but also their quantification in a biological sample which is useful to detect a pathological level of expression of these genes Indeed, these new detection means are likely to be used in ante-natal diagnosis

Thus, one object of the present invention consists in a polynucleotide corresponding to the eyal genomic sequence which is characterized by the SEQ ID N°1 that is detailed in Annex 1

The polynucleotide of SEQ ID N°1 contains the whole exons and introns of the eyal gene as well as non coding sequences localized both at 5' and 3'ends and which may be very useful to express the eyal gene or its cDNA counterparts in a suitable vector For this reason, all the non coding regions of the polynucleotide of SEQ ID N°1 are also part of the present invention

The genomic sequence has been found by the inventors to contain 18 exons named, from 5' to 3' of the strand carrying the coding sequence, respectively Q, R, S, T, U, V, W, X, Y, z, A, B, C, D, E, F, G and H The inventors have isolated three different mRNA transcripts corresponding to three different splicing patterns of the genomic sequence that leads to three different mRNAs that are herein called (as well as their cDNA counterparts) respectively

- eya1-a this trancnpt includes exons R, T, U, V, W, X, Y, z, A, B, C, D, E F G and H of the genomic sequence of SEQ ID N° 1 The resultant polynucleotide obtained by reverse transcription of this mRNA has a nucleotide sequence which is the following SEQ ID N° 2

- eya1-c this transcript includes exons R, S, T, U, V, W, X, Y, z, A, B, C, D, E, F, G and H of the genomic sequence of SEQ ID N° 1 The resultant polynucleotide obtained by reverse transcription of this mRNA has a nucleotide sequence which is the following SEQ ID N° 3

- eya7-ι this transcripts includes exons P, Q, S, T, U, V, W, X, Y, z, A B, C, D, E, F, G and H of the genomic sequence of SEQ ID N° 1 The resultant polynucleotide obtained by reverse transcription of this mRNA has a nucleotide sequence which is the following SEQ ID N°4

The polynucleotides eya1-b and eya1-c have the same translation start codon ATG, localized at the same position, and are thus coding for the same polypeptide of SEQ ID N° 8 that is described further in the specification

The polynucleotide eya1-a has an ATG start codon which is localized in exon R, but , due to an alternative splicing generating both a shorter length transcript and a frameshift, eya1-a gives rise to polypeptide different frome the polypeptide resulting from the translation of eya1-b and eya1-c he polypeptide translated from eyal is described hereinafter as the polypeptide of SEQ ID N° 7

Another object of the present invention consists in a polynucleotide comprising the cDNA sequence eya1-a which is characterized by the sequence SEQ ID N°2

The polynucleotide of sequence SEQ ID N°2 comprises notably the restriction sites as described in ANNEX 2 The invention concerns also the fragments of this polynucleotide obtainable by the use of at least one of the said restriction enzymes The polynucleotide of sequence SEQ ID N°2 is contained, from 5'end to 3'end, in the following clones HSEYA1 aNEYAK7, HSEYA1a6K7F4, HSEYA13'5' and HSEYA1 Puc3, deposited at the Collection de Cultures de Microorganismes (CNCM) respectively under the accession numbers 1-1824, 1-1836, 1-1832 and 1-1830

Another object of the present invention consists in a polynucleotide comprising the cDNA eya1-b which is characterized by the sequence SEQ ID N°3

The polynucleotide of sequence SEQ ID N°3 comprises notably the restriction sites as described in ANNEX 3 The invention concerns also the fragments of this polynucleotide obtainable by the use of at least one of the said restriction enzymes

The polynucleotide of SEQ ID N° 3 is contained, from 5' to 3' end, in the following clones HSEYA1 b5p35, HSEYA13'5' and HSEYA1 Puc3 deposited at the Collection de Cultures de Microorganismes (CNCM) respectively under the following accession numbers 1-1835, 1-1832 and 1-1830

Another object of the present invention consists in a polynucleotide comprising the cDNA sequence eya7-c which is characterized by the sequence SEQ ID N° 4

The polynucleotide of sequence SEQ ID N°4 comprises notably the restriction sites as described in ANNEX 4 The invention concerns also the fragments of this polynucleotide obtainable by the use of at least one of the said restriction enzymes

The polynucleotide of sequence SEQ ID N°4 is contained, from 5' to 3' end, in the following clones HSEYA1aNEYAK7, HSEYA1c14K7F6, HSEYA13'5' and HSEYA1 Puc3, deposited at the Collection de Cultures de Microorganismes (CNCM) under the accession numbers 1-1824, 1-1822, 1-1832 and 1-1830

Another object of the present invention consists in a polynucleotide the sequence of which shares a strong homology with the sequences SEQ ID N°1 to SEQ ID N°4

For the purpose of the present invention, a first polynucleotide which shares a strong homology with a second polynucleotide is defined as a first polynucleotide having 8Q % identity in its nucleic sequence with the second polynucleotide

The polynucleotide defined above comprises the cDNA of a new gene named eya2 the said polynucleotide being characterized by the sequence SEQ ID N°5

The polynucleotide of sequence SEQ ID N°5 comprises notably the restriction sites as described in the Annex 5 The invention concerns also fragments of this polynucleotide obtainable by the use of at least one of said restriction enzymes

The polynucleotide of sequence SEQ ID N°5 is contained, from 5' to 3' end, in the following clones HSEYA2-20R50 3, HSEYA2-20ιnt8 and HSEYA2-20F250 10 deposited at the Collection de Cultures de Microorganismes (CNCM) under the accession numbers 1-1834, 1-1829 and 1-1833

Another object of the present invention consists in a polynucleotide the sequence of which shares a strong homology with the sequences SEQ ID N°1 to SEQ ID N°4 For the purpose of the present invention, a first polynucleotide which shares a strong homology with a second polynucleotide is defined as a first polynucleotide having 80 % identity in its nucleic sequence with the second polynucleotide

The polynucleotide defined above comprises the cDNA of a new gene named eya3 the said polynucleotide being characterized by the sequence SEQ ID N°6

The polynucleotide of sequence SEQ ID N°6 comprises notably the restriction sites as described in Annex 6 The invention concerns also fragments of this polynucleotide obtainable by the use of at least one of said restriction enzymes

The polynucleotide of sequence SEQ ID N° 6 is contained, from 5'end to 3' end, in the following clones HSEYA3-z39F11 , HSEYA3-z39ιnt and HSEYA3-z39 23 deposited at the Collection de Cultures de Microorganismes (CNCM) under the accession numbers 1-1823, 1-1825 and 1-1831

More particularly, the present invention is directed to a polynucleotide which is selecting from the group consisting of

(a) a polynucleotide comprising at least 8 consecutive nucleotides of anyone of SEQ ID N°1 ,

(b) a polynucleotide differing from the polynucleotide defined in (a) by mutation, insertion, deletion or substitution of one or more bases ,

(c) a polynucleotide, the sequence of which is complementary to the sequence of anyone of polynucleotides defined in (a) and (b) ,

(d) a polynucleotide which hybridizes specifically with anyone of the polynucleotides defined in (a), (b) or (c)

An other object of the present invention are also the polynucleotides entering in the above definition and which are the following polynucleotides that are selected from the group consisting of

(a) a. polynucleotide comprising at least 8 consecutive nucleotides of SEQ ID N°2, SEQ ID N°3, SEQ ID N°4, SEQ ID N°5 or SEQ ID N°6 ,

(d) a polynucleotide which hybridizes specifically with anyone of the polynucleotides defined in (a), (b) or (c) By polynucleotide or nucleic acid according to the present invention is meant either a double stranded DNA, a single stranded DNA, including a synthetic nucleic acid, as well as their transcription products.

By polynucleotide having a sequence complementary to the sequence of one polynucleotide of the invention is meant any nucleic acid wherein the nucleotides are complementary to those of either SEQ IDN°1 , SEQ ID N°2, SEQ ID N°3, SEQ ID N°4, SEQ ID N°5 or SEQ ID N°6 and the orientation of which is reversed

By DNA according to the present invention is meant either genomic DNA or cDNA, the latter being obtained by reverse transcription of an RNA molecule

By polynucleotide hybridizing specifically with a second polynucleotide is meant that the temperature and ionic strength parameters are selected in such a manner that they allow two polynucleotides having complementary sequences to be maintained in a hybridized form.

As an illustrative embodiments of the conditions to be met to keep hybridized two polynucleotides having complementary sequences, the following hybridization conditions are used :

The hybridization step is realized at 65°C in the presence of 6 x SSC buffer, 5 x Denhardt's solution, 0.5% SDS and 100μg/ml of salmon sperm DNA

For technical information, 1 x SSC corresponds to 0 15 M NaCl and 0.05M sodium citrate; 1 x Denhardt's solution corresponds to 0.02% Ficoll, 0.02% polyvinylpyrrolidone and 0.02% bovine serum albumin.

The hybridization step is followed by four washing steps .

- two washings during 5 min, preferably at 65°C in a 2 x SSC and 0.1 %SDS buffer;

- one washing during 30 min, preferably at 65°C in a 2 x SSC and 0.1 % SDS buffer,

- one washing during 10 min, preferably at 65°C in a 1 x SSC and 0.1 %SDS buffer

Fragments of the polynucleotides according to the invention may be obtained by cleavage using one or several restriction endonucleases, the desired fragments being obtained by taking into account of the restriction sites contained in SEQ ID N°2, SEQ ID N°3, SEQ ID N°4, SEQ ID N°5 or SEQ ID N°6 respectively disclosed in the Annexes . The conditions under which the restrictions enzymes are used in order to generate the polynucleotide fragments according to the invention are described in Sambrook et al., 1989.

Another object of the present invention consists in a polypeptide which is encoded by the polynucleotide of SEQ ID N°1 or SEQ ID N°2 the said polypeptide having the sequence SEQ ID N°7.

Another object of the present invention is a polypeptide which is encoded by the polynucleotide of SEQ ID N° 1 , SEQ ID N° 3 or SEQ ID N°4, said polypeptide being named EYA1-B and having the sequence SEQ ID N°8

The eya1-a cDNA contains sixteen exons from the genomic sequence of SEQ ID N°1. The most conserved C-terminal part of the EYA1 polypeptide spans eight exons of the eya1-a cDNA of sequence SEQ ID N° 2, starting with exon A and ending with exon H. For exons D and F, 5' splicing occurs after the first base of the last codon. For exons C and E, 5' splicing occurs after the second base of the last codon For exons A, B and H, 5' splicing occurs after the third base of the last codon

The aminoacid sequences corresponding to the translation of the exons of eyal are the foliowings

Exon A RVFIWDLETIIVFHSLLTGSYANRYGR

Exon B DPPTSVSLGLRMEEMIFNLADTHLFFNDLE

Exon C ECDQVHIDDVSSDDNGQDLS

Exon D

T TYYNNFFGGTTDGFPAAATSANLCLATGVRGGVDWMRKLAFRYRRVKEIYNTYKNNVGG

Exon E LLGPAKREAWQLRAEIEALTDSWLTLALKALSLIHSR

Exon F TNCVNILVTTTQLIPALAKVLLYGLGIVFPIENIYSATKIG

Exon G KESCFERIIQRFGRKWYWIGDGVEEEQGAKK

Exon H HAMPFWRISSHSDLMALHHALELEYL

Another object of the present invention consists in a polypeptide which is encoded by the polynucleotide of SEQ ID N°5 the said polypeptide being named EYA2 and having the sequence SEQ ID N°9

Another object of the present invention consists in a polypeptide which is encoded by the polynucleotide of SEQ ID N°6 the said polypeptide being named EYA3 and having the sequence SEQ ID N°10

Another object of the present invention consists in a polypeptide which is selected, from the group consisting of

(a) a polypeptide comprising anyone of the aminoacid sequences SEQ ID N° 7, SEQ ID N°8, SEQ ID N°9 or SEQ ID N°10,

(b) a peptide fragment of at least 10 aminoacids in length of the polypeptide defined in (a), the said peptide fragment being recognized by a polyclonal or a antibody directed against the polypeptide defined in (a),

(c) a polypeptide comprising, as regards to the polypeptide defined in (a) or (b), at least one modification by addition or substitution of an aminoacid, the said modified polypeptide being recognized by an antibody directed against the polypeptide defined in (a)

A further object of the present invention are also peptide fragments of the above disclosed polypeptides that may be obtained by cleavage of the polypeptides of SEQ ID N° 7, SEQ ID N°8, SEQ ID N°9 or SEQ ID N°10 with a proteolytic enzyme such that trypsin, chymotrypsine, collagenase, clostnpaine, Myxobacter protease 3 Proline endopeptidase, Staphylococcal protease, trypsm having the lysine residues blocked, trypsm having the arginine residues blocked or the endoproteinase Asp-N Fragments of the polypeptides according to the invention may also be obtained by placing the polypeptide in a very acid solution (pH 2 5) or by cleavage using chemical reagents such as cyanogen bromide or lodobenzoate

Preferred peptide fragments according to the present invention are the fragments that are recognized by antibodies directed respectively to the polypeptides of SEQ ID N° 7, SEQ ID N°8, SEQ ID N°9 or SEQ ID N°10 Such peptide fragments have advantageously a length of at least 20 aminoacids

Such peptide fragments may be prepared either by chemical synthesis (Houbenweyl et al , 1974), from cell host transformed with an expression vector containing a nucleic acid allowing the expression of said peptide fragments, when the peptide encoding sequence is placed under the control of appropriate regulation and/or expression elements These peptide fragments may also be generated by chemical or enzymatic cleavage as described above

An other object of the present invention are polypeptides that are homologous to any of the polypeptides of SEQ ID N° 7, SEQ ID N°8, SEQ ID N°9 or SEQ ID N°10 By homologous peptide according to the present invention is meant a polypeptide containing one or several aminoacid additions, deletions and/or substitutions in either SEQ ID N° 7, SEQ ID N°8, SEQ ID N°9 or SEQ ID N°10 In the case of an aminoacid substitution, one or several -consecutive or non-consecutive- aminoacids are replaced by « equivalent » aminoacids The expression « equivalent » aminoacid is used herein to name any aminoacid that may substituted for to one of the aminoacids belonging to the initial polypeptide structure without modifying the antigenic properties of the corresponding peptides In other words, the «equιvalent » aminoacids are those which allow the generation or the obtention of a polypeptide with a modified sequence as regards to SEQ ID N° 7, SEQ ID N°8, SEQ ID N°9 or SEQ ID N°10, the said modified polypeptide being able to induce antibodies recognizing the parent polypeptide of either SEQ ID N° 7, SEQ ID N°8, SEQ ID N°9 or SEQ ID N°10

These equivalent aminoacids may be determined either by their structural homology with the initial aminoacids to be replaced or by the results of the cross- immunogenicity between the parent peptides and their modified counterparts

As an illustrative example, it should be mentioned the possibility to perform substitutions without a deep change in the immunogenicity of the correspondant modified peptides by replacing, for example, leucine by valine, or isoleucine, aspartic acid by glutamic acid, glutamine by asparagine, arginine by lysine etc , it being understood that the reverse substitutions are permitted in the same conditions

The present invention is also directed to a nucleic acid encoding a polypeptide of SEQ ID N° 7, SEQ ID N°8, SEQ ID N°9 or SEQ ID N°10 according to the invention or a polypeptide homologous to the latter or also one peptide fragment of the latters, as defined herein above

The invention also pertains to nucleotidic fragments that are complementary to the polynucleotides according to the invention, as well as nucleotidic fragments that are modified, as regards to the latters by deletion or addition of one or several nucleotides in a ratio of about 15% as regards to the length of the nucleic fragments and/or modified by substitution of one or several nucleotides, provided that the modified nucleic fragments retain their ability to hybridize with any of the O polynucleotides of SEQ ID N°1 , SEQ ID N°2, SEQ ID N°3, SEQ ID N°4, SEQ ID N°5 or SEQ ID N°6 in the hybridization conditions described above

Advantageously, a nucleic fragment as defined herein above has a length of at least 8 nucleotides, which is the minimal length that has been determined to allow a specific hybridization with either SEQ ID N°1 , SEQ ID N°2, SEQ ID N°3, SEQ ID N°4, SEQ ID N°5 or SEQ ID N°6 Preferably the nucleic fragment has a length of at least 12 nucleotides and more preferably 20 consecutive nucleotides of any of the SEQ ID N°1 , SEQ ID N°2, SEQ ID N°3, SEQ ID N°4, SEQ ID N°5 or SEQ ID N°6

These nucleic fragments may be used as primers for use in amplification reactions, or as nucleic probes

Thus, the polynucleotides of SEQ ID N°1 , SEQ ID N°2, SEQ ID N°3, SEQ ID N°4, SEQ ID N°5 or SEQ ID N°6 or the nucleic fragments obtained from such polynucleotides may be used to select nucleotide primers notably for an amplification reaction such as the amplification reactions further described

PCR is described in the US patent N° 4,683,202 The amplified fragments may be identified by an agarose or a polyacrylamide gel electrophoresis, or by a capillary electrophoresis or alternatively by a chromatography technique (gel filtration, hydrophobic chromatography or ion exchange chromatography) The specificity of the amplification may be ensured by a molecular hybridization using as nucleic probes the polynucleotides SEQ ID N°1 , SEQ ID N°2, SEQ ID N°3, SEQ ID N°4, SEQ ID N°5 or SEQ ID N°6, fragments thereof, ohgonucleotides that are complementary to these polynucleotides or fragment thereof or their amplification products themselves

Amplified nucleotide fragments are used as probes in hybridization reactions in order to detect the presence of one polynucleotide according to the present invention or in order to detect mutations in the SEQ ID N°1 , SEQ ID N°2, SEQ ID N°3, SEQ ID N°4, SEQ ID N°5 or SEQ ID N°6

An other object of the present invention are the amplified nucleic fragments (« amphcons ») defined herein above

These probes and amphcons may be radioactively or non-radioactively labeled, using for example enzymes or fluorescent compounds

Preferred nucleic acid fragments that can serve as primers according to the present invention are those of SEQ ID N° 11 to SEQ ID N° 47

Such nucleic acid fragments may be used as pairs in order to amplify specific regions of SEQ ID N°1 , SEQ ID N°2, SEQ ID N°3, SEQ ID N°4, SEQ ID N°5 or SEQ ID N°6 As an illustrative embodiment of pairs of primers according to the present invention that can be used in one nucleic acid amplification reaction are the followings

Pair l : SEQ ID N° 11

SEQID N° 12 This pair of ohgonucleotides is used for amplifying the full length coding region of SEQ ID N°2

Pair 2 : SEQ ID N° 13

SEQ ID N° 14 AΛ This pair of ohgonucleotides is used as internal primers to amplify exons E and F of SEQ ID N°2.

Pair 3 : SEQ ID N°15

SEQ ID N° 16

This pair of ohgonucleotides is used to amplify exon z of SEQ ID N°2.

Pair 4 : SEQ ID N°17

SEQ ID N°18 This pair of ohgonucleotides is used to amplify exon A of SEQ ID N°2.

Pair 5 : SEQ ID N° 19

SEQ ID N° 20 This pair of ohgonucleotides is used to amplify exon B of SEQ ID N°2.

Pair 6 : SEQ ID N° 21

SEQ ID N° 22

This pair of ohgonucleotides is used to amplify exon C of SEQ ID N°2.

Pair 7 : SEQ ID N° 23

SEQ ID N° 24 This pair of ohgonucleotides is used to amplify exon D of SEQ ID N°2.

Pair 8 : SEQ ID N° 25

SEQ ID N° 26

This pair of ohgonucleotides is used to amplify exons E and F of SEQ ID N°2.

Pair 10: SEQ ID N° 27

SEQ ID N° 28 This pair of ohgonucleotides is used to amplify exon G of SEQ ID N°2.

Pair 11 : SEQ ID N° 30

SEQ ID N° 31 This^" pair of ohgonucleotides is used to amplify exon R of SEQ ID N°2.

Pair 12 : SEQ ID N° 32

SEQ ID N° 33 This pair of ohgonucleotides is used to amplify exon S of SEQ ID N°2.

Pair 13 : SEQ ID N° 34 SEQ ID N° 35 This pair of ohgonucleotides are used to amplify exon T of SEQ ID N°2

Pair 14 : SEQ ID N° 36

SEQ ID N° 37 This pair of ohgonucleotides are used to amplify exon U of SEQ ID N°2. All

Pair 15 : SEQ ID N° 38

SEQ ID N° 39

This pair of ohgonucleotides are used to amplify exon V of SEQ ID N°2

Pair 16 : SEQ ID N° 40

SEQ ID N° 41 This pair of ohgonucleotides are used to amplify exon W of SEQ ID N°2

Pair 17 : SEQ ID N° 42

SEQ ID N° 43

This pair of ohgonucleotides are used to amplify exon X of SEQ ID N°2

Pair 18 : SEQ ID N° 44

SEQ ID N° 45 This pair of ohgonucleotides are used to amplify exon Y of SEQ ID N°2

Pair 19 : SEQ ID N° 46

SEQ ID N° 47 This pair of ohgonucleotides are used to amplify exon H of SEQ ID N°2

The primers may also be used as ohgonucleotide probes to specifically detect a polynucleotide according to the invention

Other techniques related to nucleic acid amplification may also be used and are generally preferred to the PCR technique

The Strand Displacement Amplification (SDA) technique (Walker et al , 1992) is an isothermal amplification technique based on the ability of a restriction enzyme to cleave one of the strands at his recognition site (which is under a hemiphosphorothioate form) and on the property of a DNA polymerase to initiate the synthesis of a new strand from the 3'OH end generated by the restriction enzyme and on the property of this DNA polymerase to displace the previously synthesized strand being localized downstream The SDA method comprises two main steps a) The synthesis, in the presence of dCTP-alpha-S, of DNA molecules that are flanked by the restriction sites that may be cleaved by an appropriate enzyme b) The exponential amplification of these DNA molecules modified as such, by enzyme cleavage, strand displacement and copying of the displaced strands The steps of cleavage , strand displacement and copy are repeated a sufficient number of times in order to obtain an accurate sensitivity of the assay

The SDA technique was initially realized using the restriction endonuclease Hindi but is now generally practiced with an endonuclease from Bacillus stearothermophilus (SSOBI) and a fragment of a DNA polymerase which is devoid of any 5'→3'exonuclease activity isolated from Bacilllus cladotenax (exo- Bca) [=exo- minus-Sca] Both enzymes are able to operate at 60°C and the system is now optimized in order to allow the use of dUTP and the decontamination by UDG When using this technique, as described by Spargo et al in 1996, the doubling time of the target DNA is of 26 seconds and the amplification rate is of 10¹⁰ after an incubation

The SDA amplification technique is more easy to perform than PCR (a single thermostated waterbath device is necessary) and is faster than the other amplification methods

Thus, another object of the present invention consists in using the nucleic acid fragments according to the invention (primers) in a method of DNA or RNA amplification according to the SDA technique For performing of SDA, two pairs of primers are used a pair of external primers (B1 , B2) consisting in a sequence specific of the target polynucleotide of interest and a pair of internal primers (S1 , S2) consisting in a fusion ohgonucleotide carrying a site that is recognized by a restriction endonuclease, for example the enzyme SSOBI

As an illustrative embodiment of the use of the primers according to the invention in a SDA amplification reaction, a sequence that is non specific for the target polynucleotide and carrying a restriction site for Hindi or SSOBI is added at the 5'end of a primer specific either for SEQ ID N°1 , SEQ ID N°2, SEQ ID N°3, SEQ ID N°4, SEQ ID N°5 or SEQ ID N°6 Such an additional sequence containing a restriction site that is recognized by SsoBI is advantageously the following sequence GCATCGAATGCATGTCTCGGGT, the nucleotides represented in bold characters corresponding to the recognition site of the enzyme SsoBI Thus, primers useful for performing SDA amplification may be designed from any of the primers according to the invention as described above and are part of the present invention The operating conditions to perform SDA with such primers are described in Spargo et al, 1996

More specifically, the following conditions are used when performing the SDA amplification reaction with the primers of the invention designed to contain a SsoBI restriction site

SsoBI/exo^'Bca [=exo-minus-Sca] SDA reactions are performed in a 50μl volume with final concentrations of 9 5 mM MgCI₂, 1 4 mM each dGTP, dATP, TTP, dCTP-alpha- S, 100 μg/ml acetylated bovine serum albumin, 10 ng/ml human placental DNA, 35 mM K₂HPO₄ pH 7 6, 0 5 μM primers S1 _SSOBI and B2 _SSOBI, 0 05 μM primers B1 _BSOBI and B2 _SSOBI, 3 2 U/μl SsoBI enzyme, 0 16 U/μl exo^'Bca [=exo-minus-Sca] enzyme 3mM Tris-HCI, 11 mM NaCl, 0 3 mM DTT, 4 mM KCI, 4% glycerol, 0 008mM EDTA and varying amounts of target DNA Prior to the addition of SsoBI and exo^'Bca, incomplete reactions (35μl) are heated at 95°C for 3 mm to denature the target DNA, followed toy 3 mm at 60°C to anneal the primers Following the addition of a 15 μl enzyme mix consisting of 4 μl of SsoBI (40 Units/μl), 0 36 μl exo^'Bca (22 Units/μl), and 10 6 μl enzyme dilution buffer (10 mM Tπs Hcl, 10 mM MgCI₂, 50 mM NaCl, 1 mM DTT), the reactions are incubated at 60°C for 15 mm Amplification is terminated by heating for 5 m in a boiling water bath A non-SDA sample is created by heating a sample in a boiling water bath immediately after enzyme addition Aerosol resistant tips from Continental Laboratory Products are used to reduce contamination of SDA reactions with previously amplified products

The polynucleotides of SEQ ID N°1 , SEQ ID N°2, SEQ ID N°3, SEQ ID N°4 SEQ ID N°5 or SEQ ID N°6 and their above described fragments, especially the primers according to the invention, are useful as technical means for performing different target nucleic acid amplification methods such as

- TAS (Transcription-based Amplification System), described by Kwoh et al in 1989

- SR (Self-Sustained Sequence Replication), described by Guatel et al in 1990 - NASBA (Nucleic acid Sequence Based Amplification), described by Kievitis et al in 1991

- TMA (Transcription Mediated Amplification)

The polynucleotides of SEQ ID N°1 , SEQ ID N°2, SEQ ID N°3, SEQ ID N°4, SEQ ID N°5 or SEQ ID N°6 and their above described fragments, especially the primers according to the invention, are also useful as technical means for performing methods for amplification or modification of a nucleic acid used as a probe , such as

- LCR (Ligase Cham Reaction), described by Landegren et al in 1988 and improved by Barany et al in 1991 who employ a thermostable ligase

- RCR (Repair Cham Reaction) described by Segev et al in 1992

- CPR ( Cycling Probe Reaction), described by Duck et al in 1990

- Q-beta rephcase reaction, described by Miele et al in 1983 and improved by Chu et al in 1986, Lizardi et al in 1988 and by Burg et al and Stone et al in 1996

When the target polynucleotide to be detected is a RNA, for example a mRNA, a reverse transcπptase enzyme will be used before the amplification reaction in order to obtain a cDNA from the RNA contained in the biological sample The generated cDNA is subsequently used as the nucleic acid target for the primers or the probes used in an amplification process or a detection process according to the present invention

Nucleic probes according to the present invention are specific to detect a polynucleotide of the invention By « specific probes » according to the invention is meant any ohgonucleotide that hybridizes with one polynucleotide of SEQ ID N°1 , SEQ ID N°2, SEQ ID N°3, SEQ ID N°4, SEQ ID N°5 or SEQ ID N°6 and which does not hybridize with unrelated sequences Prefered ohgonucleotide probes according to the invention are at least 8 nucleotides in length, and more preferably a length comprised between 8 and 300 nucleotides

The ohgonucleotide probes according to the present invention hybridize specifically with a DNA or RNA molecule comprising all or part of one polynucleotide among SEQ ID N°1 , SEQ ID N°2, SEQ ID N°3, SEQ ID N°4, SEQ ID N°5 or SEQ ID N°6 under stringent conditions

As an illustrative embodiment, the stringent hybridization conditions used in order to specifically detect a polynucleotide according to the present invention are advantageously the follow gs

The hybridization step is realized at 65°C in the presence of 6 x SSC buffer, 5 x Denhardt's solution, 0,5% SDS and 100μg/ml of salmon sperm DNA

The hybridization step is followed by four washing steps

- two washings during 5 mm, preferably at 65°C in a 2 x SSC and 0 1 %SDS buffer,

- one washing during 30 mm, preferably at 65°C in a 2 x SSC and 0 1 % SDS buffer,

- one washnig during 10 mm, preferably at 65°C in a 0 1 x SSC and 0 1 %SDS buffer

The non-labeled polynucleotides or ohgonucleotides of the invention may be directly used as probes Nevertheless, the polynucleotides or ohgonucleotides are generally labeled with a radioactive element (³²P, ³⁵S, ³H, ¹²⁵l) or by a non-isotopic molecule (for example, biotin, acetylaminofluorene digoxigen , 5- bromodesoxyuπdin, fluorescem) in order to generate probes that are useful for numerous applications 5

Examples of non-radioactive labeling of nucleic acid fragments are described in the French patent N° FR-7810975 or by Urdea et al. or Sanchez-Pescador et al., 1988.

In the latter case, other labeling techniques may be also used such those described in the French patents FR-2,422,956 and 2,518,755. The hybridization step may be performed in different ways (Matthews et al., 1988). The more general method consists in immobilizing the nucleic acid that has been extracted from the biological sample on a substrate (nitrocellulose, nylon, polystyren) and then to incubate, in defined conditions, the target nucleic acid with the probe. Subsequently to the hybridization step, the excess amount of the specific probe is discarded and the hybrid molecules formed are detected by an appropriate method (radioactivity, fluorescence or enzyme activity measurement).

Advantageously, the probes according to the present invention may have structural characteristics such that they allow signal amplification, such structural characteristics being, for example, branched DNA probes as those described by Urdea et al. in 1991 or in the European patent N° EP-0225,807 (Chiron).

In another advantageous embodiment of the probes according to the present invention, the latters may be used as « capture probes », and are for this purpose immobilized on a substrate in order to capture the target nucleic acid contained in a biological sample. The captured target nucleic acid is subsequently detected with a second probe which recognizes a sequence of the target nucleic acid which is different from the sequence recognized by the capture probe.

The ohgonucleotide fragments useful as probes or primers according to the present invention may be prepared by cleavage of the polynucleotides of SEQ ID N°1 , SEQ ID N°2, SEQ ID N°3, SEQ ID N°4, SEQ ID N°5 or SEQ ID N°6 by restriction enzymes, the one skill in the art being guided by the restriction maps presented in the annexes 2 to 6 of the instant specification.

Another appropriate preparation process of the nucleic acids of the invention containing at most 200 nucleotides (or 200 bp if these molecules are double stranded) comprises the following steps :

- synthesizing DNA using the automated method of beta-cyanethylphosphoramidite described in 1986;

- cloning the thus obtained nucleic acids in an appropriate vector;

- purifying the nucleic acid by hybridizing an appropriate probe according to the present invention.

A chemical method for producing the nucleic acids according to the invention which have a length of more than 200 nucleotides nucleotides (or 200 bp if these molecules are double stranded) comprises the following steps :

- assembling the chemically synthesized ohgonucleotides, having different restriction sites at each end ;

- cloning the thus obtained nucleic acids in an appropriate vector ;

In the case in which the above nucleic acids are used as coding sequences in order to produce a polypeptide according to the present invention, it is important to ensure that their sequences are compatible ( in the appropriate reading frame) with the aminoacid sequence of the polypeptide to be produced. The ohgonucleotide probes according to the present invention may also be used in a detection device comprising a matrix library of probes immobilized on a substrate, the sequence of each probe of a given length being localized in a shift of one or several bases, one from the other, each probe of the matrix library thus being complementary of a distinct sequence of the target nucleic acid Optionally, the substrate of the matrix may be a material able to act as an electron donnor, the detection of the matrix positions in which an hybridization has occurred being subsequently determined by an electronic device Such matrix libraries of probes and methods of specific detection of a target nucleic acid is described in the European patent application N° EP-0713,016 (Affymax technologies) and also in the US patent N° US-5,202,231 (Drmanac)

An ohgonucleotide probe matrix may advantageously be used to detect mutations occurring in the eya 1, eya2 or eya 3 gene For this particular purpose, probes are specifically designed to have a nucleotidic sequence allowing their hybridization to the genes that carry known mutations (either by deletion, insertion of substitution of one or several nucleotides) By known mutations is meant mutations on the eyal, eya2, or eya3 gene that have been identified according, for example to the technique used in Example 4 Specifically, probes are designed to hybridize with the mutated sequences depicted in Table 1

Another technique that is used to detect mutations in the eyal eya2, or eya3 gene is the use of a high-density DNA array Each ohgonucleotide probe constituting a unit element of the high density DNA array is designed to match a specific subsequence of the eyal, eya2, or eya3 genomic DNA or cDNA Thus, an array consisting of ohgonucleotides complementary to subsequences of the target gene sequence is used to determine the identity of the target sequence with the wild gene sequence, measure its amount, and detect differences between the target sequence and the reference wild gene sequence of the eyal, eya2, or eya3 gene In one such design, termed 4L tiled array, is implemented a set of four probes (A, C, G, T), preferably 15-nucleotιde oligomers In each set of four probes, the perfect complement will hybridize more strongly than mismatched probes Consequently, a nucleic acid target of length L is scanned for mutations with a tiled array containing 4L probes, the whole probe set containing all the possible mutations in the known wild reference sequence The hybridization signals of the 15-mer probe set tiled array are perturbed by a single base change in the target sequence As a consequence, there is a characteristic loss of signal or a « footprint » for the probes flanking a mutation position This technique was described by Chee et al in 1996

Another object of the present invention consists in hybrid molecules resulting from

- the hybrid formation between a DNA (genomic DNA or cDNA) or a RNA contained in a biological sample with a nucleic probe or primer according to the present invention,

- the hybrid formation between a DNA (genomic DNA or cDNA) or a RNA contained in a biological sample with an amplified nucleic fragment obtained by the use of a pair of primers according to the present invention

By cDNA according to the present invention is meant a DNA molecule that has been obtained by incubating an RNA molecule in the presence of an enzyme having a reverse transcnptase activity, as described by Sambrook et al in 1989 The present invention also pertains to a family of recombinant plasmids containing at least a nucleic acid according to the above teachings According to an advantageous embodiment, a recombinant plasmid comprises a polynucleotide of SEQ ID N°1 , SEQ ID N°2, SEQ ID N°4 or SEQ ID N°6, or one nucleic fragment thereof

Another object of the present invention consists in an appropriate vector for cloning, expressing or inserting a nucleic sequence, wherein the vector comprises a nucleic acid as above described in site that is non-essential for its replication, optionally under the control of the regulation elements allowing the expression of a polypeptide of the invention

Particular vectors used are plasmids, phages, cosmids, phagemids, PACs ( P1 derived Artificial Chromosomes) and YACs (Yeast Artificial Chromosomes) As plasmids, pUC vectors are preferred

These vectors are useful for transforming or transfectmg cell hosts in order to clone or express the nucleic acids of the invention

It is now easy to produce proteins in high amounts by the genetic engineering techniques by the use, as expression vectors, plasmids, phages or phagemids The polynucleotides that code for the polypeptides of the present invention is inserted in an appropriate expression vector in order to in vitro produce the polypeptide of interest

Thus, the present invention also concerns a method for the producing a polypeptide of the invention, and especially a polypeptide of SEQ ID N° 7, SEQ ID N°8, SEQ ID N°9 or SEQ ID N°10, the said method comprising the steps of a) optionally amplifying the nucleic acid coding for the desired polypeptide using a pair of primers according to the invention (by SDA, TAS, 3SR NASBA, TMA etc ) , b) inserting the nucleic acid of interest in an appropriate vector , c) growing, in an appropriate culture medium, a cell host previously transformed or transfected with the recombinant vector of step b) , d) harvesting the culture medium thus conditioned or lyse the cell host, for example by sonication or by an osmotic shock , e) separating or purifying, from the said culture medium, or from the pellet of the resultant host cell lysate the thus produced polypeptide of interest , f) characterizing the produced polypeptide of interest

The polypeptides according to the invention may be characterized by binding onto an immunoaffinity chromatography column on which polyclonal or monoclonal antibodies directed to a polypeptide among the polypeptides of SEQ ID N°7 to SEQ ID N°10 have previously been immobilized

The polypeptides according to the invention may also be prepared by the conventional methods of chemical synthesis, either in a homogenous solution or in solid phase As an illustrative embodiment of such chemical polypeptide synthesis techniques, it may be cited the homogenous solution technique described by Houbenweyl in 1974

Another object of the present invention consists in a polypeptide produced by the genetic engineering techniques or a polypeptide synthesized chemically as above described

The polypeptides according to the present invention, especially the polypeptides of SEQ ID N° 7, SEQ ID N°8, SEQ ID N°9 or SEQ ID N°10 are allowing A8 the preparation of polyclonal or monoclonal antibodies that recognize the polypeptides of SEQ ID N° 7, SEQ ID N°8, SEQ ID N°9 or SEQ ID N°10 or fragments thereof. The antibodies may be prepared from hybridomas according to the technique described by Kohler and Milstein in 1975. The polyclonal antibodies may be prepared by immunisation of an a mammal, especially a mouse or a rabbit, with a polypeptide according to the invention that is combined with an adjuvant of immunity, and then by purifying of the specific antibodies contained in the serum of the immunized animal on a affinity chromatography column on which has previously been immobilized the polypeptide that has been used as the antigen.

Consequently, the invention is also directed to a method for detecting specifically the presence of a polypeptide according to the invention in a biological sample, said method comprising the following steps : a) bringing into contact the biological sample with an antibody according to the invention; b) detecting the antigen-antibody complex formed.

Is also part of the invention a diagnostic kit for in vitro detecting the presence of a polypeptide according to the present invention in a biological sample, the said kit comprising :

- a polyclonal or monoclonal antibody as described above, optionally labeled;

- a reagent allowing the detection of the antigen-antibody complexes formed, said reagent carrying optionally a label, or being able to be recognized itself by a labeled reagent, more particularly in the case when the above-mentioned monoclonal or polyclonal antibody is not labeled by itself.

Another object of the present invention consists in a method for detecting specifically the presence of a polynucleotide of the invention contained in a biological sample comprising the steps of : a) bringing into contact an ohgonucleotide probe according to the invention with a biological sample under appropriate conditions, the DNA contained in the biological sample or the cDNA obtained by reverse transcription of the RNA contained in the biological sample having previously been made available to the hybridization reaction ; b) detecting the hybrid molecule formed between the ohgonucleotide probe and the. DNA of the biological sample.

Another object of the present invention consists in a method for detecting a genetic abnormality linked to the BO or to the BOR syndrome in a biological sample containing DNA or cDNA, comprising the steps of : a) bringing the biological sample into contact with a pair of ohgonucleotide fragments according to the invention, the DNA contained in the sample having been optionally made available to hybridization and under conditions permitting a hybridization of the said ohgonucleotide fragments with the DNA contained in the biological sample; b) amplifying the DNA c) revealing the amplification products; d) optionally detecting a mutation or a deletion by appropriate techniques. The step d) of the above-described method may consist in a Single-Strand Polymorphism technique (SSCP), a Denaturing Gradient Gel Electrophoresis (DGGE), or the FAMA technique such as described in the PCT patent application N° WO-95/07361

Another object of the present invention consists in a method for detecting a genetic abnormality linked to the BO or to the BOR syndrome in a biological sample containing DNA or cDNA, comprising the steps of a) bringing the biological sample into contact with an ohgonucleotide probe according to the invention, the DNA contained in the sample having been optionally made available to hybridization and under conditions permitting a hybridization of the primers with the DNA contained in the biological sample, b) detecting the hybrid formed between the ohgonucleotide probe and the DNA contained in the biological sample

The present invention also comprises a method for detecting a genetic abnormality linked to the BO or to the BOR syndrome in a biological sample containing DNA, comprising the steps of a) bringing into contact a first ohgonucleotide probe according to the invention that has been immobilized on a support, the DNA contained in the sample having been optionally made available to hybridization and under conditions permitting a hybridization of the primers with the DNA contained in the biological sample, b) bringing into contact the hybrid formed between the immobilized first ohgonucleotide probe and the DNA contained in the biological sample with a second ohgonucleotide probe according to the invention, which second probe hybridizes with a sequence different from the sequence to which the immobilized first probe hybridizes, optionally after having removed the DNA contained in the biological sample which has not hybridized with the immobilized first ohgonucleotide probe

Another object of the present invention consists in a method for detecting a genetic abnormality linked to the BO or to the BOR syndrome in a biological sample containing DNA, by detecting the presence and the position of base substitutions or base defetions in a nucleotide sequence included in a double stranded DNA preparation to be tested, the said method comprising the steps of a) amplifying specifically the region containing, on one hand, the nucleotide sequence of the DNA to be tested and on the other hand the nucleotide sequence of a DNA of known sequence, the DNA of known sequence being a polynucleotide according to the invention, b) labeling the sense and antisense strands of these DNA with different fluorescent or other non-isotopic labels, c) hybridizing the amplified DNAs, d) revealing the heteroduplex formed between the DNA of known sequence and the DNA to be tested by cleavage of the mismatched parts of the DNA strands

Such a mismatch localization technique has been described by Meo et al in the PCT application N° WO-95/07361 £0 The invention also pertains to a kit for the detection of a genetic abnormality linked to the BOR syndrome in a biological sample, comprising the following elements a) a pair of ohgonucleotides according to the invention, b) the reagents necessary for carrying out a DNA amplification, c)a component which makes it possible to determine the length of the amplified fragments or to detect a mutation

The discovery by the inventors of the linkage between the alteration of the eyal gene and both developmental defects and tumoπgenesis of various organs, specially kidneys, related to the BOR syndrome have allowed them to design specific therapeutic compositions for treating Eyal - defect associated disorders, particularly renal disorders, using an active principle which is selected from the group consisting of a) a purified EYA1 or EYA1-B protein or one of their biologically active derivatives , b) a polynucleotide encoding for the EYA1 or EYA1-B protein or for one of their biologically active derivatives , c) an antisense polynucleotide hybridizing specifically with the genomic DNA of the EYA1 gene or hybridizing specifically with the mRNA encoding the EYA1 or EYA1-B protein , d) a polyclonal or a monoclonal antibody that specifically binds to the EYA1 or EYA1 -B protein

The above-described therapeutic compositions are also useful to modulate the expression or the biological activity of the translation products of the EYA1 gene in case of organ grafting, and more particularly when embryonic organs are grafted Such therapeutic compositions are used to ensure a correct development of the embryonic grafted organ, in particular grafted embryonic liver or kidney These therapeutic compositions may be used in case of transplantation of allo -or xeno- organs

In a preferred embodiment of the therapeutic compositions of the present invention, the amount of the biologically active peptide component is comprised in the range from 0,1 μg/ml to 10μg/ml in the body fluid The dose-range is expressed in reference to the bioavailabi ty of the EYA1 or EYA1 -B protein

Another subject of the present invention is a therapeutic composition containing a pharmaceutically effective amount of a polypeptide of SEQ ID N° 7, SEQ ID N°8, SEQ ID N°9 or SEQ ID N°10 according to the present invention useful in the treatment of renal disorders, particularly renal disorders linked to the BOR syndrome For clinical use, the purified therapeutic polypeptide according to the present invention can be administered under the form of a solution, a gel or a dry powder It can be introduced locally, for example at the renal level or it can be administered systemically, for example intravenously

Another object of the present invention is a therapeutic composition containing an effective amount of a polynucleotide (RNA, genomic DNA, cDNA) coding for the purified polypeptide of SEQ ID N° 7, SEQ ID N°8, SEQ ID N°9 or SEQ ID N°10

A suitable vector for the expression of a polypeptide according to the invention is a baculovirus vector that can be propagated in insect cells and in insect cell lines A specific suitable host vector system is the pVL1392/1393 baculovirus transfer vector (Pharmmgen) that is used to transfect the SF9 cell line (ATCC N°CRL 1711 ) which is derived from Spodoptera frugiperda

Another suitable vector for the expression in bacteria and in particular in E coli, is the pQE-30 vector (QIAexpress) that allows the production of a recombinant protein containing a 6xHιs affinity tag The 6xHιs tag is placed at the C-terminus of the recombinant EYA1 , EYA1-B, EYA2 or EYA3-proteιn which allows a subsequent efficient purification of the recombinant protein by passage onto a Nickel or Cupper affinity chromatography column The Nickel chromatography column may contain the Ni-NTA resin (Porath et al , 1975)

In another embodiment of the therapeutic composition according to the invention, the said composition comprises a polynucleotide coding for the EYA1 , EYA1 -B, EYA2 or EYA3 polypeptide of interest

Gene therapy consists in correcting a defect or an anomaly (mutation, aberrant expression etc ) by the introduction of a genetic information in the affected organism This genetic information may be introduced in vitro in cell that has been previously extracted from the organism, the modified cell being subsequently remtroduced in the said organism, directly in vivo into the appropriate tissue

The method for delivering the corresponding protein or peptide to the interior of a cell of a vertebrate in vivo comprises the step of introducing a preparation comprising a pharmaceutically acceptable injectable carrier and a naked polynucleotide operatively coding for the polypeptide into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior of the cell and has a pharmaceutical effect at the renal, retinal or the neuronal level of the vertebrate

In a specific embodiment, the invention provides a pharmaceutical product, comprising a naked polynucleotide operatively coding for the EYA1 , EYA1 -B, EYA2 or EYA3 protein, in solution in a physiologically acceptable injectable carrier and suitable for introduction interstitially into a tissue to cause cells of the tissue to express the said protein or polypeptide

Advantageously, the therapeutic composition containing a complete or a part of the polynucleotide corresponding to the SEQ ID N° 1 to SEQ ID N° 6 polynucleotide is administered locally, near the site to be treated

The polynucleotide operatively coding for the EYA1 , EYA1 -B, EYA2 or EYA3 protein may be a vector comprising the genomic DNA or the complementary DNA coding for the corresponding protein or its protein derivative and a promoter sequence allowing the expression of the genomic DNA or the complementary DNA in the desired eukaryotic cells, such as vertebrate cells, specifically mammalian cells

The vector component of a therapeutic composition according to the present invention is advantageously a plasmid, a part of which is of viral or bacterial origin, which carries a viral or a bacterial origin of replication and a gene allowing its selection such as an antibiotic resistance gene

By « vector » according to this specific embodiment of the invention is intended a circular or linear DNA molecule

This vector may also contain an origin of replication that allows it to replicate in the eukaryotic host cell such as an origin of replication from a bovine papiilomavirus The promoter carried by the said vector is advantageously the cytomegalovirus promoter (CMV) Nevertheless, the promoter may also be any other promoter with the proviso that the said promoter allows an efficient expression of the DNA insert coding for the EYA1 , EYA1 -B, EYA2 or EYA3 protein within the host

Thus, the promoter is selected among the group comprising

- an internal or an endogenous promoter, such as the natural promoter associated with the structural gene coding for EYA1 , EYA1 -B, EYA2 or EYA3 such a promoter may be completed by a regulatory element derived from the vertebrate host, in particular an activator element,

- a promoter derived from a cytoskeletal protein gene such as the desmin promoter (Bolmont et al , J of Submicroscopic cytology and pathology, 1990 22 117-122, Zhenhn et al , Gene, 1989, 78 243-254)

As a general feature, the promoter may be heterologous to the vertebrate host, but it is advantageously homologous to the vertebrate host

By a promoter heterologous to the vertebrate host is intended a promoter that is not found naturally in the vertebrate host

Therapeutic compositions comprising a polynucleotide are described in the PCT application N° WO 90/11092 (Vical Inc ) and also in the PCT application N° WO 95/11307 (Institut Pasteur, INSERM, Universite d'Ottawa) as well as in the articles of Tacson et al (1996, Nature Medicine, 2(8) 888-892) and of Huygen et al (1996, Nature Medicine, 2(8) 893-898)

Other therapeutic compositions according to the present invention comprise advantageously an ohgonucleotide fragment of SEQ ID N°1 to SEQ ID N°6 of the invention as an antisense tool that inhibit the expression of the eyal , eya2 or eya3 gene and is thus useful in order to prevent or limit the tumor cell proliferation in certain patient organs, specifically kidney tumors

The therapeutic compositions described above may be administered to the vertebrate host by a local route such as an intramuscular route

The therapeutic polynucleotide according to the present invention may be injected to the host after it has been coupled with compounds that promote the penetration of the therapeutic polynucleotide within the cell or its transport to the cell nucleus The resulting conjugates may be encapsulated in polymer microparticles as it is described in the PCT application N° WO 94/27238 (Medisorb Technologies International)

In another embodiment, the DNA to be introduced is complexed with DEAE- dextran (Pagano et al , 1967, J Virol , 1 891 ) or with nuclear proteins (Kaneda et al 1989, Science, 243 375), with lipids (Feigner et al , 1987, Proc Natl Acad Sci 84 7413) or encapsulated within hposomes (Fraley et al , 1980, J Biol Chem 255 10431 )

In another embodiment, the therapeutic polynucleotide may be included in a transfection system comprising polypeptides that promote its penetration within the host cells as it is described in the PCT application WO 95/10534 (Seikagaku Corpporation)

The therapeutic polynucleotide and vector according to the present invention may advantageously be administered in the form of a gel that facilitates their transfection into the cells Such a gel composition may be a complex of poly-L-lysme and lactose, as described by Midoux (1993, Nucleic Acids Research, 21 871 -878) or also poloxamer 407 as described by Pastore (1994, Circulation, 90 1-517) The therapeutic polynucleotide and vector according to the invention may also be suspended in a buffer solution or be associated with liposomes

Thus, the therapeutic polynucleotide and vector according to the invention are used to prepare pharmaceutical compositions for delivering the DNA (genomic DNA or cDNA) coding for the EYA1 , EYA1-B, EYA2 or EYA3 protein at the site of the injection

The amount of the vector to be injected varies according to the site of injection and also to the kind of disorder to be treated As an indicative dose, it will be injected between 0,1 and 100 μg of the vector in a patient

In another embodiment of the therapeutic polynucleotide according to the invention, this polynucleotide may be introduced in vitro in a host cell, preferably in a host cell previously harvested from the patient to be treated and more preferably a somatic cell such as a muscle cell, a renal cell or a neurone In a subsequent step, the cell that has been transformed with the therapeutic nucleotide coding for the EYA1 , EYA2 or EYA3 protein is implanted back into the patient body in order to deliver the recombinant protein within the body either locally or systemically

In a preferred embodiment, gene targeting techniques are used to introduce the therapeutic polynucleotide into the host cell One of the preferred targeting techniques according to the present invention consists in a process for specific replacement, in particular by targeting the EYA1 , EYA1-B, EYA2 or EYA3 protein encoding DNA, called insertion DNA, comprising all or part of the DNA structurally encoding the corresponding protein, when it is recombmed with a complementing DNA in order to supply a complete recombinant gene in the genome of the host cell of the patient, characterized in that

- the site of insertion is located in a selected gene, called the recipient gene, containing the complementing DNA encoding the EYA1 , EYA1 -B, EYA2 or EYA3 protein and in that

- the polynucleotide coding for the said protein or one of its biologically active derivatives may comprise

- « flanking sequences » on either side of the DNA to be inserted, respectively homologous to two genomic sequences which are adjacent to the desired insertion site in the recipient gene

- the insertion DNA being heterologous with respect to the recipient gene, and

- the flanking sequences being selected from those which constitute the above- mentioned complementing DNA and which allow, as a result of homologous recombination with corresponding sequences in the recipient gene, the reconstitution of a complete recombinant gene in the genome of the eukaryotic cell

Such a DNA targeting technique is described in the PCT patent application N° WO 90/11354 (Institut Pasteur)

Such a DNA targeting process makes it possible to insert the therapeutic nucleotide according to the invention downstream of an endogenous promoter which has the desired functions (for example, specificity of expression in the selected target tissue)

According to this embodiment of the invention, the inserted therapeutic polynucleotide may contain between the flanking sequences and upstream from the open reading frame encoding the EYA1 , EYA1-B, EYA2 or EYA3 protein, a sequence carrying a promoter sequence either homologous or heterologous with respect to the EYA1 , EYA1-B, EYA2 or EYA3 encoding DNA The insertion DNA may contain in addition, downstream from the open reading frame and still between the flanking sequences, a gene coding for a selection agent, associated with a promoter making possible its expression in the target cell

According to this embodiment of the present invention, the vector contains in addition a bacterial origin of replication of the type colE1 , pBR322, which makes the cloning and preparation in E coli possible A preferred vector is the plasmid pGN described in the PCT application N° WO 90/11354

Other gene therapy methods than those using homologous recombination may also be used in order to allow the expression of a polynucleotide encoding the EYA1 , EYA1 -B, EYA2 or EYA3 protein within a patient's body

In all the gene therapy methods that may be used according to the present invention, different types of vectors are utilized

In one specific embodiment, the vector is derived from an adenovirus Adenoviruses vectors that are suitable according to the gene therapy methods of the present invention are those described by Feldman and Steg (1996, Medecme/Sciences, synthese, 12 47-55) or Ohno et al (1994, Sciences, 265 781 - 784) or also in the French patent application N° FR-94 03 151 (Institut Pasteur, Inserm) Another preferred recombinant adenovirus according to this specific embodiment of the present invention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or an adenovirus of animal origin ( French patent application N° FR-93 05954)

Among the adenoviruses of animal origin it can be cited the adenoviruses of canine (CAV2, strain Manhattan or A26/61 [ATCC VR-800]), bovine, murine (Mav1 , Beard et al , 1980, Virology, 75 81 ) or simian (SAV)

Preferably, the recombinant defective adenoviruses are prepared following a technique well-known by one skill in the art, for example as described by Levrero et al , 1991 , Gene, 101 195) or by Graham (1984, EMBO J , 3 2917) or in the European patent application N° EP-185 573 Another defective recombinant adenovirus that may be used according to the present invention, as well as a pharmaceutical composition containing such a defective recombinant adenovirus is described in the PCT application N° WO 95/14785

In another specific embodiment, the vector is a recombinant retroviral vector such as the vector described in the PCT application N° WO 92/15676 or the vector described in the PCT application N° WO 94/24298 (Institut Pasteur) The latter recombinant retroviral vector comprises

- a DNA sequence from a provirus that have been modified such that

- the gag, pol and env genes of the provirus DNA have been deleted at least in part in order to obtain a proviral DNA which is incapable of replicate, this DNA not being able to recombme to form a wild virus,

- the LTR sequence comprises a deletion in the U3 sequence, such that the mRNA transcription that the LTR controls is significantly reduced, for example at least 10 times, and

- the retroviral vector comprises in addition an exogenous nucleotide sequence coding for the EYA1 , EYA1-B, EYA2 or EYA3 protein or one of its biologically active derivatives under the control of an exogenous promoter, for example a constitutive or an ductible promoter

By exogenous promoter in the recombinant retroviral vector described above is intended a promoter that is exogenous with respect to the retroviral DNA but that zs may be endogenous or homologous with respect to the EYA1 , EYA1 -B, EYA2 or EYA3 protein entire or partial nucleotide coding sequence

In the case in which the promoter is heterologous with respect to the EYA1 , EYA1-B, EYA2 or EYA3 protein entire or partial nucleotide coding sequence, the promoter is preferably the mouse mductible promoter Mx or a promoter comprising a tetracychn operator or also a hormon regulated promoter A preferred constitutive promoter that is used is one of the internal promoters that are active in the resting fibroblasts such the promoter of the phosphoglycerate kinase gene (PGK-1 ) The PGK-1 promoter is either the mouse promoter or the human promoter such as described by Adra et al ( 1987, Gene, 60 65-74) Other constitutive promoters may also be used such that the beta-actm promoter (Kort et al , 1983, Nucleic Acids Research, 11 8287-8301 ) or the vimentm promoter (Rettlez and Basenga, 1987, Mol

A preferred retroviral vector used according to this specific embodiment of the present invention is derived from the Mo-MuLV retrovirus (WO 94/24298)

In one preferred embodiment, the recombinant retroviral vector carrying the therapeutic nucleotide sequence coding for the EYA1 , EYA1 -B, EYA2 or EYA3 protein or one of its biologically active derivatives is used to transform mammalian cells, preferably autologous cells from the mammalian host to be treated, and more preferably autologous fibroblasts from the patient to be treated The fibroblasts that have been transformed with the retroviral vector according to the invention are reimplanted directly in the patient's body or are seeded in a preformed implant before the introduction of the implant colonized with the transformed fibroblasts within the patient's body The implant used is advantageously made of a biocompatible carrier allowing the transformed fibroblasts to anchor associated with a compound allowing the gehfication of the cells The biocompatible carrier is either a biological carrier, such as coral or bone powder, or a synthetic carrier, such as synthetic polymer fibres, for example polytetrafluoroethylene fibres

An implant having the characteristics as defined above is the implant described in the PCT application N° WO 94/24298 (Institut Pasteur)

The present invention provides also a method for the screening of ligands which are capable to bind to the EYA1 , EYA1-B, EYA2 or EYA3 protein Such a screening method, in one embodiment, comprises the steps of a) preparing a complex between the EYA1 , EYA1-B, EYA2 or EYA3 protein and a ligand that binds to the EYA1 , EYA1-B, EYA2 or EYA3 protein by a method selected among the following

- preparing a tissue extract containing the EYA1 , EYA1 -B, EYA2 or EYA3 protein putatively bound to a natural ligand,

- bringing into contact the purified EYA1 , EYA1 -B, EYA2 or EYA3 protein with a solution containing a molecule to be tested as a ligand binding to the EYA1 , EYA1-B, EYA2 or EYA3 protein, b) visualizing the complex formed between the EYA1 , EYA1 -B, EYA2 or EYA3 protein from the tissue extract and the natural ligand of the EYA1 , EYA1 -B, EYA2 or EYA3 protein or the complex formed between the purified EYA1 , EYA1 -B, EYA2 or EYA3 protein and the molecule to be tested

For the purpose of the present invention, a ligand means a molecule, such as a protein, a peptide, a transcription factor, an antibody or a synthetic compound capable of binding to the EYA1 , EYA1 -B, EYA2 or EYA3 protein or one of its biologically active derivatives or to modulate the expression of the polynucleotide coding for the EYA1 , EYA1 -B, EYA2 or EYA3 protein or coding for one of its biologically active derivatives

In the first embodiment of the screening procedure wherein a natural ligand of the EYA1 , EYA1-B, EYA2 or EYA3 protein is to be characterized, it is processed as follows

The tissue putatitively containing the EYA1 , EYA1 -B, EYA2 or EYA3 protein bound to its natural ligand, for example the heart tissue, the skeletal muscle or the kidney are homogenized in 10 mM Hepes, pH 7 4, containing 100 μg/ml PMSF, 200 μg/ml aprotinm and 5 μg/ml Dnase, with a glass-Teflon homogenizer The homogenate is centrifuged at 1 ,000 g for 10 minutes, the supernatant is removed and centrifuged at 190,000 g for 30 mm at 4°C The pellet containing the membrane fraction is stored at -20°C until used

The cell membrane fractions are incubated first in 0 9% Triton X-100, 0 1 % ovalbumin, 5 mM EDTA, 50 mM Tπs-HCI, pH 8, with an immune serum of the invention overnight at 4°C, then with Protein G-sepharose (Pharmacia) for 2 hours Complexes are centrifuged, washed three times in PBS and three times in 50 mM Tπs-Hcl, pH 8 Then the complexes are dissociated in a dissociating buffer containing SDS in order to dissocate the EYA1 , EYA1 -B, EYA2 or EYA3 protein from its bound natural ligand Immunoprecipitates are analysed by western blot following the technique described by Gershoni and Palade (1983, Anal Biochem , 131 1 -15) The anti- EYA1 , EYA1 -B, EYA2 or EYA3 protein monoclonal antibody produced by the hybridoma clone 1-4 was used to detect the EYA1 , EYA1 -B, EYA2 or EYA3 protein and a panel of candidate antibodies, for example antibodies directed against different sub-units of integπns are used ( at a concentration of 1 5 μg/ml) to identify the ligand that was previously bound to the EYA1 , EYA1-B, EYA2 or EYA3 protein in the tissue extract IgG peroxidase-conjugated antibody (Bio-Rad, 1/6,000 dilution) is used as second antibody The blots are revealed by chemiluminescence with the ECL kit (Amersham France)

In a second embodiment of the ligand screening method according to the present invention, a biological sample or a defined molecule to be tested as a putative hgand of the EYA1 , EYA1-B, EYA2 or EYA3 protein is brought into contact with the #uπfιed EYA1 , EYA1-B, EYA2 or EYA3, in order to form a complex between the EYA1 , EYA1-B, EYA2 or EYA3 protein and the putative ligand molecule to be tested The biological sample may be obtained from a cerebelum or a renal extract, for example

When the hgand source is a biological sample, the complexes are processed as described above in order to identify and characterize the unknown ligand

When the putative ligand is a defined known molecule to be tested, the complexes formed between the EYA1 , EYA1-B, EYA2 or EYA3 protein and the molecule to be tested are not dissociated prior to the western blotting in order to allow the detection of the complexes using polyclonal or monoclonal antibodies directed against the EYA1 , EYA1-B, EYA2 or EYA3 protein

In a particular embodiment of the screening method, the putative ligand is the expression product of a DNA insert contained in a phage vector (Parmley and Smith, Gene, 1988, 73 305-318) According to this particular embodiment, the recombinant phages expressing a protein that binds to the immobilized EYA1 , EYA1 -B, EYA2 or EYA3 protein is retained and the complex formed between the EYA1 , EYA1 -B, EYA2 or EYA3 protein and the recombinant phage is subsequently immunoprecipitated by a polyclonal or a monoclonal antibody directed against EYA1 , EYA1-B, EYA2 or EYA3 protein

According this particular embodiment, a ligand library is constructed in recombinant phages from human or chicken genomic DNA or cDNA Once the gand library in recombinant phages has been constructed, the phage population is brought into contact with the immobilized EYA1 , EYA1 -B, EYA2 or EYA3 protein Then the preparation of complexes is washed in order to remove the non-specifically bound recombinant phages The phages that bind specifically to the EYA1 , EYA2 or EYA3 protein are then eluted by a buffer (acid pH) or immunoprecipated by the monoclonal antibody produced by the hybridoma anti- EYA1 , EYA1 -B, EYA2 or EYA3, and this phage population is subsequently amplified by an over-infection of bacteria (for example E coli) The selection step may be repeated several times, preferably 2-4 times, in order to select the more specific recombinant phage clones The last step consists in characterizing the protein produced by the selected recombinant phage clones either by expression in infected bacteria and isolation, expressing the phage insert in another host-vector system, or sequencing the insert contained in the selected recombinant phages

Another subject of the present invention is a method for screening molecules that modulate the expression of the EYA1 , EYA1-B, EYA2 or EYA3 protein Such a screening method comprises the steps of a) cultivating a prokaryotic or an eukaryotic cell that has been transfected with a nucleotide sequence encoding the EYA1 , EYA1-B, EYA2 or EYA3 protein, placed under the control of its own promoter, b) bringing into contact the cultivated cell with a molecule to be tested, c) quantifying the expression of the EYA1 , EYA1 -B, EYA2 or EYA3 protein

Using DNA recombination techniques well known by the one skill in the art the EYA1 , EYA1-B, EYA2 or EYA3 protein encoding DNA sequence is inserted into an expression vector, downstream from its promoter sequence, the said promoter sequence being described by Cohen-Salmon et al (1995, Gene, 164 235-242)

The quantification of the expression of the EYA1 , EYA1-B, EYA2 or EYA3 protein may be performed either at the mRNA level or at the protein level In the latter case, polyclonal or monoclonal antibodies may be used to quantify the amounts of the EYA1 , EYA1 -B, EYA2 or EYA3 protein that have been produced, for example in an ELISA or a RIA assay

In a preferred embodiment, the quantification of the eya1-a, eya1-b, eya1-c, eya2 or eya3 mRNA is realized by a quantitative PCR amplification of the cDNA obtained by a reverse transcription of the total mRNA of the cultivated eya1-a, eyal- b, eya1-c, eya2 or eya3 -transfected host cell, using a pair of primers specific for eya1-a, eya1-b, eya1-c, eya2 or eya3 of the kind that are described in the PCT application N° WO 93/07267 (Institut Pasteur, HHS)

As an illustrative example, a pair of primers used to quantitate the reverse- transcribed eya1-a, eya1-b, eya1-c, eya2 or eya3 mRNA is the following Primer 1 5'-CTCAGCCATGTGCTCTGTATAATTAAGAGC-3' Primer 2 5'-AGGCACGGCACATGAATTTGTGCACGTGCT-3' 1% The process for determining the quantity of the cDNA corresponding to the eyal, eya2 or eya3 mRNA present in the cultivated eya1-a, eya1-b, eya1-c, eya2 or eya3 -transfected cells is characterized in that

1 ) a standard DNA fragment, which differs from the eya1-a, eya1-b, eya1-c, eya2 or eya3 cDNA fragment, obtained by the reverse transcription of the eya1-a, eya1-b, eya1-c, eya2 or eya3 -mRNA, but can be amplified with the same ohgonucleotide primers is added to the sample to be analyzed containing the eya1-a, eya1-b, eyal- c, eya2 or eya3 -cDNA fragment, the standard DNA fragment and the eya1-a, eyal- b, eya1-c, eya2 or eya3 -cDNA fragment differing in sequence and/or size by not more than approximately 10%, and preferably by not more than 5 nucleotides by strand,

2) the eya1-a, eya1-b, eya1-c, eya2 or eya3 -cDNA fragment and the standard DNA fragment are coamp fied with the same ohgonucleotide primers, preferably to saturation of the amplification of the eya1-a, eya1-b, eya1-c, eya2 or eya3 -cDNA fragment,

3) to the reaction medium obtained in step 2), there are added

- either two types of labeled ohgonucleotide probes which are each specific for the eya1-a, eya1-b, eya1-c, eya2 or eya3 -cDNA fragment ant the standard DNA fragment, respectively, and different from the amplification ohgonucleotide primers of step2),

- or one or more labeled ohgonucleotide pπmer(s), specific for the eya1-a, eya1-b, eya1-c, eya2 or eya3 -cDNA fragment and the standard DNA fragment and different from said ohgonucleotide primers of step 2), and one or more additional amplification cycle(s) with said labeled ohgonucleotide prιmer(s) is/are performed, so that, during a cycle, after denaturation of the DNA, said labeled ohgonucleotide pπmer(s) hybrιdιze(s) with said fragments at a suitable site in order that an elongation with the DNA polymerase generates labeled DNA fragments of different sizes and/or sequences and/or with different labels according to whether they originate from the DNA fragment of interest or the standard fragment, respectively, and then

4) the initial quantity of eya1-a, eya1-b, eya1-c, eya2 or eya3 -cDNA fragment is determined as being the product of the initial quantity of standard DNA fragment and the ratio of the quantity of amplified eya1-a, eyal-b, eya1-c, eya2 or eya3 -cDNA fragment, which ratio is identical to that of the quantities of the labeled DNA fragments originating from the amplified eya1-a, eyal-b, eya1-c, eya2 or eya3 - cDNA fragment, respectively, obtained in step 3)

Primers and probes hybridizing with the eya1-a, eyal-b, eya1-c, eya2 or eya3 -cDNA fragment and used in the above-described quantitative PCR amplification reaction are described in the PCT application N° WO 93/07267 (Institut Pasteur HHS)

More technical details regarding the performing of the quantitative PCR amplification reaction are found in the PCT application N° WO 93/10257 (Institut Pasteur, Inserm)

Finally, the invention also pertains to a method for screening a gand molecule capable to bind to a polynucleotide according to anyone of claims 1 to 6 comprising the steps of a) bringing into contact a polynucleotide as defined hereabove with a ligand molecule to be tested, 23 b) detecting the complexes formed between the said polynucleotide and the ligand molecule

Such screening techniques are described, for example, by Kadonaga et al in 1987 and by Kageyama et al in 1989

In order to better understand the present invention, reference will be made to the appended figures which depict specific embodiments to which the present invention is in no case limited in scope with

D8S1060, D8S1807, D8S530, D8S279), YAC end clones (876D10L, 201 A6L, 942C8R) and internal Alu PCR product (456H3ιnt) in the region PAC clones are indicated in italics and P1 clones in standard print On this Figure, the 5'end of SEQ ID N°1 is on the right side whereas the 3'end of SEQ ID N°1 is on the left side

Fig. 2 Sequence of the EYA1 cDNA The potential N-glycosylation sites (ammo acid positions 30, 166, 171 , 176 and 220) are circled The protein kinase C phosphorylation sites (ammo acid positions 42, 57, 259 and 414) and the cAMP- dependent protein kinase C phosphorylation site (ammo acid position 263) are boxed The leucme zipper is underlined and, in the 3' non coding region, the two polyadenylation sites are boxed

Fig. 3 Detection of a deletion by Southern blot analysis in a sporadic case of BOR syndrome a, Hybridisation with a probe containing exon D resulted in a signal of reduced intensity in the proband's DNA as compared to the unaffected parents b, Hybridisation with a probe containing exon C resulted in two bands, one of the normal size range (2 3 kb) and the second of a lower molecular weight c, Hybridisation with the Sp6 insert end of P1 4405 resulted in a band of equal intensity in DNA from all three individuals

Fig. 4 A gnement generated by the PILEUP program of the ammo acid sequence for EYA1 with the deduced ammo acid sequence of the murine orthologue Eyal , and of the human homologues EYA2 and EYA3 Residues highlighted in grey indicate

boxes indicate similarities The start of the eya homologous region (eyaHR) is indicated by a triangle

Fig. 5 Northern blot analysis of the expression of eyal -a and its mouse orthologue Eyal Human a, foetal and b, adult poly A+ northern blots (Clontech MTN7765-1 and 7760-1 ) hybridized with a 278 bp PCR product derived from the 3' untranslated region (beginning at the stop codon) of the eya1-a cDNA Mouse c, embryonic and d, adult poly A+ northern blots (Clontech MTN7763-1 and 7762-1 ) hybridised with a 256 bp product derived from the 3' untranslated region of the Eyal cDNA (beginning 9 bp before the stop codon)

Fig 6 In situ hybridisation to parasagittal (E13 5 and E19 5) and transversal (E14 5 and E17 5) sections of mouse embryos a, Hybridisation of the antisense RNA probe to the neuroepithelium (NE), and to the mesenchyme of the otic capsule, of the inner ear at E13 5 b, Hybridisation signal in the cochlear neuroepithelium (NE) 3θ and in the spiral ganglia (SpG) at E19 5 c, Hybridisation signal in the olfactory epithelium at E13 5 d, Labelling of the metanephπc mesenchyme surrounding the just divided ureteπc branches (arrows) near the medullary region, at E13 5 Progression of this hybridisation signal towards the peripheral cortex at e, E14 5 and f, E17 5 (arrows)

The general techniques used within the framework of the present invention are described below

A - P1 and PAC library screening

Nineteen P1 and 3 PAC clones from the Genome Systems libraries (P 1-2535 and PAC-6539, Missouri, U S A ) were isolated by PCR using primers flanking the 7 markers from D8S1060 to D8S530 (see Fig 1 ) End clones of the P1/PAC inserts were isolated (according to the manufacturer's directions) and primers corresponding to six of them were chosen to rescreen the two libraries, resulting in the isolation of a further 3 P1 s and 13 PACs These 16 clones were positioned relative to the initial 22 clones using the aforementioned probes A contig of minimum overlap was constructed using 3 P1 and 3 PAC clones The corresponding end clones were isolated and used to confirm the overlaps by Southern blot analysis

B - Subcloning, sequencing and analysis

Approximately 15 μg of DNA from P1 4405 was sonicated and separated on a preparative 0 4% LMP agarose gel DNA within the size range of 7-10 kb was isolated, blunt-ended by T4 DNA polymerase and Klenow, phosphorylated by T4 polynucleotide kinase and ligated to dephosphorylated Smal digested pBC SK+ (Stratagene) Approximately 250 subclones with a confirmed insert size of 7-10 kb were amplified in LB medium containing 30 μg/ml of chloramphenicol End clones were directly sequenced, using the T3 and T7 primers, on an ABI 377 sequencer In the first instance, sequence data was compared to the EMBL and GenBank protein and nucleic acid databases, and also analysed using the GRAIL program⁵⁰ to screen for the presence of potential exons Subsequent to the detection of sequence homology to the drosophila gene eya, translated genomic sequences were compared to the deduced ammo acid sequence of the eya cDNA Once all the ends of the individual clones had been sequenced, the sequences were assembled using the computer programs Phred (B Ewmg, unpublished) and Phrap (P Green, unpublished), and edited with Consed (D Gordon, unpublished) This resulted in the construction of 31 sequence contigs covering approximately 90% of the P1 insert Using the orientation and distance constraints of end sequences from subclone inserts of a determined length, a scaffold of these 31 contigs covering the entire P1 clone was determined, as well as the size and position of intercontig gaps (method adaptated from ⁵¹ , R Hei g et al, manuscript in preparation) Individual subclones from the scaffold were then selected for sequence extension to bridge the gaps between contigs C - cDNA isolation

Oligo dT and random primed cDNA populations were generated from a) nine week total human foetus polyA+ RNA and b) embryonic day (E) 13 total mouse polyA+ RNA, and amplified using the Marathon cDNA amplification kit (Clontech) according to the manufacturer's recommendations. The reconstructed EYA1 cDNA sequence was verified by PCR amplification of the full length coding region, and sequencing, using the primers 5'GSP.F (5'CTCAGCCATGTGCTCTGTATAATTAAGAGC3') and 3'GSP.R (5ΑGGCACGGCACATGAATTTGTGCACGTGCT3') Sequence analysis was performed using the GCG Sequence Analysis Software Package⁵² SAPS (sequence analysis of protein sequences)⁵^ and ppsearch (protein pattern searching) software (derived from 54,55).

D - Mutation detection

Southern blots containing EcoRI, Mspl, Pstl or Taql digested DNA from both familial and sporadic BOR affected patients were prepared according to standard techniques. The eight exons, exon z and exons A to G, were amplified using P1 clone 4405 DNA and the flanking intronic primers listed below The resultant PCR products were random labelled with 32p and hybridised to the Southern blots. A search for sequence mutations was carried out on the 8 exons in 42 unrelated individuals. The exons were amplified and sequenced using the following primers derived from intronic sequences: Exon z exz.F 5ΑGGCTAATCTTGGCACCATGG3' exz.R 5OACTGCTGTTTACGTAGCAGG3'

Exon A exA.F 5TGAATAACAGCTTTCTCAGCC3' exA.R 5'GACTATATAGTTCTTCTCCATTT3'

Exon B exB.F 5OTTTCAGCCTCTCCCAATGC3' exB.R 5ΑCCAACAAACTCCTGTCTCAC3'

Exon C exC.F 5ΑCCTACTGATTGACATAGTTGA3' exC.R 5ΑCTATAAAAGGGAGATGGTCAC3'

Exon D exD.F 5'GTGACCATCTCCCTTTTATAGT3^*

- exD.R 5TGCTGAGGTACTGGTGGTAA3'

Exons E & F exE.F 5ΑAATCTGGAGGCTGGTATTC3' exF.R 5ΑGAGTACTGCACATATTCATCA3' exG.R 5TGCTGTGGCACATACAACCC3'

Exons E and F were coamplified in the same PCR product and sequenced with the external primers exE.F and exF.R, and the internal primers, exE R (5ΑTGAACAAGCACGAGCATTGC3') and exF F

(5'GCAATGCTCGTGCTTGTTCAT3'). All PCRs, and subclonmg of PCR products into Hindi digested M13, were carried out as previously described⁵⁶ 3>V E - In situ hybridization

A 256 bp PCR product was derived from the 3' untranslated region (starting 9 bp before the stop codon) of the murine cDNA. Hybridisation of this PCR product to Southern blots containing mouse genomic DNA resulted in a single band The 256 bp PCR product was then subcloned via the EcoRI and BamHI linkers into the EcoRI/BamHI digested pGEM-4Z, Promega, (creating pM3UTR). The sense and antisense RNA probes were labelled with dιgoxigenιn-11-UTP and the hybridisation signal detected by sheep anti-digoxigenin antibodies coupled to alkaline phosphatase. Whole E9.5 and E11.5 Rj:SWISS mouse embryos, parasagittal cryosections (7 μm) from E13.5 and E19.5 embryos and transversal sections through the kidneys of E14.5 and E17.5 embryos, were treated and hybridised as previously described⁵ -⁵⁸.

Examples

Example 1 : Construction of a P1/PAC contig

Initially 7 markers mapping proximal to (D8S1060), distal to (D8S1807, D8S530) and within (876D10L, 456H3int, 201 A6L, 942c8R) the translocation-associated deletion '⁶ (see Fig. 1 ) were used to screen P1 and PAC human genomic libraries 19 P1 and 3 PAC clones were isolated. In order to fill the gaps between these clones, chromosome walks were initiated by using the insert ends to rescreen the libraries. A further 3 P1s and 13 PACs were isolated. Of the 38 total clones, 3 P1s and 3 PACs (see methods) were used to construct a contig with minimum overlap (Fig. 1 ).

Example 2 : Sequencing and identification of a candidate gene

P1 4405 (see Fig. 1 ) was chosen as the starting point for DNA sequencing of the deletion interval. Initial sequence data showed homology to the 3' coding region of the drosophila developmental gene eyes absent (eya). Further genomic sequence data was then directly translated and compared to the deduced ammo acid sequence of the eya1-a cDNA (eya protein). Seven putative exons (A to G, Fig. 2), flanked by consensus splice sites, were identified. The corresponding ammo acid sequence was found to be highly homologous to an eya protein fragment that finished 26 amino acids before the C-terminal end. In order to search for an exon(s) encoding the C-terminal end of the putative human protein, DNA sequencing was continued into the adjacent P1 , 10910 (see Fig. 1 ). An additional potential exon encoding a sequence homologous to the last 26 ammo acids of the eya C-terminal end (exon H), and containing a stop codon, was identified The 271 aa peptide encoded by exons A to H showed 69% identity and 88% similarity with the drosophila eya protein. This proposed human homologue was therefore named eyes absent-like 1 (EYA1 ). Databank comparison of this human sequence with a C elegans expressed sequence tag (EST) database, resulted in the detection of an EST (C07779) encoding a 99 aa peptide showing 43% identity and 64% similarity over 72. This peptide was 38% identical and 58% similar to the eya protein. Finally, no significant homology was detected upon comparison of the EYA1 deduced amino acid sequence with the amino acid sequence derived from the complete coding sequence of S. cerevisiae.

Example 3 : Determination of the cDNA sequence

RACE PCR was used to isolate the eya1-a cDNA. Successive rounds of amplification were performed on 9 week total foetus mRNA resulting in a reconstituted 3698 bp cDNA sequence containing a poly A tail (see methods). The translation initiation site was identified by the presence of an adequate Kozak consensus sequence (GTTCAGatgT)^''⁸, preceded by stop codons in all three frames. The cDNA sequence contained a 138 bp 5' non coding region followed by a 1677 bp open reading frame and a 1883 bp 3' non coding region (GenBank accession number Y10260) (Fig. 2). An additional 3' non coding region of 1164 bp, was also amplified. Sequence comparison with genomic sequence from P1 10910 demonstrated that both were transcribed from exon H and resulted from the usage of two distinct polyadenylation sites (Fig. 2).

Example 4 : Mutations in BOR affected patients

To test for possible DNA rearrangements within eyal in BOR affected patients, Southern blots containing DNA from 21 familial and sporadic patients were hybridised with probes corresponding to the exons A to G, and the exon immediately adjacent to exon A (exon z) identified by comparison of genomic sequence from P1 4405 with the eya1-a cDNA sequence. In individual 4 (a sporadic case), hybridisation with exon D resulted in a signal of reduced intensity suggestive of a deletion; this reduction was not seen in the proband's parents (Fig. 3a). Hybridisations with the exons A and B resulted in signals of normal intensity (data not shown), C resulted in a band shift (Fig. 3b), and E, F and G resulted in bands of reduced intensity (data not shown). Hybridisation with a probe corresponding to the Sp6 insert end of P1 4405 (located between exons G and H), resulted in a band of normal intensity (Fig. 3c). According to the assembled genomic sequence of the P1 4405 subregion, the deletion in individual 4 was estimated to span 5.8 - 7 kb.

Exons A to G and exon z of 42 unrelated affected individuals were sequenced (see methods) and seven mutations were detected (Table 1 )^{1 9}. A premature stop codon was detected in exon z of individual 1. A replacement of a T with a CC insertion was detected in exon D in individual 11. The affected family members of these two probands carried the same mutations. In individual 23, a 1 bp insertion was detected in exon z. The phenotypically normal parents of this proband did not carry this mutation. In individuals 22, 34, 40 and 48, four mutations were detected consisting of a 4 bp insertion in exon G, a 1 bp insertion in exon E, a 5 bp substitution/insertion in exon E and a donor splice site mutation of exon F, respectively (Table 1 ). No other family members of these four individuals were available for testing 3^

Table 1 : Mutations in BOR affected patients

Type of Individu Nucleotide Effect on coding Exon case al change sequence

Familial 1 823C→T R275X z

11 1251T→CC Frameshift D

Sporadic 4 del-7kb Deletion C,D,E,F,G

23 755ιnsC Frameshift z

Unknown 22 1555ιns-4^a Frameshift G

34 1359ιnsC Frameshift E

40 1372T→AGAGC Frameshift E

48 1498+2T→G Aberrant splicing F

^aTTGT insertion Nomenclature denote mutations as described in ref 19 ^b The nucleotide positions given are referenced to the eyal -a cDNA

Example 5 : Amino acid sequence analysis

The 559 ammo acid sequence of the EYA1 gene product was compared with the drosophila eya protein A high degree of homology between the two proteins was restricted to the previously mentioned 271 aa C-terminal region encoded by the last eight exons, A to H This region was named eyaHR for eya homologous region A lower yet significant degree of homology was seen over 125 aa (ammo acid position 130-254) in the N-termmal part of the EYA1 protein which was 21 % identical and 79% similar with the eya protein The deduced ammo acid sequence of the EYA1 protein has a molecular weight of 60 3 kDa +/- 10% Sequence analysis failed to detect a signal peptide An a-hehcal segment composed of four successive hepta repeats forming a leucme zipper, was predicted between am o acids 431 and 458 (Fig 2) The a-hehcal segment was preceded by a 30 residue basic segment suggesting the possible existence of a bZIP domain The drosophila eya product also contains a basic stretch of ammo acids followed by four successive hepta-hke repeats, in which three leucme residues (ammo acid position 635, 642 and 656^{"1 7}) are replaced by three other residues, an isoleucme, an alanme and a methionine respectively, which also have a positive hydropathy value²⁰ Five potential N- glycosylation sites, four potential protein kinase C phosphorylation sites and one potential cAMP-dependent protein kinase C phosphorylation site were detected (Fig 2) Example 6 : Evidence for a novel gene family

The EMBL and GenBank databases were screened for sequences homologous to the eya1-a cDNA sequence Three human and two murine ESTs, showing significant homology to the eyaHR, were detected The human ESTs H07988, R72695 and Z39529, consisted of two overlapping ESTs assigned to chromosome 20 and an unmapped human EST, respectively The two murine ESTs detected (W34432 and W83314) were unmapped Using primers specific to H07988, the cDNA sequence was extended in a 5' and 3' direction by RACE PCR using 9 week total human foetal mRNA A 1929 bp cDNA sequence with a 198 bp 5' non coding region, a 1614 bp open reading frame with an initiation site within a strong Kozak consensus sequence (GTGGAAatgG), and a 117 bp 3' non coding region, was obtained (GenBank accession number Y10261 ) The 538 aa coding sequence predicted a 59 2 kDa protein Due to the extensive homology between the deduced ammo acid sequence of this cDNA sequence and the EYA1 protein, the corresponding gene was named EYA2 The 1207 bp human EST clone Z39529 was sequenced and mapped to chromosome 1 by hybridisation to a panel of somatic cell hybrids (data not shown) The cDNA sequence was extended in a 5' and 3' direction by RACE PCR A 2860 bp cDNA sequence with a 113 bp 5' non coding region, a 1716 bp open reading frame with an initiation site within an adequate Kozak consensus sequence (GTCCTCatgG), and a 1031 bp 3' non coding region, was obtained (genbank accession number Y10262) The 572 aa coding sequence predicted a 62 kDa protein Likewise, because of the extensive homology with the EYA1 protein, the corresponding gene was named EYA3 The overall homology between EYA1 , EYA2 and EYA3 ranged from 45-68% identity and 73-84% similarity with EYA1 and EYA2 showing a significantly higher degree of homology with one another than with EYA3 (Fig 4) The eyaHR of EYA1 was highly conserved in both the EYA2 and EYA3 proteins with EYA2 protein being 83% identical and 99% similar, and the EYA3 protein being 62% identical and 96% similar Also within the eyaHR, EYA2 and EYA3 contain a basic ammo acid stretch followed by an a-hehcal domain in which the third leucme residue (ammo acid position 448 of EYA1 ) has been replaced by a histidine (in EYA2) and a threonme (in EYA3) (Fig 4) With regards to the drosophila eya protein, EYA2 was observed to be 65% identical and 90% similar, and EYA3 58% identical and 90% similar, for the region corresponding to the eyaHR, and the two proteins showed the same lower degree of homology for the N-termmal region, as was seen for EYA1

Using primers specific to the murine EST W34432 and E13 total mouse mRNA, the cDNA sequence was extended in a 5' and 3' direction by RACE PCR This led to a reconstituted sequence of 2482 bp containing a 233 bp 5' non coding sequence, an open reading frame of 1659 bp with an initiation codon within an adequate Kozak consensus sequence (TCAGCCatgT), and a 590 bp 3' non coding region (GenBank accession number Y10263) The 553 aa coding sequence predicts a protein of 60 3 kDa The deduced ammo acid sequence of this cDNA was 99% identical to EYA1 for the eyaHR, and 91 % identical and 97% similar to the remaining region The homology between the extended sequence of W34432 and the eya1-a cDNA sequence extends into the non coding region and was 85% in a 46 nt overlap at the 5' end and 73% in a 594 nt overlap at the 3' end, compared to a homology of 90% in a 1659 nt overlap in the coding region The high level of homology led us to consider the corresponding gene as the murine orthologue of eya1-a (Eyal ) The deduced ammo acid sequence of the 436 bp murine EST W83314 showed a higher homology with the EYA2 protein (99% identical) than with EYA1 (85% identical and 90% similar) or EYA3 (64% identical and 80% similar) These results suggested that this EST sequence could belong to the murine orthologue of EYA2 and it was not further characterised

Example 7 : Analysis of the expression pattern of eya1-a and Eyal

By northern blot analysis of human foetal tissues (Fig 5a), a probe specific to the 3' untranslated region of the EYA1 cDNA (see legend of Fig 5) hybridised to a 4 4 kb transcript that was highly expressed in kidney, less in brain and weakly expressed in lung An additional weaker 3 8 kb transcript was seen in foetal kidney In adult human tissues (Fig 5b), a 4 4 kb transcript (accompanied by a fainter 3 8 kb transcript), which was highly expressed in heart and skeletal muscle, and weakly expressed in brain, was detected The size difference between the two transcripts is likely to be explained by the use of the two distinct polyadenylation sites The total size of the two transcripts is indicative of additional 5' non coding sequence A faint band of 4 6 kb was seen in adult liver No transcript was detected in adult kidney By northern blot analysis of total mouse embryos (Fig 5c), a probe specific to the 3' untranslated region of the Eyal cDNA (see legend Fig 5) hybridised to a 4 4 kb and a 4 2 kb transcript that was highly expressed at E11 , and less expressed at E15 and E17, no transcript was detected in E7 mouse mRNA In murine adult tissues (Fig 5d), a 4 4 kb transcript was seen that was highly expressed in skeletal muscle and weakly expressed in brain No transcript was found in murine adult kidney

In situ hybridisation was performed using an antisense probe from pM3UTR (see methods) on whole mount mouse embryos and parasagittal and transversal cryosections On whole mount E9 5 and E11 5 mouse embryos, a specific weak labelling of the otic placode (E9.5) and the otic vesicle (E11 5) (data not shown) was observed? No labelling of the branchial arches was seen By in situ hybridisation to cryosections, at E13 5 a strong signal was detected in the cochlear and vestibular neuroepithelium as well as a weaker signal in the mesenchyme of the otic capsule (Fig. 6a) and of the middle ear, and in the statoacoustic ganglia (data not shown) At E19 5 there was a strong signal in the whole vestibular (utπcule, saccule and semicircular canals) and cochlear neuroepithe a, and in both the vestibular and spiral (auditory) ganglia (Fig. 6b) At E13 5 a strong signal in the olfactory epithelium (Fig. 6c), and a weak signal in the surrounding nasal mesenchyme, was seen The labelling of the olfactory epithelium persisted at E19 5 (data not shown) At E13 5, within the metanephros, a strong labelling of the mesenchyme surrounding the just divided ureteπc branches, located nearer to the medullary zone, was observed (Fig 6d) and no labelling of the peripheral mesenchymal blastema was seen There was no signal in the medullary zone, the ureter, the mesoπephros or the mesonephπc duct (data not shown) Kidney sections of E14 5 (Fig 6e) and E17 5 (Fig 6f) mouse embryos showed that the mesenchymal hybridisation signal 3* accompanies the progression of the uretenc branching towards the peripheral cortex This was concomitant with the disappearance of the previously observed signal nearer to the medullary zone leaving only a weak signal in the S-shaped bodies At E19 5 only a signal at the limit of detection was observed in the peripheral cortical region of the kidney Finally, the sympathetic chain comprising the cervical, thoracic, lumbar and sacral ganglia displayed a strong hybridisation signal at E13 5 (Fig 6g,h), which persisted at E19 5 (data not shown) No signal was ever detected in the eye

Conclusion eyal seems to join the group of developmental genes with a normal activity that is close to the threshold level for the appearance of clinical defects, such as PAX6²¹ >²²-²³, GLI3²⁴ SOX9²⁵ and Sonic Hedgehog²⁶-²⁷ (for review see 28) Such threshold dosage effects may explain the variable expressivity and incomplete penetrance of BOR syndrome within the same family

The three compartments of the mammalian ear are derived from different embryonic structures and are generally considered to develop independently⁹ Development of the inner ear begins with the otic placode (evident in the mouse at E9) which subsequently forms the otic vesicle The ventral wall of the otic vesicle gives rise to the stato-acoustic ganglia which will eventually differentiate into the vestibular and spiral ganglia From E12, the otic vesicle itself enlarges and two pouches emerge which differentiate into the cochlea (ventral pouch) and vestibular apparatus (dorsal pouch) At E13 the cochlea extends through ¹/2 a coil and by E18 has reached the full number of coils (1 3/4 coils) The differentiation and maturation of the inner ear neuroepithelium is a long and complex process, that is completed at 20 days postnatal The ossicles of the middle ear are identifiable at E13 The first branchial arch appears at E8 and all arches have disappeared by E12

According to the branchial and ear anomalies associated with BOR syndrome, expression of Eyal was expected in the branchial arches and in the developing middle and inner ear Within the inner ear, as a reciprocal interaction between the epithelium and the mesenchyme of the otic capsule is known to be essential for its development²⁹, Eyal expression was expected within either one or both of these tissues Likewise, the sensoπneural deafness would be consistent with an expression in the neuroepithelium and/or in the spiral ganglia By in situ hybridisation to mouse sections, Eyal expression was detected in the otic placode in the developing membranous inner ear (i e the cochlea and vestibule) as well as in the surrounding mesenchyme of the otic capsule While the cells of the organ of Corti stop dividing at E14, expression persisted concomitant to the maturation of the sensoπneural epithelium Gene expression was also detected in the associated vestibular and spiral ganglia Assuming a similar pattern of expression of the murine and human homologues, these multiple sites of expression thus account for all the inner ear anomalies of BOR syndrome The expression pattern of Eyal suggests a direct role for this gene in the development of all components of the inner ear (whether derived from the otic placode or the surrounding mesenchyme), as well as in the maturation of the neuroepithelium The observed expression of Eyal in the middle ear pπmordium is also compatible with the defects of the ossicles, and of the middle ear cavity, seen in BOR syndrome In contrast, no expression was seen in the branchial arches, which is most probably attributed to a transient expression of the gene in these structures or a level of expression below the sensitivity of our assays Rather intriguing is the observation of a high Eyal expression in another placodal derivative, the olfactory epithelium This could be indicative of a general role for this gene in the development of the sensory placodes although, to date, in contrast to the olfactory and inner ear epithe a, we failed to detect a signal in the developing lens

Consistent with the renal (hypoplasia, dysplasia, aplasia) and collecting system (duplication or absence of the ureter, bifid or extra pelvis, blunted or distorted calyces) anomalies of BOR syndrome, Eyal expression was expected in the ureteπc bud and/or in the metanephros Eyal expression was not detected in the ureter, in the kidney collecting system nor in the non condensed metanephπc mesenchyme Our data show that Eyal expression was restricted to the metanephπc cells surrounding the just divided ureteric branches and accompanied the progression of the induced clustering of the metanephπc cells from the juxtameduilar to peripheral cortex This expression was barely detectable upon completion of this inductive process (E17-E18) and undetectable in the adult kidney Eyal expression thus seems to be a marker of the newly condensed metanephπc mesenchyme The temporal pattern of expression of Eyal indicates that the activation of this gene may be dependent on induction by the ureteric bud According to the anomalies observed in BOR affected patients, this gene plays a crucial role in kidney morphogenesis, as well as in the growth and branching of the developing ureter and collecting system

Hox-1 6^0,31 Paχ2⁸² and some components of the retmoic acid pathway (reviewed in 33), have been shown to be essential for murine inner ear development Signalling molecules required for kidney morphogenesis have been identified based on in vitro studies and genetic evidence (for review see 10, 34 These molecules include the transcription factors Pax2³²-³⁵-³⁶, Pax8³⁷, WT-1³ HNF1³⁹, BF-2⁴0₍ the secreted glycoprotein Wnt-4⁴¹ , the tyrosme kinase receptor c- ret⁴², and several retmoic acid receptor genes⁴³ How the developmental step(s) controlled by Eyal fits into the cascade of events controlled by these other regulatory genes remains to be determined Of particular interest is a possible relationship in kidney morphogenesis between Eyal and Pax2, Pax8 as well as WT- 1 These three genes are expressed in the condensed metanephπc mesenchyme, albeit not exclusively, and for two of the three, Pax2³² and WT-1³⁸, a direct role in kidney morphogenesis has been shown by studies of knockout mice

This work has also demonstrated the existence of a gene family comprising at least two other members, EYA2 and EYA3 This family is defined by a highly conserved 271 aa C-terminal region which is also present in the drosophila eya protein, the remaining part of the protein shows a lesser degree of homology that decreases towards the N-terminal end The actual size of this newly identified gene family remains to be determined, a task which will be greatly facilitated by the high conservation of the C-terminal domain shared by the EYA genes Interestingly, the high conservation between the human eyal and murine Eyal genes extends into the 5'and 3' non coding regions This conservation, which was not found with EYA2 and EYA3, strongly suggests an important role for these untranslated regions The conserved 271 aa C-terminal region is likely to confer a similar molecular activity for the encoded proteins As the drosophila eya protein was known to have a nuclear localisation, the identification of a putative bZIP domain within the eyaHR of EYA1 was particularly appealing due to the association of this domain with various families of transcription factors However, this possibility needs to be considered with caution as EYA2, EYA3 and eya do not entirely fit the recently determined criteria for a bZIP domain⁴⁴

ANNEXES

Annex 1 : Sequence of the eyal gene

Annex 2 : Sequences IDN° 2 to SEQ ID N° 47

Annex 3 : Restriction map of the eyal -a cDNA

Annex 4 : Restriction map of the eya1-b cDNA

Annex 5 : Restriction map of the eya1-c cDNA

Annex 6 : Restriction map of the eya2 cDNA

Annex 7 : Restriction map of the eya3 cDNA

ANNEX 1

The polynucleotide of SEQ ID N°1 according to the present invention is hereafter presented. In order to facilitate the reading of SEQ ID N°1 , it has been divided in five subsequences that are presented below, begining with the 5'end non coding region that is localized upstream the eyal gene, and ending in the 3' non coding region localized downstream the eyal gene.

SEQ ID N°1-A : This sequence corresponds to the Contig Exon P. This contig is contained in the S1C6 clone (subclone derived from the P1 11083). The sequence contains a 5' non coding exon that spans until the nucleotide in position 794. This exon is contained in the in eya1-b (SEQ ID N°3). The sequence SEQ ID N°1 -A is contained in the clone HSEYA1 S1 C6, deposited at the Collection de Cultures de

Microorganismes (CNCM) under the accession number 1-1828. The SEQ ID N°1-A is the following :

GAAAAGATAACGXGTAATTAXTCCCCAAAGATCTTAAXTGGTAACTACTGTTGCA

AGAGGACGCGTGTGTGTTGACCACGTCTCAGAGAAGTTATTCTCACACAGCTGT

TTATTCAGATAGCGTGGGGCGCGTTCATTXACATAATTTTTTTTTTTTTAAACAGC

AAGGTTGAGTCAGACAGAGCAGTGCTCCTTTCAATTGTTGCATTTAAACCAATAA

GGTTAGGACAAGAGMTAGCTGTGGTTTGCGTTGCAAAAACCAAAAAAAAAAAA

AAAAAAAAAAAGAAAGCCCCGAGGCTCCATGGGCAGACCTACAAGGCTGCGCA

AACAAATCGAGGGATGAGATTCTGCTGTTTCTTTGTCTAGGGTTCTCAGATGCTA

TCTGCCGCTGCTGTTTGGTGGGGAAGGAGCGCTGGGCGCAAAGCTGTTACCAA

ACAGAACGGTGGGAGCTGATGGCTCCGAGTTTGGGGCGAGGTAGAAACTCTCC

AGTGCCACTTCCGACTTTAAGCCTTCCTGTTGCCGTCCACTGTGGCGGGTTTCT

TCCTGGGGAACACGTTTTCGCTCAGTCGCTCGGCAGCCCGAGCCTGCGGCAGC

GGCCAGGCGCCTGCCCCCTGCGCCGAGCTTTCCCCTGCAGAGGCGCTCCACT

CCCAGAAGCGCCGCGGCTGCACCAGAGCGCCTGAGAGCCCCCGCGCGTACCC

ATCCAGGAGCAAAACTATGTCAGGAATGGAGGTTTGCTAACCCAGAAAATTCGA

AGGAACACATTAAACTGGTGGATGCAGCAGATGTAAGCGCTGTAAGTACAAGCT

GACTGTTTGAAAAATTGTAGTGACTGACAACAGCTGTCCTGTGGGCAAAGCCCA

AGCTAACAGGAGCGCTTTTATTATTGTTTTGATTTGTTTXCAAAGAAAAACATTAT

GGCATGGGGAAAATTATGACACTTTGTACCTAGTACTTTATGAGCCACAGGGTT

TCATCGTAGAAGTAGAATTCCCAACCTCCAAATTATGAGCGATCAGGATCATTAA

XAAATAATTACAAATTAATTGTTAACAATTACTTTTGTTATGCTCTAGATAAATGTA

ATGCAXGACCATATTCTTGCAGGGAGATGACTTCATATCTCTTTTCTGAATTGCT

GCACAAATCTCAAATATATTTGTGTGGTATTGAATAAAAGCXGTACATATATGTTC

ATTGGC TACATXAGTT CCTTTTACACCCTTTGATATGAAAAAAATGAXCXTT

TAXCXTTTC

SEQ ID N°1-B : This sequence corresponds to the Contig exon R. This sequence is contained in clone B4CL34 (coming from a classified lambda phage library wherien the YAC 953H7 was subcloned). This sequence contains the first exon, exon R, the translation initiation codon of eyai-a (SEQ ID N°2) being at the nucleotide in position 378 of the sequence. The end of exon R is localized at the nucleotide 402 of this sequence. Exon R contains a potential splicing site at the nucleotide in position 402 of this sequence (fusion with exon P) that is used by the eya1-b form (SEQ ID N°3) and by the eya 7 -c form (SEQ ID N°4) In eya1-b and eya1-c Exon R is spliced to give raise to exon Q Exon Q spans from the nucleotide at position 353 to the nucleotide at position 402 of this sequence The sequence of SEQ ID N°1_B is contained in the clone HSEYA1 B4CI34 deposited at the Collection de Cultures de

Microorganismes (CNCM) under the accession number 1-1837 The nucleotide sequence of SEQ ID N°1-B is the following

TTATTTTTTTCCAAGCACCTTTTCCAAGCTGCCTGGGCAAGGCTGCTTTGCATGT

TGATTGGGGGAGGGGTAATCCCATTTGCAGCTGTCATAGGCCAGTGATGAATCA

CTATCACGTCATTTGGCTGCCTTCCCCCCCTGACAAGGGGAGGAGGAGGATAG

ACCTGCAGGAGGAAACAACAGTTGAGACCTCAACTTAAAGACAATATGCCTTTC

ACTGCTGCAGTATCCTCCTTTTTCTCTTTTGGTTAAAAGAGAGCATTGTCGTTAT

CAGCCATGTGCTCTGTATAATTAAGAGCTGACACTGAAGCAGAGTAACAACATAT

TCTAATTTTTTTACCCCTGATCACAGGTGCAAACATCTCAAGCCAGTTCAAATGT

TGCTGTTTCCTCAAGTTGCAGGTAAGGATATTGTTGATATTTGACATTTTTGGAG

ATTACCTTTCTGTGACCCATATCTATTTTTGTCTAGTGTTTCTTAATTATTTCTATT

AATAGTAACAGCCTCTGTTGGAAATTACTTAACAGTTTGTGTGTGTGTGCTTGTA

AAACATTTTAAGTATGTAGAAAGGGAATAGGAGCCTCGGTTTGAAAATAATTATG

GGCTATAGTTTCTAGTGTTAGGAAAACTGTGAATGTGTAATGAGAATCAGGAGA

GAAAAATTGGATTTTAGACCATTTCTAGTTTGAATTCAGAATATTTGGGGAAGTC

AAAGCTCACTTTAGAAGTAAAATGGTTCATGTGAAGCTGTTTT

SEQ ID N°1-C : This sequence (Contig S) contains exon S, which is an additional exon that is ppresent in eya1-b and eyai-c This sequence is contained in clone

B4CL34 Exon S spans from the nucleotide at position 638 to the nucleotide at the position 765 of this sequence The polynucleotide of SEQ ID N°1 -C is contained in the clone HSEYA1 B4CI34 deposited at the Collection de Cultures de

Microorganismes (CNCM) under the accession number 1-1837 The nucleotide sequence of SEQ ID N°1-C is the following

TXCAGGXXCGACTCATAXGTTTAAGGGGCATTGTTCAGGGAAACAGGGGACAAT

TAAGTAGTTGCCAGAGTAAACACAGGCATCAGGATXTCTCXTCCTGTCATAGACT

ATAGGTTTCAAGGTTTATTTGGTTGGAGACCCATTAAATTACAACTGGGTGGTAA

ATTATAAGGGAGTACCTTAGTGCTAATTTATTAGATGAAACAGATTCCACAGAAG

AGTGATCTATTTCCATTTTACTTCTTAACATAGTTGCCTTTTCTTTTAGAAAAGTA

ATGCCTTTGGTCC I I I I I AATATAAAAAGACAACGATGACTGTATTTTTTCTTTGA

AAATTATAATAGATGTCATTGTCTCACATTAGACCTTGTGACTTGATGAACTGTGT

AAGAGAAAAATGAAATTTTAAAAGCTTCATGTGACTGGCTGTATAGGTAAAGCAA

GTGACATCTTTTGATAGTGCATATGATGAAGGGATACCTGAAGTCAAAATGTGA

GATGAATTATTTTGAGTCTAAGAAATAGATACAATGGGACTTTTGTGCAAGTGTG

TTTCAAAGGTGTCAAGTCATTAGCGCATTAAATGGTGGTGTTATGAATTTGGTAC

ATCTGTCAAATGGAAGGCTTCTTGTCTTTAGGTCTATGGAAATGCAGGATCTAAC

CAGCCCGCATAGCCGTCTGAGTGGTAGTAGTGAATCCCCCAGTGGCCCCAAAC

TCGGTAACTCTCATATAAATAGTAATTCCATGACTCCCAATGGCACCGAAGGTGA

GTGTCTGCCTTCTCACTATTTACTTTGCACCTTGTTTCTCCAXCAGTATATGCTTT

GTTATTAGGTGGTTATGATTTCAGTTGTCAAAATCATAAAACTACTCTTATATTTC

CCCTTTAGGTAATAATGTGACTTACCAATCAGTACTCATTGTTAGGAGCTTCATG

TTTCCAGTTATTGTAACATTGCGTAGATTGGTTTCGTTTCACCAAATGCATATACA

TATATACACATGTATATTATACATATTTGTGTGCATAACATTTTTAACCTTTGAGT GTGTTTTATCTTCTTATTGATCATGTACACXCTGCAGTATCAATTTGTGTTGTXTT

GTTGTGTGACCAGTGAAAACTTGGAATXATATCCXTTATTTTCCCXTTTGTGGTT

AAATATAGCCXAATTTGTTTAAATGTACACXTXGGACTGGATTGTTTGTAAATATC

AAXGAAATAATTTGTGTGCCXAXTGTTCTTAAGGCTTGGCTAGAAAATAXAAXAA

TTTTAAAAAGTATGTTTATGAAXTAXCCXGCXTTTAGGTGATAACXCCXATTATXX

CTXAAXATAGGTCTTAAATGTXAACATXCATATTCTXGTATTAAACCAGXAXATTA

CXXAATTTAATAATCXAATCCXCTCGAACCTA

SEQ ID N°1-D : This sequence contains exon T. Exon T spans from the nucleotide at position 1056 to the nucleotide at position 1133 of this sequence. The sequence is contained in clone S3B12 (which is a subclone derived from the P1 11083). The polynucleotide of SEQ ID N°1-D is contained in the clone HSEYA1 S3B12 deposited at the Collection de Cultures de Microorganismes (CNCM) under the accession number : 1-1827. The nucleotide sequence of SEQ ID N°1-D is the following :

TTTGATTAAGAGXAXAAGGGAAATAACCXTTTTACCAACAAAAAAGCXAGGGGTT GGGAXAAAATTTTTTGGGAXGGGAGGGXCAAXTTTTXXACCCCAGTAAGGTT

TTTTXTXXAGGGXAAAAACCAAAAGGGAGGGXAAAXTTTTXTTTTXXTGXAAAAA

GAAXATXTTAXAAATTTGGGAGGGXAACAAAGGGXCXGGATAGTGXXXAAATTXT

ATTGGCTTGCCTTXTTTXGTTAGGGGGAACAAXAAAAAGGTTXACCXATATTCAG

GGGAGTGGAXGGCTXTTAAGXGAATXXGXXGCATAAAATTTTAAGAGXTTXTTTT

CACAGCACAAAGGGACTTGACCCAAATATCAATGTATGTATTTCCACAAAXAGTC

TGCXATXCAATAXXGTXTTGAAATAGAAAACTTTGTTTTGTGGTXTCATAAACATT

CAGTTATGTTTGXTGGTACGAAGCATAACATAAAATATTGGGTGAATTGGCAGTC

AGATATGGXTGGGTGGTAATGGATGGTGCATTAAAATCAAGCCCTXGGTTTTTTA

GCCATGCTGGAGAACCCAGAAGGTGTGTGTGTGTGTGGTGGGGGAGACXTTGC

CCTTGAGCATCAGGAAGACCTGCTGGTGTCGACACCCACTTACTTCXTGCTTCC

CCACATCTTTCTGTTCTATCCTTTXGACTCTGACCGAATAACAGATTTTTTCCACA

ACCCCAGGAAATGATTTTATATCACATCATGTAGATTTTGAGAGATAATTTATCAA

TAAGTAATTCAGTAAATATGTAAAACAATGATGXATTTAATTAXTTTXACAGTGXT

GCTTTTAAAGGGTAACTTGTGACAATAGATTGTTTCCTAAGGGGAAAGAACTTAA

TATGTGAAI I I I I I I AAAAGGAGTCTATTTATGCTTATGATATATGTTCAGTTAGG

GAAAATGTTTTTTGAAATGATTTTCTTGTCATTACTCTGAATGTTATATCTTCTCTT

TCCATGTCTTTACAAATAACTATTAATTCAATATCGATGTATGTTGTTTATTTTTGT

AGTTAAAACAGAGCCAATGAGCAGCAGTGAAACAGCTTCAACGACAGCCGACG

GGTCTTTAAACAATTTCTCAGGTTCAGGTAAGGAATAAATATTAAATTTTCACATC

AACACCAAGCA I I I I I I CTAGTTTATTCATGTAATGTCCCTGTGTATGTATATGTA

TACATGTATATGTGTATATATGTGGGTATATACGTACTTATGTATATATACATATA

AAATAGCTTTGCTCAXAXTCACATATTACCAATXCTGTTTTXATTCATTGTGAAAA

AGTGGTTGCTGAAATGTTGCTATCCAAXAAATGGGGTATAAAAAGGGTGTTGTT

CTTCTCCTTTCTTCTACTAATTATATTTACTGTGCTGGTGACTCCXCCCCCGTTCT

CTCTTAXATGTATTATTTTTCCAGGTCTXCAAATTTGGTXAGGCXAAATTTGGTGT

TTTACXCCXCTAAGCAAACTCGCCAAXTCATCTTGCTAAXTAACTGTCAXATCXX

CTATAGATCTAAATXAAATACACTCTGTAAAGCAXCATTAAGTXCTTCAGTTAXTT

CTTAATCTCCTCCACATTAATAAAACCXGCCTCCAATAAXXCAXAATAXCCAACTT

TCACAAGTGTCACAATGAACTACCGACXAGAAAATGACTGTGATCXXAXGGTGG

TXGTXAXAACGTTACXACATGTTTGCCXXGTCACCCACCCCCATTTTGGXAATAT

XXCATTCAXAATXTATTTACXACCCAXGGTAACCTCAGAAATTCXACCTXTTTCXC

AACXCXTGCCTXCTAACXTTXCAGCCCCCCCCCTAATTXGXAAAATXCXATAACT TCCACGTAAAXTCGTXAATTGAXTTTAATCCXAACCTCCCCTGGAATCXTTTCXTC

TCAATACCAXTTXTCATXCCCCCTAXAXAACAACTTXCTCTTXTXXXCXAXCATTX

AAAAXXXGCTAAAACAXCACXCACCCTCCAAAXAXTTCACTGAATTCTCACAGCT

TXGAAXAACCGAAAXTXCAAAXTTTXTXCCXCCGGGGCXTTCAXTCCCCCCCTTT

TTGTTTCCCCTTCCTXCTAAAAAAXAX

SEQ ID N°1-E : This sequence corresponds to the Contig 4405-9480 corresponding to the assembly of the sequences derived from the P1 9480 and the P1 4405 that are publicly available at the Genome Systems library, Missouri, USA.. This sequence contains 13 exons of eyal which are respectively localized at the following nucleotide positions:

- Exon U : nt2606 to nt 2675; - Exon V : nt 2995 to nt 3140; - Exon W : nt7185 to nt 7322;

- Exon X : nt 25141 to nt25223;

- Exon Y : nt 25628 to nt 25814;

- Exon Z : nt 52945 to nt53084;

- Exon A : nt55019 to nt5102;

- Exon B : nt80417 to nt80235;

- Exon C : nt107808 to nt107866;

- Exon D : nt107979 to nt108140;

- Exon E : nt 109103 to nt109217;

- Exon F : nt109323 to nt109444;

- Exon G : nt113575 to nt113675.

The nucleotide sequence of SEQ ID N°1 -E is the following :

GAGGATCCTGAGAGTATGGATGGAGTATGCAGGACAGACCAACAGGCAATTCT

GGATTGGGGGTCGGGGAGCCACCTGAG

TCATGAAATAGAGGGGAAAACAAGATGGCTGTGAAGTTCTAATTTCCACGCAGG

GGGTCGGAAGACAGTATGCTTTGGTG

GGAAAAGTTAGGCAGACCTGGGTTCAATTTGCTTAGATACGTTACTTTACTTCTA

TCAGCCCTTAGTTCCTTATATGTAA

AATGGAAACAGAAAATCCAACCTTGAAGTATTTTGTGGGACAGATGGTGATGCA

GTCCACTGAGTAGAGTTGAAAACACA

GAAAGAAAAGACAAGTGTGGGGTAAAAGTTAGTGGGAATCTGGGAAAATTATCT

CAGTCTTCATGACAATCCTATGAGGT

GTAGGTAGTATTACTGCCATCATTTTATAGGAGAACATATGGAGACATAGAGGG

AATAACTTGACTAAGCCCACATAGAA

GAAGTGGGATTTGAATCTGAGCTGAAATTCAATTCTGATTCCAGAACCTAGGCTT

AATTCCCACGTTCTGCAGCCTCCTA

AGTCTTTCATCATATGTATATACACACATACTGTGCATATCCTCTAAATCAAAGCC

TTATGGGACTTCCTGTACTCTGTG

TAGTAACCCATAACCCTGATATCTTTTATTCTGTTGTTTGTTAAAAAGAAAGAAAA

AAGAAAAAAATTGTTGGGTACAAC

CTATTGAGTTTATG CTTTTCATGAGCCATGACCCACAATTCAAAAAACATAGA

CATAGAGCTAGGAAAGAATAAATGG

GGTTTCTGTGGCTTCTGTCAGCAAGTAGCATTGCATTTTTTTGTTGTTTGGTTTT TAACCAATTATTTTGAACTCCAATT L S

GTCCCAGGAAGAGATTACTTCTCCAATAATTCTGCTGGGTAAGTCCATGTTACC

CTTGGCCTGTTTTCTGTCTTAGAGGA

GCAGAAGAGGTTTTAGTTGCAGATGACCTGGGCAAGTGGTAGTGCTCATAGTCC

AAGTGTTCTCTCACTAATCCCTCTCT

TCTATAAACTGCAGTCTTTAACTGACCATCCCACAGTTCTTTATGTGTCACCCTT

CCCATGTGTATCAGACAGATCTAGT

CTTTGCACCTTCGCACGTGCAGCATCTAGCACAGCACCTGGAACAGAGTAGTG

GCTCAGTATGTGCCAGATGATGGGGTA

TACCAGCTGGGCCGGCTTGCCATCTCTTCAGTACAGATGCTGTCTCTGCCAAGC

GGCCAGCCAGATGTTTGAAGTCTGGG

AGCTTGCAAGAGTGGTACTAATCTAGATCTACTGTTCCTGGACAGGCCTTGTTT

GTTTTACAAGGAAAAGATTACTCATA

GATTTGAAGACTCTAGTCCAATTCTACCATTGTCATCCCAAACACAAGCTAGAAA

ATTAAATTTGAGAAACATTAATGTA

CCAGTGTGACAATGGGAAAACTATATGAAACTTTGAGTACATGGAACCTCCTAAA

AGGATTTTAGTGTAGAATAGAATTA

ATAAATAATAGAGCCTCCGAAGATAGTAATTATTGGTAGAAGAATAAAATAAACAT

TACATCTTATAATAATTGCCCCCA

AGGAATATGCATCTTAGCCCGATAGAGATTAGAGAATTGGAGGAAAAAAATATGT

ACATGTATACCCATAAACGTAAATA

TGTGTAGATATATATGTATGTATATATACACACATATATGTAGATTTATATATATGT

AGATTCACTGTGTTTACAATGAA

AGAAGCTGAAACTGCCATTTTCAGGACTTTGTTAACATAGGTTTTTGCTTCTTCA

GTTCAAAATTAGAGCACAAGAACAT

TTGCTTGGACTTCAGTGCATTATTTAATCAAGTAAACGTGATTAAGAAGTAAAGT

TCTGCTTGAATGCTTAAATACCTCT

TTAAAAAATAAAGGCAAACC I I I I I I I AGTTGAACATTGAAGTTGATCACATATAA

CTAAAAGGCAAAGGAAGACCCAGA

TCAGCACAGGAAAAAACAGGCTTAGCTTGCAACTTGAGAAATTTAAGATAGTTAT

AGGAACGCATTTTTACTAAAAACCA

TTGCTAAATCAATGCAGGCAAATGAGGATCTATCCTGTTTCTTCTCACCCGAGAG

AAGATTAGACCATGTTTCTTGGTAA

TACAGCCTCAGGTTTGTGTGTTCTTGCTGAGCACAAACAATAGGCCAGCTGACC

TTTCAGATTCTCACCCAATCCTGCAA

TTCAGCTTCATACCTCCCCTTTCAGCACAGCTTTCCAAACAGTAGCCAGGACTC

CTTGTACTCTTTAAGTATTGCCACTG

TAAAAACTCACAGCATGCCAGCAGGTATTTGCGAAGATTGTGATGACGTTTTCTA

AGAGTTCATTTAAAACTAGAGCCCT

TAATCAGTTTCTATTCATTCCTCTTTATAGTTTAACTTTCTGTTAGGTGGAACCAT

ATGAAATTTCCATCTCCGCAGGTC

ACAAAGACCAAATATCTGAAATTTCATATGGCCAACCTATAGTTGAAAATATGTCA GTGCTTTCTATATATTTTGTGTTT

TGCTTGGATTATTGTGCATTATTTTGTTTTGTTTTTGTTTTCCAGCAATTGGGAGC AGTAGTTTCAGCCCACGACCAACT

CACCAGTTCTCTCCACCACAGATTTACCCTTCCAAGTAAGATGTATTTTCTCTTA ATCAATAAATAACTTCTCTGAGTAG

ACTTTTATTTATTTAAAGTCATGTCTGTGCCCACATGTTCCATCTTAATTTAAATG AAAGTGCATTTCATAGAGATGTAA ACATATTGATACAAATTTATTTTGAGCATCATGTAGTGGAGACACTGAAGTATTTC

AGTGCTTTGTCAATTCTGTGTGAA

AGTTTTACTTTAAGATACAATATTTAGCTGGAATTTGTGATGTGGTTGTTAATCGG

TTTGCATGTTTCCATCTAATGCAT

GGGCTTTCTATTTTCTGTCATTTCTGACAAATAGCAGACCATACCCACATATTCT

CCCTACCCCTTCCTCACAAACTATG

GCTGCATATGGGCAAACACAGTTTACCACAGGAATGCAACAAGCTACAGCCTAT

GCCACGTACCCACAGCCAGGACAGCC

GTACGGCATTTCCTCATATGGTGAGTAACCTGCAACTGTAGTGGTGGCGTTATC

GCATGGGCATGCGTGTATTAACATGT

TTGAGTGGTACTTAAAGACCCAATCTTTGGCCAATTTAGAACGTGTTGTCACCTT

CTGTGTCTAAATTCTTAGTATGATT

GTTTCTAAAGAGCATTATAAGAACTGGTAGCTCCTTATCTTGAGGTCAGAGGTAG

CGAAAAGATTATGTGGTTCGTATGC

CTTCTTGCTCTCATTTTTCTCGATTTACATCTCAACCCTGAATTTGCCATTTGAGC

ACTGGGCTCTGGAATCCAAAATAT

TTTTGTGCATATTTGTGATAAAACGTCTGTGAATGAAGTCTTTTGGGGTACAGAT

TCATTTCCTATTTATCAGATGCCTA

GCTGTGATTAGGAACGTACAAGTAGAATTCAACAAACTCTTGCTTTAAACTCGAG

GGTGGTAATAGTCTCAAATTTCAGC

ATTATCAACCATTAGGAAAAGTGATCTATATATCACTGCCTGGAGTGTACTTTGA

AGAGGCAAATCGGGAAACATTTAAG

GCTCACATGGACATTTGTTCCTTCCCACCTGGAGATTATCTTTATCTTGGTGTAA

TTTAACTCACTTTTTATGGGTCAAT

ACCTGTAAATTAAAAATAAATCCAACAAAATCCCAAAGCTTAATTAAAAAAATTTC

AGAATTTCAAAATACCTTTTCTTC

CATTAAATTGTCATTGCAATTCTAAGTAGATTGAACTGAATAGAAACACTTGAATT

ATTGGCATTTTATTAAGAAAAGAA

TAAAAACTAGTGATCAAGGAATAGTACATGGACACTTGGAGGAAAGGATTGATA

GAATAAGTAATTCTGTGCAACAAGTA

CAGAATTTTTTGTGACCAGTATTCTAATTGTGCTTCTGCTTGATACTGTTTTGTAG

TATTCTTTTCTGTGGTATATAAAA

AGAATTAATGCTCTTCTTTAAGTATTTCATTAATAATTAGAAGGTAAATTACTTTAT

TCCTTTTAATTCTATCTCAAACA

AAATCAACTGTATATGAATTTCTTATAAAATATATACAAATAATCTAAATTATGTAC

GACTTCAGCATTTATAGTTTTAT

TTTAAAACCATCAGAACCAATGTTTTCTAGATAGGTATTTTTAAAGTAGACTTTAG AGACAGCCCTAAAATGGAA I I I I I

AAAACTATCTCTCTTTTCCATAACTTTTATTCACAGTTTCTCATAATAATATTAGCT ATGGATTAAATTTAGCTGGAATT

TACTTTCAAACTATTCTTGGACTACAAGAAGAAAAAGGATCAGCTCCCATTCTAT AATAGAAGTCAAGGCAGTCTTTGAT

TTACGTTCTCAAAATTTATTTGTGAGATGGTTATTTGAAGTATAAATGTATTTTCC AGTCCACACAGTTCTAAAGTACTG

TCAGCCTACCAGTGCTGCCATTGGAAGGTGCCATTCCCATCACTGCCCTGGCTA CCAGCCACACATCTTTCTTCTTCCTT

CTCCTCCCCATTCAGTCTTGGTGGGACCTGGACATCTCCCTTTGCCAATTTCTG AGCTCTGGGTTAGGGGTTCCTCCAGT TCTAAACCATTAATGAATGTCCTTGTCTGAAATAGCTGTTCAAAGAGTGTGAGAT

GAGCAAAAGATCTGGGAATAGAATA

AGAAGTAGCAGAGTGAGGATGGGGATTTTAATGGGATAGATGGGGATAAGGGA

GGGGGAGAGAGCGAGGCTCAGTGTGAA

GGGCTTTGATTCCAGAGTTTTCCATGGTTATAAATTCCCAGGGAATAATTTTCAG

TTCTTAGCATTTTCCTAATAACCCC

CATCTTCTCTTTTGGGAACTTGAGTAGAAGGAGATGCCCTGCGTACTTCTGATC

CAAAAGTTCTATTTTGGGGGCCAGTG

CATTGAAAAATATGGCATCCTTCTCCCCTTTGCTCATTCATGAAGGAAGAGCACA

TAGCTTGGGTTTTAACTGCTGATAG

TCAAGGCTCAGATGGTCTTACAAAACTTTCTCCAAGGACTTGTACTCTTGACTTT

TTATTTAGACCCTGTAAGAAAAGGT

TGGTGTATCTTTTTATCATGAGAATGATAGATCAGTGGGGAAACACAAATTCATT

AGTCTGCCAGTGAAATGAGTTTGCCACCTTGTAGGTAGAAGTCACAGAGATAGG

TGTGGCTATGGGTGCTTTCTACCTCT

TTCTGCCTTTCCTTACATGTTCTTATCACAAAGATCCCCATCTGGAACCAATAGG

CTAGAAACAGAATAAAAATACATGA

CTTAGGCCAGGCGCAGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGGCTG

AGGCGGGCGGATCATGAGGTCAGGAGA

TCTAGACCATCCTGGCTAACACGGTGAAACGCCGTCTCTACTAAAAATACAAAAA

ATTAGCCAGGCGTGGTGGCGGGTGC

CTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCATGAACCTGGG

AGGTGGAGCTTGCAGTGAGCTGAGATT

GCGCCACTGCACTCGAGCCTGGGTGACAGAGCGAGACTCCGTCTCAAAAAAAA

AAAAAATACATGAATTATTGGTATTTT

ATTGGTATAGGTGAAGGCTGGAAGCAGATCAGTATAGTCTCAAATGCAGCAAGA

GGTGGAAAATCCCTAGGGGTGACAAC

ATGGAAAGAAAGGGATTTTCTAAAGCCATCATGCCACCTGTCTGGGGCAGGTGT

ATTTCCAGGGACCATTTCTATGTGTG

GGTTATTCATTGGTTTGGGATCACCTTTACAGGGAAAATCACATTTTTAAAATGT

TAAGGTTGTGTTAAGACTTAAATGT

TAAGGTCATTATGAAGTTATTTAGAGATACTGCAGGAATTAAAGTAGGCACTCTC

AGCCAAGAGTCAGATTCTCCCACTG

CCTTAAAACAATTACCTAGCTGTACTCTGCTGATGAGATTCCCCTAAAATGGAAA

TAAACAGTAAGACTACTGTGTATTC

TAGATCCTAAAGATATCATTTATCTTAAGTACGAAGCAAAATTATTAAAAAATGTG

AATTCCCTCGGCAGAAA I I I I I CT

TCCCTCCCTCTGTGAGAATGAAGGAGAGTTGTTAACATGTATGACTGTCTTTAAT TGTAATTAATTAAAGACAGAGCTGA

GCCCCATACCTCAGTGCTGCCAAATCCAAGTCCCAAAATTACGATGTTAGCCTG GTGTTTGGGGAAGTATTTGGGGGCCA

CAGATACATCATTTTTATATTATCATATGATAAAAACAAAGGTACTTTAGACTACA TCGAAAACAATTATAGTCAAATAT

TTCACACTAACAGGGACAAAAGGAAACTGCTGGTCAACCTCACTTGTTTATAAAT CACTGCTGTTTTATAAGATTCAGCT

CATTAGTGTTCTGCTTCCTTTGTGCTTATTTTCTGATAGGACCTAGATATTCAGG AAAAGCCTTTCCACCATGTTGTTTA t,8 GTTCTGTGCCCAGTATTGCTTCTTTGGACTTCAAAATGAGTTGGTACTGAAATCA GCTTTGTAGTAGAAAGAGTAAAGGA

GGGAAAAATACTGTGATGGTATAGGATCTAATTAATATTTTTAATATAAAAAAGGA TTTTATTTTTAACACTAGTCAATA

TAGCAACTTTGGGAACATTTAGCTAAAATTTAATTATGGCTATTTCTTTACTGTGA AGGAGATGATCTTCAACTCTTACA

TAATTAAAACAACAAAATTAAGTATGAAAATATATCACTGGAGTTACATTTTATAG AAGGGATATGTCTTGAAGTGCAAT

AAAACAAGAGAGAATAATGTGAATAGACATTCAAATCTGTAATCCTGGATTTAAA AAACTGAAGTTGTACTTTATGGTAT

TTCACTGAATGCATGTATACCCTTTATTTTTGAGATAAGATTGGGGAAGCATTTG TAGTTAAACGGTTGTGGAAGTGTAT

GTTTTTGTATATTTACCAGCTTTTGAAAATGGACAGATAGATAAGCTATCACTAAC TGATTTAGGTGCATTGTGGGCAGG

CATCAAGACTGAAGGTGGATTGTCACAGTCTCAGTCACCTGGACAGACAGGATT TCTCAGCTATGGCACAAGCTTCAGTA

CCCCTCAACCTGGACAGGCACCATACAGCTACCAGATGCAAGGTCTGTATATAC ACATTGCTATTTTCCTTAACCCTGTA

GGTTGATAGTTAACTTCTAAAGTAGAACCTGATGTTCCATGTAAATAGTAAATGA TGATGGCAACTGGATTGGGCTTAGA

GGCAGGACACCTGCTTCTAGTTTGATTCATCACTTACCTATTTTCTAGGCCTAAA TTTCCATATTTATAACATCAGGGGG

TTAGACTGGATGATCTATAACATTCTTTCCAAAAAGCACTAAGATTTTTTGTGAAG TTGACA I I I I I GCCTTTGTGTCTT

CTGTCAGTTATAAAAGAAATGAAAAAGTCAAAAGTCAGCCACGTAGGAGGTTGT GATATGGCATGTACTAAAAACTTACG

CACTTGGTATTACCATCCAACTTTTCCAAAAGGGCTGTATTCTTTATTTTCATTAT GAAGTGAAGCTTGGGGAGTGACAG

TCATAGCTGTAGAAATAAAGTATGTGATAAGTGTTGAATAATGATCTCCCAAAGA ACAGCTTGATCAGATTTACAGACAC

CCAGTGAATCTTTAAGAAAATGCTTGTAAGAATCAGTCCCTGGGTTCTGTTTTTG AAAATATAGACAAAACATAATGTAA

GTACAAGTCAAATGGAATGACAGCTGTCATTGTCAAAGCTGGTCTCAAGAATAC ATAGACATTACTTAAAGTTAGCACAT

TGTTATCTTGCAAATAGATCTTTAAAGTGCTTGAGAATAGTTCTCACATAATTTAA ACATTTTCCTAATATCTTGTCTTT

ACTACAGTTCAGTTCAACGAATCATATTTGGCCTCCTAATAGTGCCAATCCATAG TTGAATTAAATCCCTGATGATCTCT

TTTTGATTGAAGGGGTTC I I I I I ATAGTTGTTATTGTTTTTGTATATTTTAGCTTAT TCTTCATTACATTAATAATATTT

GTTTATCACAGACAAATTAGAAAATACAGAGAAGCAAAAATATAAGTCTCATACAT TTATTATTATCATTATGATATATG

TTTGTCTAGATATACAGCATGGGCTCATTATTTAATACAGTATTGCAGCCTGCTG TTTTTAGTAACAAGTACATTTTAAT

CATCTTTTCACATCAATAAGTTCTGTTCTATGGTATCCTTTTGATGGCTACATGGA ATTAGCTAAATAGGTATATCTTAC

TTGACTAATTCCCTAAATAGATATATCTTATTTACCTAGTTCCCTACTATTTGACA TTTAGAGCAATGCTATGCATATCT TATCTACACACCTTTAATTATTTCCTTTGAATAAGTTTAAGTATAGAATTTCTAAGT AAATTGTTTTATGCTCCTTTAAG

GATTTCCTATATATTTACACAAGGAAGGGCTGTGTGTTAAAGTATACACATTTGA TACCTTTCTTTGAAAAAAGTGTCAT

TTTGGTAGGTACAAGATTTTGTTTTTACACACATTTAGAAAAAACATAAAACGTTT TTTCTTCTCTTCAGTGGTTGTCAT

CTTTATCTTATTATTTTTCACATTAAAGATCTTAGCCTTTTTAAATGTCTTATTAAT ATGTTTCCTTAAGAATGA I I I I I

TTTTATTTATCATTTTTAAGAGTCTATTTTCCTGTATGGCAAACTACAATTTTAGTG GACAAGATCATTAACCTTTTCAT

ATTTGATTTTGCATTTGATTCAAAAGGCCTGAACTGTAAAGTTTTGACATATTCAG TGATTATAAAATAAAAACTATTTT

ATAACAGATAGCATTTATTGGTAGAAGGATATTATTTATGTGCCTCATTCCAAAAC TTTTATGCAGCCTTGGAAAAGAAC

TGTTCTGCACATGCTGAATATCAATTGAGTAAAGGAATATAAAGAACAAAAAGTG ATGGAAAGACTTTATTAATGAATTT

AACAGTCCTAGGCTCTAGTAATAAAATGGATCATAAGGCAGACAAGGTCTGTTT GCTCATAGAGCTTTCATTCTTGCCAA

AGAGAGAGACAGTAGACAAGCAACCAACTAAATTATGCAATTGCAGATTCCTCC GGGAGACATCAGGAAAATAAAGAGTG

CTGGAGGGGTCCCAGGGTCTAGCAGGGTGCCCGTGGTATCCAGGTCTCCTTGA GGAGAAGTGGATCCTAAAAGTGGGAAA

GAGGCAAGTGAAGTGCCCTGCCCAGAGCAGAGCTACAGAATTGTATGGTTCTG GGCCTGCGCCCATCCACAGCCTTGCCA

TTACCCGTCCACCAAAAGCTAAAGCTAAGTACAGAAACTGAGAGTTAGCATCTA GAAACTTTCATAGTCAGTCAATAATA

AATTTTATGTTTATTGAATCAAATAATAAT I I I I I AAAATGAGACTTATACTGTCTC TTTTTTTAGAAAA I I I I I I I I CT

TTTAG GTACATTACTTTGTATTTTGCTCAGTTGTCAGTCTGTGAAGGATTGGA GGAAAAAACCAATCCTGGACCTCAG

ATAGTTTGAGAAATATTGTGCTTGAGAGTTACTTTCATCTTGCTCTTCTCCCAGA TCAAAACTTATGCAGCAGGAACAAT

TCAGAAAAAGACAAACCAGCCATTACAGTATAGTGCATAAAAAGGGGAAATGAAT GAGTAGAAAAGGAAATGAATTTGGA

AGAAAATAAAATTATCATTTAAAAGAAATAAGGACATGTATAGCACCAAATTAGGC ACTGCTTTTCTAGCCATAGATTTA

TTATCATCTGGGCTTACTGGATATTGCATTAAAACTTTTTGTGTGTTTTTATTCAT C C CTTAGTTGAATGGCCTTTCTGT

GGGACATTAGGACCCATTTCCCATGACTGAGGCATTATTTCACATGACTATCTTT ATAATGATAATTTACTCAGTTTTAG

ACTATTGGATATACAGGTTGAGTATTCCTAATCCAAAAATCTGAAATCCAAAATG ATTCAAAATTTAAAACTTTTTGAGC

ACTCACGTGATTCTCAAAGAAAATGCTTATAGGAGCATTCTGGATTTCAGATTTT TGGGTTAGGAATGCTCAACCAGTAT

ATAATGCAAATATTCCAAAATTTGAAAAAAATTCAAAATCCAAAACATTTCTGGTC C CAAGCATTTCAGATAAGGGATAT

TCAACCTGTAATATGACTTCCACATATTAAGCCAGATGATCATATACTATTAATTT GAAAATTAACTTGCCAAAATGATG SO

ATGGATAATGCTATATAATTTGCTACAGAATGTATATGGCCAGATTTGGGTACTT

AATTGTGATTAATACGTAGTTCTTA

AATATTAAATGCAGTATGTATACTGCTTCTCATTATTCAATGATTTTGTGCACCTA

GTTGCGGGAGTCTGTTGACCTTTG

AGTGTCTTTTTAATACATGTTTTTTAATATAGCAATTTCTACAATGTCTAAAATTAT

TTTCAAAATTCTCTCAGTTCTTT

TAATCCTTAATGAATAACAGTGATTATTTGCTTTTATCAATTTCATCTTATAGCTAT

TAAAATGTATTTGAATCCTGGAT

GAGAATATATTTTGGCAATATGGGGGATATTTTTGCCTAGCTTTATTTCCGGGAA

ATCTTTTCAACTTTAAAAATATAAG

GTATGATGACTAGTTATTCAGAGATTTACCTGTTCAAATTTAATATCAGAATTTTA

CAATGATAGAAGAACTATTCTAGT

AGCCCAGGGGAAATATTCTAGTAGCCCCAAAAACTGAAGTGTGACATGTATGCA

TCCTCTAAGTGTTTTGCTGATTAATT

TAAAGAAAACTTATGATGATCAGCTCTTTGGTTCTATCAGACTTAGAAAGACTGG

AGTAAAGGTACAGTGAAATCTGGCT

TCCTGGTTAGCAGTATGGGTTCTGGAGTAAGATTGGCTGTCAGCAGTTCTACCT

TTGCTTTCTGTGGGCCTTCAGCATGC

AATGACTCCTTCACATGTGTTTCCTTATCTGGAATAGGGGAATGAGATCTACTCC

CACAAAACTGTTGTGAGATATCATT

GAGATTGTCTGTAAATCACTCTGACCAAACTGTGGTACATAGTAAGAACTCAAAT

GATTGTTGTCAATTTAACTGCTCTC

ATTGCAGTGGTGATGTGATTACTATTATTTTCATTATTATCATTATGGTCCAGCTT

TCTGGAATAGTTCCAGATTAGGTA

GCATAACCTAAATGCTCCTTCTCTTCCCCCCACAACACACAAAGTGTAATTAAAA

TTTTAATATGTGTAATAATATATAT

AACAAAATATATTTTAGCCATGAAAGAGAACTTTAAGAAACCAATAACCAAATAAG

CATAGCAAAAAAGGAGCCAGATGT

AATTAATATGCTGGCATGGAAGGAAACTTACTATCAACAAAGGTCAAGCTTTTTG

TACATGATACCGAAAAAGCGGGGGA

GAAACAACATGAAGAAAAAAATCAGGAGAGAGGAAAAGCTGTATGGCAGCAGTG

GTGAATGGGGTGTTGGAAAATGAGAG

TGAGGAAGCAAGGAAGAGCCGCTGGCAAAAAATAAACTCAGCTTTGCAGTGGT

GGTGG.GAGAGAGACAGTCATCAACCAA

GTCCTTGTTGGTCATGGGCCCTGCAGTCACATCTCTGAGTGCTAAGAATGGGTG

GAGAAAGAGGTCTCCACCAGTCATTG

AAGCATCAAATTGTTTAGTTCAGAAAATTAGGGAAGAAGATGGGAATGTGATAAG

CTGTGCTATGTAGAGATGGAAAACT

CTAGTGCCCATACAACAAAATAAAATAACAGTTGACATTTATTGTTTACTTTTTAT

ATGCCAAGGACTAACCTAGTTATT

TCCCTTGTGACATCTTATTTAATCTTTACATAACTCTGAACAGTAACTTTGAAAAG ATAAGAACCTATGGCTCACGCCTG

TAATCCCAGCACTTTGGGAGGCCGAGGTGGGCAGATCACCCAAGGTCAGGAGT TTGAGACCAGCCTGGCCAACATGGCAA

AACCCCGTCTCTATTAAAAATACAAAAATTAGCCAGGTGTAATGTCACGTGCCTG TAATCCCAGCTACTTGGGAGGCTGA

GGTAAGAGAATCTTGGGACCGTGAGGTTCAGTGAGACAAGATCGTGCCACTGC ACTCCCGCCTGGGCAATGGAGTGAGAC TGTGTTTTCAAAAAACTAACTAAATAATAAATAAATAATAAGAACCTGAAAATGCT

AAAGAATGCATAAAATTAGTAATT

TAGTTTGTATGATATCAACAGTTAAAAATGGCTACTCAAAACGGAAAATGGCTTA

GAAATGTCAGAACTCCTTGTTGTAC

ATACACCCAAATTACAGAGCTCACAGGAGTGGCTTTTAAAGAAAACTACAAAAAG

CCAAAATGTTGTTGCTTGGTCTTTA

AATTTTTCTGAAGGGCAGGCTTGATCGTTAGAACTAAACCATTGCATTACATACT

TAGTTATATAACTAGTCAAACACCA

ATTCATGAAGCTCTAAAAAAAACTTCTTAGAAAACTCATTTCTGAGCACTGTCAGT

GCTTTGACACTTGCACAGCCTTTT

AAGAGCAGATTTGGCAATTTACGGCCATTTTGCCACTGTCAAACCTTGTGAACT

GGGCCAGCCTTACTTGGTGCCCTCAT

TTGCTCCAAGTGATCCTTCTGGAATCACCCTTTCAGCTCCAACTTTCAACACTTC

TGTCCTGCCAAGCAGGCTTCTGTGA

ATCTGACACATGCCATTTTAAACTAAACAAGGATCAGATTTTCGTCGTGCATGCT

CTAATACTAAAATTCCATGAAATAA

AACCTCAGTGATTGGGAAATCTGATTGTTTTCACCTTTGCTGTAAGATCACTGGA

AAATTCTATTCATGCTTTAA I I I I I

CTTTTGTGCACCACTAAAAGATTAGAAACTTAAGTTCAGCTGAGAAAGACAAAGT

GTTCTTCTTCATAAACAACTGTAGA

GTTCAACCTGAGGAGGTGAAATCTTTGCTTATTCATTTTTGACATTTTCCTGTTC

CGCAGTGTTAGCTTTGG I I I I I CTG

GAAGAAGTTTTGGAATGGTCTGAGGGGGTTTTTCTTCACACACCATTGACAAAG

CTGATGCAGATGAGTTAGCTGAATGG

GTGGGTGTGGATGGACAGAGTGTGCCTGGAGACCCTTGGGTCAGTTCTGCAAA

CAATATGTTTTTACTGCTGCTTACCAA

TCAAAGGGCACTGGCCACAATAGTGAAACGGGGTGTTGTGTGTGAGTTTGTGC

GTGTGTGCACGCATGCATGAAAGGTAA

GAAAACTGCTTAGCAGAAGCAGCTTCGGCCTCTGATTGCTTTCACAGAATATCT

CCCCAGTGTGTCATAAATAACCTTTA

TTCTCCTTTTATGTAAAAGAAACAACTAACTTGAAATCAAACCTTTAAAAATGCAA

GGTCTGTACCTATCACTGGTGTCG

CTATCCATTTAACTCCACATATAATATAGATTTATGGTTCCATCTAATACAGAAAA

CAATGAGCTATCTTTATAATGGTG

GTGCCCAATCATATGAGGAAAATAAAATTTTGTTTAGCAATTTGGCTGAGGTAAG

GCAGTCTGTAGTTCACGTGCAAACA

ATTTTCACACACCTACAAAACAAAACCATTAAAATTAAGTTGTTATTTAACTGGTT

AAGCCTCAGCTCTGTGGAAAACCC

AGCTTTCCATAATAGCTTAGTTCTAGTGATTCTAGATTATTAAAGGTAATGAACAT

CTTTAGTCATATTTTAATCTCCAG

TATCTAGTATTATGTCTAAAATATACAAATGTTTGTTAGAAGTGGAGAGGTCTATG

TCACAATGAATGTTAGAAATTAAG

GTTCATAAAA CTCTGATTTTCTTCATTTGGGGAAATGAATTTGGAATGTAGCAT

AATAAAAATTCAGTTAATTCACCA

TTTAGAAGAGCAAAGACAAAGTTATGGAATCAACCTAAATGCCCATCAATGGTAA ACTGGATAAAGAAAATGTGGTACAT

ATACAC CATGGAATATTATGCAGCTATACAAAAAGAATGAGATC ATGTC CTTTG C AGGAACATGGATGGAGCTGGAGGCC ATTATCCTTAGCAAACTAACACAAGAACAGAAAACCAGCTACCATATGTTCTCAC

CTATAAGTGGGAGCTAAATAATGAG

AACATATGGACATGTAGAGGGGAACAACACACACTGGGACCTGTCAGAGAACG

AAGGGTGGGAAGACAGAGAGGATCAGG

AAAAATAACTAATGTGTACTAGGCTTAATACCTGGGTGATGAAATAATCTGTACA

CCAAACCTCCATGACACAAGTTTAC

ATAACAAACCTGCACATGTACCTCTGAACTTAAAAGTTAAAAATATATATAAAACA

TAAATAAGACATTTCAGCTAATTT

ATTGTCATCGTATGTTTAATGCCATCTTTCCTTGTGTTAATACTCGTAGATAGAG

CTGGTTGGAGACATCACAGAATCAT

CACGATGGTGGTATAGAATGCCTTTCTGAGTGTTTTATTGTCCCAGAATATCAAT

AATGGTATAGGACATTCCCATCATC

CCACACCAGGGAAGGTGTCAGGATCTGCCATGATAGATCGAAGGGGCTGCACA

GATGTACATGTACTAAACTCAGAGGGC

CCTGGAAACCCTGCTTTGTTTATTTTCCTTTAGGTTTTATTGCTCTCACTTAGAGA

AGAAAATAACACAAATTCTCACTC

CCTCTTTGTGTATAATGTTGTTTGTTCTGCTAAAGAGATTGGGGTTCAAACTAGT

AAGGAAAGACAATAAATCAGGGCAG

AGGTTTGTTAAAGGGTACGTGGAAGAGGCACAGCTCTGCGACCATCACACCTCA

TGCATCCCTGCCTCATTATGAAGTGG

GAATGATCCAGTAACATCACCCTTGTTTCCTTTGATCTGCCTCTCTGTAGCTTTT

CAAATGTACGCCTGAACCATTTAAG

TTTATTAGTCATTTATAACCTAAAAATTGCTTAACTAAATATGCTGTTATGCTTTAA

AATTTTTTTATTATCTATAGAAA

AGGTATTTTTACTTTAAAAGATAGTGGTTTTAGGAAAATATACATTAAGCTTAAAA

AAGTAATCTGTGATAAAGCATTTG

AGATTTGTACTTTCTGACGTAAAATGAAATTGAAGAATAGCTGTTGCAGCTTGCA

CTGATGTGCAGAATTGCCCCTTAAT

TGCTTTGCCACTCTTCTCATATTTA I I I I I TAGTTCTTGGCATTTTATGTTCTAGA

TTTTTTTTTTGCTTAAATTGATTG

TATAAGTAATATTTTGTTTCACTTCTTTTGGCATCTTGTATATTTCAGGTGTTAAA

AACTGGATAAAAATTTCTTGACAT

CTAGGCACAGCAATAATTAGTGACCCATACCAATATTGTTTTCTTCATGAGTTGT

TTTTAAT AAAATATATAGAAATAT

ATCAAGACAGGTAAATGTAAAGGCTTTGCAGTCTTTATAACCTGTGTTAAAAGTT

CTCTTTGGAAATAAAAACTATTACT

GGGACAAAAAGGAAAACTTGGTCATTTAAAATCTATCTTCTAAGGAAGGGGGAA

AATCCTGGTTTTAAATAGCCCATCTG

GCCTTCGAAAGCTTTTCCCCAGGGAGCTGCACTAACTTCTGCCACATAGATCGT

GCTTGATGATGGACATAGCCCATGGC

AAAGAGGCCAGAGATGGAAAGCAAATGGACTTCTGTGAAAATACACGAGGGACA AACAGCATCCTGCTTGCCCAGTAAGT

GCTAGAAAATGCTCTAAAACATGGCAAGTACAGACAGGCCCCAGCAGGTTCCAG CATTCTTATGTTGGCTGGGATTCTGG

TTTTTAATGTGAGTCCTTGTTCTCTGTGAAGCAGACCTTTGACCTACTTGATACT c CTTTT i π 1 1 1 T i TCTTAAAAAAA

AAAAAAAGCT I F T I GAAGTTATAACTGGGCAGTATTATCCAGGTTAAATCATACA CAGAACTTTGATTTAATATTTTAAG TAATGTTTAAATCGACATAGGCTGTGTTCTTAACTACGAAGAGGTAGGGAAGAT

GTATTAAGAGAAGTTTGGAGGGATGG

GTTTGTTTTAAAACTAAAATTGCTTAGCACTAACAGAACTGCATGTCCCATATAG

GATCTTCAAGCAAGACTGTTTCTTG

TTTGTTATGGTGACAAGGCCACCACTCTTTTTCTGTAGGAAGCCAACATTGATAA

GAAGAAAGCAAAGCAAAATCTTTGT

GATGCACTGTCCCTGGGACACTTTATGTAATTTACTTTTAAATTTACTAGCCTAAA

ACGGAGTTACAATGAAGAAGTCCT

TTAAAAATGGGATCATTTGATTCCAAATGTATTTATTAAATGTCTTAGGTATGATT

ATTTCTGCCCTTAGTGTGTCTATG

ATTTTTGTGTAGCCAAAAATTAATACCACTTAATACCAATTTGCTTTCAACATTAC

C G ATTCAGAAAATCTAAAAGGTAG

ATTTGGCATGACAGATTAGAAAACCATAACCTGGCACCAGAGATAATGGTCACC

TTATCCATCCTCTACTTTTTGAATGA

AGACGAGGACCCTGCCTCAGCCATCTCACATGATGATGTTGGAAATAACCTCAG

AAAATGGATGTCGAAAGATTGAACTA

TAAATGTACTCTAAAGCCATAGGATGTTATTGCATTCTGAATTTGAAGTTTTTCTT

ACACAAGCAAGACTTGTTTTCATT

AATATACCTGTGATATGCCAATGTCTGAGCTATGAGAGTGAGTTACCTTCTTAGG

AGGGCAGAATCTGGACCATGCGTCA

GTGTTCTGTTAGGATCCTGACAGCTGGACTAACAAGGTTAACATCCTAGCGGCA

TGCTCCGATGGTATGTGGATGCGAAG

TTCTTGTACCATGTCTGGGGTCCAGTGGTATTCATCGCTCAAATAGGTGAGATTT

TATGAGGTAAAAATCCATGGATACT

TAATCAACTTAAAATTTTATTTGATTTAAAGTCTGCTGGACTATTTACTGGAGATT

CAAAAATATTTTAACTCCTTTCAG

AAGGTGCTTACAGGTCCAGAATTTAACTTTCTTTACAAAAATTAAATCTATAGTGA

TTATAATCTGATTTCAAAGATAAT

CCTTAAAACATCAAGTAGTTGGAAAGAAGCCAGTCAGCCGAGTGTGGTGGGAC

GGACACGCTTGTAATCCCAGATACTCA

AGAGGCTGAGGCACGAGAGTCGCTTGAACCTGGGAGGCAGAGGTTGCTGTGA

GCCAAGATCATGCCACTGCACTCTAGCC

TGGGTGACAGAGTGAGACATTGTCTCTAAAAAATAAAAAAAACCCAGCAAAGTGT

TCAGATAAAATCTGTTAAATATCAA

AGTTCTCCAGATAAAGTCTGAGAAATAAGCTCAATTATTATTTATCTTTTCTTTCC

CTACTCAC I I I I I I AAATTGATGG

AAAAAGTTGAGAGACCTTCTTTTCTCTCTTAAAATCTTTTGTGGGCCTTAAAGTCA

TTCTTCCAAACTGTCCCCCTCCCC

ACCAACAAAAATCTATGGAAAGGTCCTCACCTGTCCCCACACCTAATACTTTATG

ACAATCGTGTTCTCACTAAATCCTT

TAGTCACTTACCACATGGATTGAAATTATCAAGCAACAGTACAGTTCTGGCTCCC ACTCCAGCCTAGAAGAAGCCTTCAA

AGGCATGAGTCAGACACGGTTTATCCATCTCTATGTGAGGCAGACAGGATAAAT ATAGAAAATGAGCTTAAGGAACTTAG

GTCAGAATGAAAACAAGACTAAGGGGGACTTGCTTAGTACTGGTCTAATGGAGA GGTCAATTACCTGCTATCAATTATTA

GGGAGCTCAGGTTTCCAGAAGATAAACTCAGTACAAAGAGTAACAGTCAAGGAA AAGCTTATTGGACAGATGAGACTTGG GTGAGGCCTTGAAGGATGGAAAATATGGAGTGAGGGAGGGAAGCATTAAGAGG AGCAAGACTGAGCAAGGTGAGTTTCCG

ATATAGCAGTGATCACATCCTAAAGTACCATGGTGAGCTGTGCATGGGGAGCTG TAGGCAACAGCCTGGTTATAATATTC

TCCTGAGCACCCCCATCCCAAACCCCGAGTGGCATCTCCAGGCCCAGATAAAC ATTTGTATCCTTGCATTTGTAAGGGTT

CTCTAGCTTTTCCCCCACCCCCGGCAATTCAGAATCGTAACATTTCACTGGTCC CAGTATACTAATGATGATTTGTTGGA

CATTCGGATTCTTGCATTTTGCCATTAAAGGATGCCATTTCTTTTCCTGAGCTGT TTTTCTTAAATGATCAAGAAACTTC

CATCACCCACGATCAGTCACACTCCTCACCCAACTTCCCAAAAACTTTCTATTCT CTGATCTTCTTTGGATAGTTTATTC

TCACCCTGTGCCAGGAGTGAGTACACTTCTTCCCTTTTTGGGGAAGCATGTGCT TTCCTTCCCAGTCTAACCAGGTGTCT

TCATGTCATTTTGCTGAGCACCTCCTTAGCAGGGCTGTGGCACCCAAGTGAGTA ACACAGTGAGAGCTGTGACACCTGGG

TGGGTGATGCAAGGTGATGTTCTTTGTGTCATCTTCATGAGTGTGTGGGATTTT CAAACTTTCTTCGAGGGATAGGTTCA

TGATGCCCACTTCTTTTGCATCACAACAACCTCTAGAGTTGTACCTATAAAGAAT GCCAGTAGATGCTGTTAATGAAACT

AACTTGAAAAGCTGTGTGTGTGTGCGTATGCGTGTGATTAGAGATATAGTATCTT CCCACTTAGAATATAAAAATAATCA

ACTTTAAAAGACAGCAGTGGAAGGACAAATAATGCTGTATCTTAAGAGATGTTGT GAAGGGAAAAATTTTCCAGTTTCCT

GTTGTGGAAATGAGTGTCTCCCCTCCATGTCAAAAAAAAAATAATAATAACCATG ACTATGATTGTTAACAATTATTTAT

TTATTTATTTATTTATTTGAGATAGAGTTACACTCTTGTCACCCAAGCTGGAGCG CAATAGCATGATCTCGGCTCACTGA

AACTTCAGCCTCCCAAGTTCAAGTGATTCTCCTGCCTCAGCCACCCAAGTAGCT GAGATTACACATCCGGCTAA I I I I I G

TATTTTTAGTAGAGACAGAGTTTCACCATATTGGCCAATAGTTATATAACTAGGTA AGCTTTTCCTTTTAACCAATGAGT

AAGAAGGTATCCCCACTCCCACCCCAAACTCTATTGAGAATTATTGAAGTTTCAC ATGAAG.CAATCTCTGCCAAACTTAT

TGATTGAGAAATAATGGGTTATAATGGGAATTCTTTCTACTTTGAGGAATTTTCAT GATATAATGTGATGTAAAACCTTG

CATTATAGGATGTAGCGGCTCTATTTTATTTATCTGGAGGTGTCTTTTGATGTAA AGTCATTTGTTTCTAGAAAAGAAAA

TACTTCTATATTATTTTGATTAAAGAATTCCAAACATTACAAATAATTCTGTGACCA AAAGTTGTAAGTGAAGACCCTTG

CAAATCCAACAGAAACTCTCAGTGGATTGATGTTAAGGGAAGGAGGTCTGTTTT ATCCTGGTCATGTCTTATCCTGTCTC

TCTCTAGCATCCAGGTTCAAGCCTGGTGTGTGACAGCCTTCTACACAAATGTGC CTCTCTTGCTAAGCCTGCAAGTGAGT

CTAGCAGCCCATACACTGAAGCTTTCTGGGGAGCATGAGCATTGTTGTTTTGCC TTTGCTTTGTGGCGTTCTAAACTTCA

GTGAAATCCATCTCGTAGTGTTGGCACTGAAGTCTCCATTGAATTAGCTATCAAA GTGGAGCCCCTTACTCCAGATGATC TTTAGCTCAGCTCAAGAACCAGAAATACCCCACACAGATAGGCTCTGAGAAGCT

TCAGCTCTGAATGCTTGAAGAGAATG

ATTACTTTCTTAGTGTATAAGAAGAAAGGTTATACTAGTTACATATTTTTGAAGCA

ATTGCTATAACACCTCCTGAATTG

GTACTGAAAATCAGACACTTATAGGTATTTGGTAAACATGTGGATTTTCAGATCA

CACCCTAGTCCTTCTGCCTCAGTGA

GACTGATTTCTGTGTCTGAAAGGGAAGCCCAGGTATTTGCCTAGGAAGAATAAG

CACTTCCTAATGACTCTGAGGCAGTT

TGTCAGGCATATTTTGGAAATGGTCATAAAAAAGGGAAATAACTCACAGACAAAA

ACCTTGCCATTCTGAGATGCAATTT

ACTATAGAGATTAAGAGCGTCAGTTCTGGAGCCAGGTGCCTGGGACTGAATCCC

AGCTCTGCCACAGTTGCTTATTACCT

CTGGCAGTTTCTTCGTCTATAAACGGGATGTAACAGCATCCACCTCCTGGGGTG

GTTGTGAGGAATCGGTGAGTTCTAAT

AGGCAAAATGCTTTATTTGGGCTGGCTCAATGTAAGCACTCAAAAATGTTTAAGT

ATTATTATTTGTTCTTGTCACTTCT

ATTTTGCTTCTAACAC I I I I I AATATAATTATAAAATAATCCCTGAGATTATTTACA

AGATATAAATTATTTTAATATAA

TTGTTTTATAAAATAATGCCTGGGAAAACTTTAGTGTATATCAGAACTTC C C ATTA

TACAATATAGAATCGAGCAAGAGG

AGGCAAGACAGCATTGTATAGACCTTAAAACCAGACAGATCTGTGTTTGTGTTTC

ACCTTCATTCCTTGGAGCTGTGTTA

CTGTAACCTGACCCTCCATTTCCTTATCTGCAAAATGGGAATGATCATACACCCT

GTAGTATCAAGTTGTTGTGGAGTAT

AGGTGAAGCATGTACGGGCGAGCCTACTTCCTAACATAGAGAGAGTACTCAATG

GGTATTTTGTTTTATTGTTATTATAT

TTTGCACTCTGGGGATATTTGTATGTTCCAGTCTTGGAAGTAGCATTTAGTCTGG

AAAGTCACTTGGGTGCCATTTGGAG

TACTGAATTGTGATTTACTTGGTCAGGCCCTAAGTCCTATGTGAAGGCAGGTGG

AGCGGCCCTGCGAACTTGATTCTGGT

CGTGAAAAATTAATATTGCTTGAGCCTGCTTCTCTAGTATGTACACCTCTAGTAT

TGTTATTATATTTTGCACTCTGGGG

ATATTTGTATGGGTTAGGTGTAAATATCCAA I I I I I TATAGGAATGTCGAAGTTTT

TTTAGTTGTTTTGTAGGGGAAGGA

CTAGTTTACATATAATTAGTATTTGTTAGTTTACAGGTCAATTAGTACTTGGCTTA

AAATACTGATTAGTAAGTCATGGA

TGTGTGTGTGTGTGTGTGTGTGTGTATATCAAATGTACGTACATCACAGAGATTG

ATAAACACACACATATATCATGGAG

ATAGATGGACAGATGATAGGTGGATTGATTCAGCAACCCAGGTTTAGAGGGAAA

GAGGAGAGAATGGGGATTCAGGGAGG

CTTTTCTGAGAAAGCGGATTTCAAATTCTGCTGAATTGTGAAGTGCAATCAAAGT

TAACCTGGATTTGACAGGAGGAAGT

AGGAGCTGTAAGGAGTAACATTAGAAAGCCTGCTGCATGTTGGTTTACTAGGGT TAGGCTGGAATGTATGCCCGGGTGCA

TTGGAGTCACAGTTGCTGGGAAAAGACCAAAGAAGTTCAAAAGATAAACTGGGT TTAGACCAGCTAAGGCCTAATACATA

AACCCTAGTAAGCAGTTTGAATATTATCCTGAGGGCAACAGGGAGCCATTAAAG GTTACTTTTTTTAGCAGGGGAGTGAC AAGATCAGTCATGCACATTAGAGAGTCTACTGACTTTGCAGAGAATGGGCTGGA

CAGTACCCAAAGATGGGTAAAGAAAC

CAGTAGAGGAGGCTGCTGCAGAAGTCCCCTGCTGTGGCCTGATCCGAGTTTAG

CAGCTCTGTTTGCCTAAGTCCTATGTG

AAGGCAAGTGGAGTGGCCCTGCGAACTTGATTCTGGTGGTGAAAAATTAATATT

GCCTGAGCCTGATTCTCTAGTATGTA

CACCTCTCCCTAAAGTAAGATCCTTTTGGAAGTTGCATTAAGTCTCCAAAGCCAC

TGGGGTGCCATTAAACTCCTGTTTC

GGCATACTAAATGGAATGGAATCTCAGCCTGCAGTCAGCCAGCAAATGAGTATA

ACTTATGTTGAATTCCAGCTAATGTA

CAGTCTTGGAAGTAGCATTTAGTCTGGAAAGTCACTTGGGTGCCATTTGGAGTA

CTGAACTGTGATTTACTTGGTCAGAC

CCTCGTGGCCTGTGTGTGCCAATATAGCTTCCATGGTCTTTCCTACTTCATTTCA

GGGGATAACTTATAGAATGAAAACA

TCCATTTTAAAAATATATTTTCTATATCTTATTCATTTTCTTGGTGGATAGGGATTA

TTGAGGTCAGTATTTATTGCTTA

AAACTCTGTCATTTAAATGAAAATAGCAAGTCGTAACCTAAAAATGTATAGTTTAA

ATGAAAGTAACGGATCATAACCCA

AGGTAATTTTTCCTCTAAAATGCATATAATAGCTGAAGTTTCTTTTAAAAAAGCAT

TGATATCCACATCTTTATCCAAAA

GTAATTTAAAATTTCTTTTAC GGCATTTTTAAATGTTCATCATGTATAAGTTTGC

TTTATATATAATATCAATAATAA

ATTGTTCACTGATATGCACGTAAAGAATGAGTCATATGCCTGTAAGGGACAATGT

TTAAAAAAAAAACAAAAACTGGTAG

TCTGATAAGCCCTGTGAGGTCTGAGGCTATGTCTTAGTCATCCTTTTAGCCCCA

TGTAATTGTCACCATGAGCCTGGCAT

TGAATATATGTTTCCTTTGCATGTAATTACCACATGCAAATGAACATTAATATGTG

AAGCCTGTCTATTGATGGCTAATT

GTGGTGGCTACTAGCACTGTGAGAGCTCTCTGCCTATCTGAGATAGGTGTTCTG

GGAACTTCAAACAATTGATATTCCTG

AAATATACAAATGAAGAAACCTCAAATAGAGTCTCTGTGATAACTAAATTTAACAC

TATTTTAGGATATAGATTCCCTTC

TTAATAAAAATAATTCTTGGCTCAAGCTTTTAAAGTTCATAGATGCTTGATAAGTA

AATAGGAATAGAATTTTAGATATG

TAAAGAGATTAAGTCATATTTATCTAATTCGGGAAAGTAAAAGAGGGAAAAGATA

CGTAAATAGAGAATTTTACAAGTGC

TTGCTGCCATAAACCATAATGTGCCTAATGTTTATGTGATATTGTAGAGTGTCCA

TTATGCTTTCCAAAACATTACCTCA

TCTGATCTGTTAGGAACACTGCAAAGTAGAGTTTTTGCAGATAAGGAAATTATGT TTTAGAGTGGCTGGGAGACTTGTCC

AAAGTCCAAATAGCTCATAAATGATGAACTGGAAACTCTGTTTTCTGACTCTTAA GCCAATACTTAAATGTTCAGTAGCA

AGTAAATAATTGGCATATATTACCACATTTTATACCTGGAGCCATCTGAATAGTG AGTTCACATTGAAGAGATTCCTCCT

TGAACTGCCAACACATTTATGGCCTATACTGCTCATTTGCCGTAACACTGTAACT TTTTAAAAAATGGCTTTTATTTCTA

ATTATAGAAGCAACATAGGTTCCAAGTAGAAAACTTAAAAATGTAAATTGGCTTAA TGAATGCTATGTGACCATATATCA 5

AATCCTTCAGAGACAGCATTTTTTAGAATTTTGTGCTTTTTGAAAATTTGTCATGC

TTTAAAAATATTTTAAATGAGATC

ATCAAA TACTTTTTGTTATTTTAGTTTTTGCTTTCACAAAATAATATGAAAATTT

TTCTCATGCCATTAAACATCATT

GTAATACCGTTTAAACAGCTTCATAGTATTCCATTTTCTGAATAGTACCTGTTATG

GAGTTGGGGCTGCTTCCCTAGGTT

TTCTTATTAGAAGCAACACTGAAGAATATCTTTGCTATCTCTTTGTATAAATCTAT

AATTTTTTTTCTTAGAGTAAATGC

CTGAAATAAAATTGCTGGGTTAAATGGCATACGAAATTTCTCAATCATAAGCCAA

ATAGCCAAACAGAAAGATTGTGCCT

ATGTAAATTCCCATTTTGCAGTGTATGAATGCCGTTTTTTATATATTTCCCAATCC

CTGATCTTACCTTCTTAAAAAAAT

TTTTGCCAATATGATTATCAATGGCAGCATATTATTGTTTTGCATAGCTTAATAAC

AAAATAGTAAATCATGTGAAAGAG

GGAACCCAGCTTTGCTCAGGTGTTGTATCTTACACAGCGCCTACTGTTCATTGT

TTAACAATGTGGACATTCAACAGACA

GAACATTATCTTGACAGCATATGTAACAGATAAAACATTAATCATATTCTTCTTTC

ATCTTTGTTGTGTCAAATTTATCA

GGTGCTGCCATTCATTCTTCTGTGAAATGAAATGGCTTTTTCCATACGCCATTTT

AATGACCATATAATACAAGTCTGAA

AGCATTTTAAGTTCTTGGAAGTAGTATCATAGTTTTTGAGCTAGCAAGAACCTTA

AAGATCATCTAATCTGACCCCATCA

TTTTACATTATGTGAATTTAATATAAAGACATGAAAAATCTTGGTGTGTTTTGCAA

ATTGAAAAGTTCATTGTCTAATAT

TTTTACACAAATGTATTATTTTGTCATTAGTCTATATAAGTTACTAACATCCTTGG

AAATTTAAACCTCTTTG I I I I I I A

ATTTTGTAGTAAAAATAAAATCATCAACTATGTTAAGAAAACTACATTTTAGACAT

GACAACAGATTCTAGTGTCGAAGA

TAAACCAAATACCAATTCTGCCTTTTCTAAATGCTATTTTCCTGTACCCACATTAT

TCCACAAGCCTGGTATAATCGTAT

TTCTTTTCATTTTGTTTTAGGTAGCAGTTTTACAACATCATCAGGAATATATACAG

GAAATAATTCACTCACAAATTCCT

CTGGATTTAATAGTTCACAGCAGGTAATTTAAAAAGCCAGTTTAACTTCTGAAAA

ATATCT TAAATGCAACATGATTTT

CTTATGTAAAGAAAGATCTGTTTATAAGTGAGAGCTTTCCTAAGGCTTTTAGTAG

TTTTCTTTTAAAATTCACTTTAGCA

GTCTTTTGTGGTGAGTTGTTTGGTTTTCAGATTGGTTTAATTTTACCTAGTATTGC

ATCATAGAAAAGCAAAAATATGAA

GAATGTGTATAATCTTACATAAAATTTGTTTGCAAGTCTGAGCTCTTTAGTTAATG TGTACTTGTATGCCCCCGTGTGAA

TTCAGAAAAGGCTCAGAAACCCAAACATACTTACAGTTACACTTTGATATGTAAG AGATAAGCAGCAGTATTTTGACAGA

CAATTCTTGTGTGGTTTGATTTGGTAGGACTATCCGTCTTATCCCAGTTTTGGCC AGGGTCAGTACGCACAGTATTATAA

CAGCTCACCGTATCCAGCACATTATATGACCAGCAGCAACACCAGCCCAACGAC ACCATCCACCAATGCCACTTACCAGC

TTCAAGAACCGCCATCTGGCATCACCAGCCAAGCAGTTACAGATCCCACAGCAG GTAATTATGTGAATATTATTAGTCCT GCAGGTGATATTTTCAATGGTTTCAATGCATTTTTCTACAGTCATATATTCATGCA

GTGGTTGCACAATTTTGGCAGCTT

TTGGCAAGGACTAAATATAAGTCAAGCAGCATGACTGTGGCATTCATTTTTATTT

ATTTTTTTATTTGCTTGAAATTTTA

AAATAAAGTATATAAACATCAAAAGCACATGAAATAGAGATTTTATTTGTAAATAT

ACTCAATTTTTAAGATCTGTGTAG

TTTTTGAGATGCATAATTATAAATACTTAGCTATAACTATAATTCATTGGCACCAC

ATATAGAGCATAAAATGGCAATGC

CTAGAATTTGGCATTTATCATTATTCAATATTTAGTGTTTTTCATCAAAGTAATATC

TTGCTGATAGTTTGAAAAACAAT

CAAAAGTGAGCCTCTGCTTTCCCACATGTGGCCTGCTGTGAATTTCATGACAAT

GTGTCATGAGTTTGGGCATTTGTTAT

GCTTCTGCACAACTCCCTTGCTGGTCTCTTTCAAATGCCAGCCTCTCCATGTGG

AAATCTTCCTACATGATGTCTTTGAT

ATCTCTTCCCCTCATTGATTCTTTCTTTCTAGAACTCTTAATGGTGTCTGCCTGAT

TTCCTGAATTGGTCTTTACTTAAC

GTTTCCTATACTTTCACATACTTCTCACCTCCTTGCCTTAACTTTTTAAAGAGTTA

ATCATACCTTTCATGGATTACAAA

ATAAGCTATTATCCTCAAAGAGTCAAAGTTTTCTTCCTCTTGCCTGTTTCCAGAG

CTAAAGCCCACTCCATTGCTACAAG

TCAGGTTAAAATGGTCTTAACATTGAAAAAATTACCTCTCTGATATTATAATCATA

GTATGCATTCTAGGTTATCCTAAA

TCATGAAAAAAAGTTCTGTAT TAAATGCTAATTTAATTTTTGACTATAGAATTAT

ATTTTAAATATTAATGGGTGATT

TCTCCCCCAAAAGTCTTCAAAATTTTTCATGTTCCACGTTTTTTTCCTTTTAAAAA

TAAGTATGCAGACATTGCTTTATT

CTAAAGTTTCATCATTCTTGTCTTTCAATTAACTTCTAATGCTCATTATTTCTGCT

GCACATTTTAACTTTAAATATTTT

TGTCTTTGATAATTGTATTCCATGTATCTTTATTTTCCAAAAACTAAGCTGATTCC

TCATTCAGTTTACATGTGTACATA

CAGTTTTCATAGAACTGAGTATACATATCTCTCTCTTCCTTTAATTAGATTTCACA

TCTATATAATGACACTGAAGACAA

ATTTTATTATAG TGCATTGAATTTATTATAGGAGTTTTTATAGAACTTTCTCTAC

TTACATAAACTTCATATAGGCAT

CATGAGGAAAGGTAATGGCTTGTCCCAACTTTAGGGAGATCTTGCCTTTTCATTT

GAACCACCTGTGTCATGCAAGCTTC

CTGAAAACTAAAAGGAGTTTTGAAATGAAGATCATTATGTGAATGAGATGTAGCA

AACCTCTTTTAGGATCTAGAGAGTC

ATAGAAAGAGAAGAGGAGACAGCGAATAATATTATTTAATTTCTGATATTTTGGC TGTGAATACTACATTGTAAATAAAC

TGTTGAAGACTTAATTGTGTTTCTTAAATTAAGGTTGATTAGAGCTTTTTTGAAAA GGCCATTTGGTCAGATATAAGTGT

TTGTCTCTGAAACCAGACATTGTTCTAAGTGAGTATTTTTCCTAAAAGGGATGCT ATGTGAGAGAGTGGGGATGGTGGAC

TATAAAGCCAGGCCACATGGAAAACAATGAAAAATTCTAATTTCAAAATCGGTCC ATGGTTTTAAGAGTGAATGATGTAT

TTTAGCTTGTATTGTAAAAGTAAAAGTGCTTAGTGTTTTAGGTACTTATTTTAATT TCATTATCTTATGGGAAAAGAATA GGAATGAGTTGAACCGAGAAAAAAATAATACTTTTCAGATTTCTTATACATACCTC

TTCCAACTAAAGCGAGTATGTCTG

GATCAGCCTTTCTTGTGTTGCATAATTAGAAGTGGAAAGTTCACTGAGTTTCCAC

ATTGAAAAAGAAATGGTTTTCTTTA

AAAATTCAAGGAAGATCTAGTCTGGGTATTTTGTTTTCATTCTTTAATTTGTGAAA

AGACCGCAGATTCAGGCTAATTTT

TTGGCATTAACTTGTCTTTCTTAGTAGTCGTATATAAAATAATAGACTTACTATTC

TTGATGAAAAGGAAGTTTTGATGG

TAAAGTACATAAAACAGAAGAATTTATTAAACTAAATAATTAGAAATGACTTATTAA

AAACAATTTCTGTTTTGAGGTAG

AATTTTTTACATTGTTAAAATAATGAATGTCTCTTTTTGTTGGAATAATTTCGGGT

CTTTTTTTTTTCTTTTACTTTTAG

TCTATTGGGTTTAAAGTTAGTATTTTATAATACTGGAGAGTTAGACTCTAGTCATT

TTCTTATCTTATGTATGACCTGGT

TTCTGCCTTTCAATTTCATTATATTATGCAGTAGGGACAAATCTAGGTATATGTGA

TATTTATCTCATGCTTCATGATTT

CTGTTCCTGCATTGTTCTTAGAATTGTTGTGTCACAATGGACATATAAAGATAATT

TTGTAAGGGTTTTTCAGGGCAAAC

ATGTAAGGACTCATTTGTGGG I I I I I I GTGCCTTTTTAAAGTTACTTTTTAAGTCT

GTGATGGTGTGCAGTATTACTTGT

AAACGTAATTCCTTATGAATTTTGAGTTCAAAAATTATTTTAAAGTGTTCATAATG

AAATATTCAAAGCTTTATTACCCT

TTGGATGGATATTTTAATACGTTAAAAATAATATAACTTGAATCTGAAAGCTTCCA

AAATTTTACTCTATTTTAAAAACT

AAAAACAGTGCCTTTGCATTCATTCTTTTAAAAATGAATTTGTTAGCTCTTTCTTC

ACTATCTTAATTAAAAAAATTCAC

ATAATCTGCAGATATGTTAGAATCTACCTTGAGAAATATAGATATTTTCTAGTCAA

TGAGATTTTAATTTATTTATTGCC

TACTCATGCCTACTCTTCAACTAGCCATGCCTTAAACTCATGCCTACTCTTAAAC

TCATGCCTACTTTTCAACTAGCCAT

GTCTTTAAACTCATGTCTTAAACTCATGCCTACTCTTCAACTAGCCATGCCAATC

ACCCCTCACCGCCAATTGAAGGACA

TCTTGGTTGCTCTGAGTTTCTGGCAGTTATGAATGAAGTTTCTATAAACATTTGT

GTGCAΘI I I I I I I AATAGATATAAA

TTTTCAACTCCTGTGAGTAAATACCTAGGAGTGTGATTGCTGGATCATATGGCAG

CACTACGTTTAGCTTTAGAAGAAAC

TTCCAAATCATCTTCCAAAGTGGCTATACCATTTTGTATTCTCATCAGCAATGAAT

AAGAGTTCCTGTTGCTCCATATTC

TTGCCAGAATTTGGTGTTGTCAGTGTTTTAGATTTTGGACATTCTAATTGAGAAG

TAGTGCTATCTTGTTGTTTTGTTTC

CTAATGACATATGATGTGAAGCACCTTTTCATATGCTTATTTGCCATCTGTATGT

CTTCTTTGGTGAGGTGTCTGTAGAG

ATATTTTGCCCACTTCTTAAACAGGTTGTTTGTTTTCTTATTGTTGGGTTTTAGGA GTTCTTTGTTTATTTTGATACACA

TCTTTTATCAGATAGGTGTTTTGCAATATTTTCTCCCAGTCTATGGATTTTCTTTT CTCTTAGCAGTGTCTTTTGCAGAG

AAGTTTTTAATTTTAATACTCCAGTTTATCAGTTTTTCCCTCATGGATCATGCCTT TGCTCCTGTATCTAAAAACCTATT GCCAAAACCAAGGCCATCTAGATTTTCTCCTGTGTTACTATCTAGAAGTTTCATA

ATTTTGCATTTTACATCTAGTTCTC

TGACACATTTTGAATTAATTTTTGTGAAAGATGTAAGGTCAGGGTCTAGATTCAT

TTTTTTGCATATGGATGTGCAGTGG

TTCCAGATTAATTTTTGAAAAACTATCTTTTCTCCATTATGTTGCCTTTGCTGCTT

TGTCAAAGACCACTTGACTGTATT

TAAGTGGGGCTATTTCAGGGCTCTCTCTTCTGTTCCATTGATCCATTTGTCTATT

TTTTCACCAATACCACTCTGTCTTG

ATTACTACAGCTTTATAGTAAGTCCTAAAGTCAGACAGTGTCTGTCTTCCAACTT

TTTTCTTCTCCTTCAGCATTGTGTT

GGCTATTCTGAGTCTTTTATCTCTCCATATAATCTTTAGAATCAATTTGTCAGTAT

CCACAAAGTAAATTTCTGGGATTT

TGGCTGGAATCACGTTGAATTTATAGATCAAATTGGGACAAACTGATGTCTTGAC

AATATTGAATCTATCCATATGCATG

GAATATCTCTCCATTTATTTATATCTTCTTTGATTTCTTTTATAGTTTTTCTCACAT

AAAGCTTATATATAATTTTTAGA

GTTACACCTCTTTCTCTCTCTCTCTTTCCCTCTCTAGTATTGATGGAAATAATGGT

GTGTTTTTTTTGTTTGTTTGTTTG

TTTTTTGAGACGGAGTCTCCCTGTCGCCCAGGCTGGAGTGCGTGGCGCTATCT

CGGCTCACTGCAGGCTCCGCCCCCCGG

GGTTCACGCCATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGCGC

CCGCTACCTCGCCCGGCTAA I I I I I I G

TATTTTTAGTAGAGACGGGGTTTCACTGTGTTAGCCAGGATGGTCTCGATCTCC

TGACCTCGTGATCCACCCGCCTCGGC

CTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCCGGCCGGTGTGTT

TTTAATTTCAAATTCCAACTGCTCATT

GCTGGTATATAGGAAGGCATTTGACTTTTATCTATTATGTTATATCCTGCAACCTT

CTTCATTGGTTTCAAGAGTTGTTT

GGTTTTGGGGGTGGGTATTTGTTGA I I I I I I I I I TTAATATTTTCTACATAGACAA

TCATGTTTTTTGTGAAAAAAGACA

GTTATATTTCTTCCTTCCCAATTTGTATACCTTTTATTTTCTTATTGCATTAGTAAG

GACTTGTACAATGTCGAACAGGA

GTGGTAGAGGGGACATCTTTGCTTTGTTCCTTATCTTAGAAAGGAGACATCTAG

TTTCTQGCCATGAAGTATAATGTTAG

CTGTAAGTTGTTTTTTGATGTTCTTTGTCAGCTTTAGGAAGCTCTCTCCCCTTTC

CTAGATTACTGAG I I I I I I IATTAT

GAATGGGTATTGGACTTTGTCAAATGCTTTTTATGCATCTGATGATTTGATCATAT

GGTTTTTCTTCATTAGCTCATTGA

TGTGATGGATTACATTAATTTTCAAATGTTGAACCAGCCTTATATATCTTGGATTA

ATCCTACTTGGTCATGACATATAA

TTCTTTTTATACATTGTTGGATTCAGTTTGCTAATATTTTGTTGAGGATCTTTGCA TTTATGTTCATGAGAAATATTATT

CTAGTTTCTAGTTTTCATATCTTGTAATGCCTTTATCTCATTTCAGTATTAGGGTA ATGCTGGCCTCATTGATGGAATTA

CAAAATGTTCCCTCTGCTTCTGTTTTCTGGAACAGATTGTAGAGAATTGTAATCA TTTTTTTTTTTAAGTATTTGATGAA

ATTTACCAGGGAACCCATCCTGGTTTAGTGCTTTCTGTTTTGAAAGATTAATTGT TGGCTCAATTTTTTTAATAGGTACA GACCTATTCATATTATCTATTTTTCCTTCTGTGAGTTTGGGTAGACTGTCTTTCAA

GGAATTGGTCCATTTCATCTAACT

TTGTCTAATTTGTGGATATAGAATTGTTCATAACATTTCTTTATTATCCTTTTAATG

TTCATGGGATTGCTAATGATGGC

TGTCTTTCATTTCTGATATTAGTAATTTGTGTATTCTCTGTTTATCTTGGTTAGCC

TGCGTAGAGGTTTATCAATTTTAT

TGATATTCTCAAACAGCCAATTTCATTGATTTTCTCTGTTGTTTTCCTGTTTTTAAT

TTTATTAACTTCTCTGATTTTCA

TTATTTCATAATTTTTTTCTGATTGCTTTTGAGTTAACTTGCTTTTCTTTTTATGGT

TTCCTAAGAGTGGTGCTTAGACG

ATTGATTTTTAGATCTTTCTTTTCTAATATATGTGTTCAGTGCTGTTAATTTTCTCC

TGAGCGCTGTTTTTGTGGCATTT

CACAGAATTCGATGGGTTCTGCTTTTTCATTCTCATTTACTTCACAATATTTTAAT

ACTTATTTTTGTTTTTATTACCAC

TGGAATCCTCCCTTTTAATGATGTATATATTTATAAACATTTTATTGGAGAGAAAA

ATCTTAGAAATTTCATAGTTTACA

GTAAATATAATTAGTTACAGTTACCCAGATATTAACATCTTACAGATTAAATGGCT

TTCTCTTATTAGAAGATATGTGTA

ATTTATGAGTAGAAGGGTATTCCATGGTAAATTCAAATTTCACTCTCTATAAAATA

AAATTTTGATCAGAACCAACTAGT

TTTCCTTGTTCAATCAAAGACTGTAAGACAGTGTCACAATGAAATCCATGATTAC

TTTAAATAATAAATTACAGATTTCA

CTCATTTTTA ITl I lAAAAACCCATCTTTTACAAAGCCAATGATTTTTTTTCTATGA

TCAAAATAGAGGAAGGGAAAAAT

AAATTTGAAGGTGTTTTCCTTTTGTGCTAATATCTAAACTAGCATTTTATC ATC C C

CTAGCCACTGAGGTATAAAGTAAG

TTATCATCTTGTACTGATCGTAAAAGCTTAAGTCATATTACTACTAATCTAACTAA

ATATATTCTTTCAAAAGAGTGATA

AAAATACT I I I I GAGTATTATTACGAAGAAAATTGTAATTGACTTTTCTCAAAAGA

TCACTTAAACCA I I I I I ATCATAT

GATATTTTTCATTATATGATTCATGTGTATAATCAAAATTTAAACAATCATCTATAA

TC C AATTACTGTGCTGTATTTAA

ATAATTTGGCCTGACAACATATTTTTTATCCCAAATCTCTTAGAATATCTCATAAT

TTTGATAGCTATCATCACAATACA

TAATAAAATACAGTCGAACACAATTGTTGTGCTTAGAGCTCTACATTCATTACCA

CATGTAATCTTACAATATACCATGA

GATAGGTACTATTTGTTCCATTTTACAGATAAGGAAAATGAGGCACATCACTCTT

AAATGCTCAATAAGTGTTAGGTATC

ATTAAGTTAATCAAGAATGTTGAGGCTAAATTCTTAAAAAAGCACTATTTAGCTAT

AATATTGTA I I I I IAAAATAATAC

AAATATAAAGCAGTGAGGGACAAAGATAAAAGTGAATGGTGAACGAAACATTTTA GATTAATGTGAAATAAAAGATAATT

AGTTTTTAGGAATAAGATTTAGTATGAATGCCGTAGAAAAGTTTGTTTTTCAAAG GATTTTTTCATTTGAATTTCTTACA

TCAACTTGATGTTGAAATCTACCTTCACGTGTGCTGATCACATGCAGACAATTTA CTGGTTAAGGTGTTGGACGGGAATC

TTGCCGATATTGAATTTCAAGTGACAGAGGAAAAATCAGTATAAGAGGGAATTCA ATATGCATGGGAAACCATTTGTTCC CTCTGCTTCTTTGATCCCTGCCAAGGCACAAGAAAGTAGAGCAAAGCGCCTGAA

AAAGGGGAATGTTCTTGTTCAAAGTT

GCAGAACTCTGAGCCCTCAGAGGAACTCAGTGTGACAGGTTAAAAAACTAAACT

CCCGGCTATCTTCCCTGTCATGCAGG

ATATTTGAGTCTCGTCTGTACTTGCTAACATGCTTATGTCTGTTCATTGTGGAAA

TGATCCTGAGGTTTGCAATAGCAAG

CGTCTTGATGCTTCCATTTTACTAAGTGAACGTTTTGGAAAATCTGCAGGCCATA

CTTGCTCATTTTCACAGATGCCTTG

TGGACTTTGGAAAAGTTTGCAATTTGTTCAGCTCAAAAATAAACTATTTCAGCAAA

ACAATTTTAACTAATGTTTTGGAA

ACATTTGTCGGCAAAACAAATATTTGTATCTGAAATAGCTAGGCTATTAAGATGT

GAGAAAACAAAACAGATGTTTGTTT

TGGGTTGTTGGGTTTGTTTGTCTTGATGGCATTTGTTTTCTTTCTTTCTTTCTTTC

TTTTTTCCCAGAGGATATATAGTG

GTATAATAAGATTATAAAATTCATGGAAATAAAGTTCACTCTTTGTTTTGTACTGT

GAGAAACGTTATGAAAATGAGTTT

TGTTACCTGTGAATTTTGGACTGTATTTCCAAGAAAAGAAAAATTCAGTCTTCTAA

GCACTTAGCCCTTTTGTGCCTTGT

TACTGGTTTCCCTAA I I I I I I CAATTACTTAGCCATATGTCAGGATGCCTTATAGA

GTCAGTTTTAGTACACTGGATTTA

AATATGTAATTCTTTCTTATATTTATACATAACAAGATTTTTATATTAATCAAAATAA

TGCATATATTATTGAGCTAATT

AAATTAGTTTATTAACCCTCCCAAACATTTATTATCTTCTTTTGTAACTAATTGATT

CATTATTTGAAAAACTGTTACAT

ATTAAGTGTCATATGGGACCAACACATATCAGTGTAATAATTTCTCGTATTTAAGT

TGGAGGACATCGGCATTTACTGGT

TCAGTTTTTTAATTGGAGATGGTTGTAAAGAGGACAAAGTTATGAGTGTTTGCCA

TCTCTTCACCTAATTTAATTGCGAT

TTCATCGTTTGGTAAGTGGCTTTTGGATTAAGTTTTAAATAAAGGTAGAACTGAT

GGATTAATGGGGCTACCAATGTTAT

TTCTATGTTGATAGTAAGTATTGTTTGATAAATTTTTTTACTGAATAATAACACATG

AACATGGGACAAAATTATAAAGG

CATAGATGTGTTTACAGACAAGAATAAACCTCCCTCCATGTTCTATACTCGGCAC

CTTGGTTTCATATTCTTTGCTTATA

TAAACATATACTTAACAACTGTCCCCCGCCTCTCTTTTTTTCCACATTCAAGTATG

TTTAGC I I I I I GGTCCTTCACAAA

TAATAGAGTACTATACATGCTGTTCTGCACCTTACTTTTTCACTAGACAATATACA

GAGAAAATTTCAGTAAGTA I I I I I

GTTGTGGGTTATATATACAGATTTAACAGTGGCTAATATTTTTTGAACATTTACTA

GTGCCAGGTATTGTTCTAAGCACT

GTTCATTCATTAACTTAGATAATTCCCACCCAGCTCCATGAGATAGAGTTGTATT

GTCATTGTCCTCATTTTACAGATGG

GAAAACTAAGGAAAGGTCAGTGACAACACTGAAATTAAACCTCTGGAAGCTTGA

CTTCAAACTTGAGTTTTTAATCAGTA

AAGCATGCTGCTGCTTGAAACCACTGTGTCTGAAGTTTCAAAGGAGCAGAAAAA TGAAAATGTAAACAGAGAGTCCTAAA

GGTAGTAGCTGCGTAGTAAACATCTTCTTTAATCAGCACTCAGCAAAAGCATATA TTATGAGGCATTCAGTTAACTATGT GTTAAATGTTTGAGCTCTACTTGCTGATAAAATAATGTAGGTCTGAAATTACAGG

GAAGAGGTATGTAGTATATTTTGGG

TTATTATGTCATAAGTATTCACTGAATTATAAAAGCAGTTTGTGGATATCTTGGAT

AATCCAACAGCTGATTGCTCCCAT

TCTCCCTCATTTTCCACTTCCTGAATCAGTTCACTCCGTGCTTCCCCTGTATCCT

ACTTAAATAGTCACCTGTCATGGAC

CTGCTTAGGGAACAACTTGCCACATCTCTACCATTTCCCTCCCCTTCCTTCCAG

GGACCTCCTTGCATCTTGTCCTCCTT

TTTTTCTTTATCTTTTAAAAAAAATTTTAAATAACTCAAATGGCAGCTCATGGATG

ATGAACACTAGGTGTAGGAATGGT

GACGATTCTATCAAGGAAAAGGGAAGCTATTCGTTTTAAAGTGAAGGTGGGGAT

CTGAATCCAATACCATCACCTTCTAA

CACTTTTAAAACTTATCTGACTCCTTCAGAAACTTCAGGGACCTCTTGATTAATTT

AAATTTCATTTCTTCATTATCATT

ATTTAAGATAAGTTTTAACTCTTTCAGTTTTATGTGACTGCTGATATTTAAGGGTA

TAATTCTCTGCTATTTTGCATTAT

ATTTGTGCAGTATTTCACCTGGATTATTGATTGATTAGGTAAGCATGAATTGGTT

TGGAAATCATGTCTGACATCGTTTC

TGTGAGAAAATAGCTTCTGAATTTCAAACAATTGATTTAAACATATTTAGAACGTA

GCATATTTGTAAATAGACTACTCT

CTTATATCTACAAATCCACTTGCGTAACCTTAGCAGGAAGCAGCTGGGCAATAG

GGAAGCTTTTAATTTTCGTTTAGCAT

CGACTGTATAGATTCTTTTGCAGAAGACCTTCACCTTACTGAATATAAATAAGGA

ATCCAGAAAGATTAAA I I I I I CCTA

AGCAGCACAGCCTGCCATTGGTGACATCTCTGAATGGGACACTAGAGTGTCAG

CCTTGGGAGACAGAAGGGTGTAGTGTG

GCCTTTCCCATACTTCCCTGGGTAGCACAACTGTCTCCCAAAGATGGAGATGAA

TATCCTTTTATCCTGTAATGAGTTCC

CAGGTGGGAGCAGTTAGGGATACAAGTTATCATATTTAAAGTTATTGGAGTTTCT

CTACTTTAAGTCTTTTCTGATTTTT

GATATGACAGTACTCATTGAGAATTGAGTGTAATGTCCAATGTTTCCCAGTCATA

CCTGGCTGGGGTTGGGTACCACTTT

CCACCTGTGCTTCCCCCAACACCCCATCTGAGAATGTCTATGAATGTCTCTTGA

CCAGCACCCTCCCTTTGAATACAATT

AGGAAAAACTGGAGTGGTGGCTAGGGATTTGGAGTTGGGCTAACCTGGGTTTG

AATTCTGGCTCTGCCACTCACTTGGGA

AGGCCACTTAGCTCCTTGGTGTATTATTTCTCTGTGGAATGAGCATAATACCACT

CTTGTGTATAGGATGACTATGAGAA

TGAAATGATGTAAGTGGCTAGCAAAGTGACTGATACATATTAGGCCCTCAAATGT

CCATTTCTTCCATTCGGGCATTCAA

GAGATATTTATCAATGCTTGCTGCATGCCAGGCACTGTGCTTGGTGCTGGGAAT

ATGGGGGTGACAAGAAAGGCAAGGCC

CCTGGCCTCAGGAAGCTGATGTGTTCTTCCCTCTTGAAGTTCATTACTGTTGTAC ATTGTGGATTGTGGTTGTTTCAGAC

ACTAAAGCCCCAAAAGTGTGGCTCACCAGTTTCTTTCATGTAGAATGACTGTGG GGAGCTTAAACCAGTGGTTGAAATGT

GTAAGAAGMT TTAGAGGTTTTTACCAGGTTATCCAGAGATTACTGGAAAGTG GATAATGGTCATATTTTAGGAGTGA TGAAGCTGTCTTAATTGAGTTACTAGACCTTGACTAATGAATTAATTTACCTAGA

GACCTCGATTAGTGCCTGCGTGAAC

AGGGAATAATGGCTATGGAATACAGGTAGAGAAGTCAAAGCAATTCTCTAAGAT

TAATTCAGTTAGTGTTTATTGGTCTC

GGACTACTGGTTATCCCTATAAGGCTAGTCAACAAGTTTACTTGCCTTTAGTTTA

TTTTACAAAAGTTTTTTGTGGGAGA

TTTCTATGACTGAAAACAAAATTATTACAATTCCTTTACATTAAATCTTTTATTCTG

CTAAGGTAATATTCACTGAGAAT

CTAAACTAATATAATGTAATTTATTCAAGGTTTTGCTGAGGTCCTTTTTTTTCTTTT

ATGCCAAAATCTAAAAAAAAAAT

TTAGGCCAAGCGGGGTGTCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGA

AGAGGGTGGATCACGAAGTCAGGAGTC

CAAGACCAGCCTGGCCAAGATGATGAAACCCTGTCTCTACTAAAAATAGAAAAAT

TAGCCAGGCGTGGTGGCAGGCACCT

GTAATCCCAGCTACTCGGGAGTCTGAGGCGGGGAATTGCCTGAACCCAGGAGG

CAGAGGTTGCAGTGAACTGAGATCGTG

CCACTGCACTCCAGCCTGCATGAGAGAGTAAGTCTCTGTCTCAAAAAAAAAAAA

AAAAACTACCAATATGAAATTATCAT

GGTCACACTATCACACGTAACTTTGAAGGTTTGATGACCCATAACTTCATAGATC

TGTGTGGATCCCAGGTGA I I I I I GA

GGAATATCATTGGCCTTCTTTCAGGACTGTGGCCTTCTACGTGGGAATCTTCCC

TGATTGGGCCTGTAATTTTCATAGGA

AAATGTCTATTTCTATTCTCAGCACAGCTAACTTAGTAAAGCCTATGAGTTTCCC

CCCACTCAACCAAGCAGTAACACCT

TCGCATACCCTCTTACCTCACTGCCACTTAAAACTCAAAGGAGAAACTTGTTATT

GTCAGCTGGCATAACAATATGTCAT

GGCTCCATGCCTCCAGATATTTTCAGTTTTACCCTTAAACAAGATGCCTGATTCC

AGGGTTCTGCACTCTGCAGTAGTGA

ACTCCAGAGTTACAGAGTCAGCAGTAATCAGCCTTAGAAGAAAGAATCTTGTGA

CACTTGCTGTTTTATTTACCACGAGG

GTGTGCTCCTGGGATTCCGTAGCATTCCTCAGTGAGCAGTTCTGTGAGGTGTTT

C CATC CCATCTCTATAATGGGAACAC

CTACA I I I II l ATTTTAAGTTCCGGGTTACATGTGCAGGATGTGCAGGTTTATAC

CTGGGTCAACGGGGCACACCTCCAC

TATTGTGGAGAAGTTGTTTGTCACATCTCATGCACACCATGAGTCATAGAACTGT

TCCTGCCTTTGCCCTGCATGGACTG

CAAGAAGACCCGTGTGGTCCATTTCCTCACTGGCTCCATTTTCCCCTTCTGATC

CACATTCTTCTTCTAAGGTACTCATT

CATTTTTTAAAAAGTTGACTGTATAGTTATTATGTATATATTTGGTGGGTTATCTT

AAACCCTTCTTTTAGAAAAGTGGG

ATATCAATTATTCAGAAACTAAAATTAAGAATTAAAAGGTTGTATTCAACAAGAAT

AAGAACCAGAGAACAGACATGCTC

AGGAAGAAATTACCTTGCAAAAACAATAAGAACCAGAGAACAGACATGCTCAGG AAGCACTTACTTTGTTAACAACAACT

GTTTTGGAAACTTTTTGGTTAATATTTGCACAAAAACCTGATATGCACAAAAAAAG CCTTTTGGGTTAATATGCACTTCA

TGTTTTCATATAACTGTAGTCTCCCAAGAGCACATGCCTCTTTATCATTTCTTCTC TTTGCTCAAAGTTCCAAACCTACT TGTATAATGAATAGGCAGGCCTGTTATTCGTTTCAGTTCAGCTATTTCATGTATTT

TTTGAAAAGCATGCTGCCCATGGT

AAAATCTCTCCCAATGTAAGGAGTACACTTTCTCTGTCATGAATATTGTTTTCAG

CTTCTGCAATTTATTTTATTTCCCA

TGCCTACCACTTCTTAAATGTTCTCCCTTCAAACATTCTAAGAATTACAGGATTTG

CTTGGAGTCTCTCCTTTCTAGTTT

CTC ATTTAGTGTTTTTCTTAAAGTATATGTTTC C CTACACTTGTTCATCTAATTACT

TGCCTTGATTCAGCTTTTATATT

GATAAAGGTTACTAAGAAAGCACACTTCTCTTGAGATATATTTCTTAATTGTGCTA

TTAAATTGCATCCCCTGGTTCCCT

AATTGAAGGCAAAGTAGAAAACGCAGTTTCTCCTACTGACATATGGAAATGAGAT

TTTAATGATGTTCGTCAGCGTTTCA

TTACCAGCCATTGAGCATCTCTTACTTTCAAAGTACCATTGGGTTCTATGGAGAT

ATAGAAGACAAGAGGGTCATTCACT

GTCCTCAGGAGCATTTTGCCACTAAAGAAAACTATGTGTACAGCCAAACCTAATA

AAATTTCCA I I I I I AAAATGCCATG

CTAGAAGAAACAAAGTCCTCAGGAAGAGTGCAACAACAGATTCCTTACATAAGT

GTTTGAAGAAGAAATTAACAAAGAAG

GTGAAATTCTCAGCATTGGAGGATGTGAAAGTGCGAGGGACAGAGGATAGGGA

AGGGATATTTATTTGAGGCTAGAGAAA

TAGACCACTTTAAATGGGCTCAATGACAGGATATCCTTTCCATCAGAATTCGGTT

AAGTTATATTTAAATTTTTTAAAGG

ATGAAAGCATTTTTGAACTGGAATATCACTTTATCATCATCTTTACAAATAAGGTA

ACAGCCCAGAGGGGTAATTTTGAA

CTATTGACTTAGAGCAACAACACAAATACACTTGTATAAAACACTTTAGCATGCAT

TATTTGATGCAATCTTGACAATGG

TGCTGTTCCCATTTTACTGTTGAGGAAACTGAAGTCCAGCAAAAGTCCAGATAAC

TGAATAAATTATAAGCTATCAAGGC

TGCTCTGGAATGTATGCAATCCTAATGTGTTAATTTGTAGGTTATTCTGTCCACT

ATGGTGAAATTTGAATGAATCCATT

TTCTGTGTTATGCCCTGTAAGTTAGAGAGGATATGCAGATGTACCAGAATGTGC

AAGATTCAGCTTCTCTGCAGGTGGGT

TATTTTCCCAAAGAGTGAAAGGAAAGAAGGTCCCCTTACTGGAGTCAGTGAGGG

TTCCATCACCCAAGCAGCCAAGCTCT

GGTTGTGGGCAGCCCCTCTCCTTTAGGCCCAAACCTCCAGAAGAAGGTGGCAG

CCAAGGGTCCCCAGGCAGGTACCCATC

AGAGGCTGCACAGCCTGTCTCTGGCCTCCTCATTGACAAGTGAGACTTACGGCT

TGGAGAAACAATGCTGTTTCTGAGGC

CAGTCAAGTCTGTAGGGCGTCTGAGGTGGGAGCAGGCAGGTGGAGTTGGAGG

CTTTAGAGAAGACAGGCCTGGGTTCAGA

TCCTGATATTGGTACAGCCTAGCTGGCAGACCTTTATTTTGATTAACTTCAATCT

TTTTATCTGTTAACTGGGGACAGTA

ATACATCCCTAAACAGATCTGGGTAGAAATAATCCTAAACCTAGAAGAAGCCCTG TTCTCTCCACTCTTCCATCTCTCAA

CCCTGACCCCAGCCTCAAGTTTCTTTTGCTGTGATATGTCTTGCCTGGCTTTGC ACTGTTTCAATCAGTTGAGACCAGAG

GATGGGATAGTGAACCAAGTGTGTTTGAGTTCTCCTGTCAGAGAAATGCTGTCC C CTAATGAACATGAAGAC ATTGATAA GTATTTCCCCTGTGACACATCCCTCAAGAGAGAGGCTAAACAGACCCTTGGAGA

TTATCAATGATCATGTGCATTTTGCT

GTGTGAACCAGAGATGCTCCACATGCCAGGTTGTGGAAAAAAAAAAAAAGTTTA

ACGAGCTTCTGATATGTTCCAGGCAC

TGTTCTAGACATAGGGGATTCTGCAGTGAGCAAAAATCAAGCCTCTGCCGTCAG

GGAACTTACATTCTATTGGAGGCACA

GATGATAAGTACATATAAAATGCATCTGGCAGAGTGGCTGGAAGGATGAACAGC

TGCCTAAGGAGAATAGCGTTAGGGCA

GGTAAACTGTTTCGGAAGAGTTGTCCATCGGGTGACACTTGAGCAGAGGCCTTC

AGGAAGGGAGGAGAGAGCCACCTTAA

TATCTGGGGAAAGAATTCTGGGGAGAGGAAACAGTAGTTGCCAAGGCCTGGAG

GTGGGGCTGTGCTTTTTGGATTCAGCA

AGAGCAAAGAAGTCCCATAAGGCATTGGAGCCTCTGGGAAAGGAAAGTGGGAG

GGTAAAGGCAGGGAGGAAGAGAAGCTG

TAGTAGAAGGCCTTGTGGGCATGAGAAGGATTTGGGATTTTATTTTGAATAACAT

GAGCAGTGTTTAAGCAGAAGCATGG

CATGATTTCTTACACCTGTAAATAATTTCTCTAGTCATGGACAGAGAATAAGTTG

CAACAAGGCAAAAAAAAAAAAAAAA

ACAAGGTGGTGTTGAAGAGATCAGAGCTTCTGCTTAGCACATGTGAAGTTTGAG

CTGTCTGTTAGAAATCTAACAGGAGG

TGGTGACTCAGTCATTGGATAAGTAAGTCTAATGTTTACAAGACAGGTTAGGGC

CTAAACTTGTGAGTCTTCAACATCTG

GAAGCCACTGAAAGCTGTGTGTGTGGATGAGTTAACCAGGACTGTAGGAGCAA

ACACAGAAGAGGAGACAAGCACTGAGC

CCAGGGCCTTCCCACGTTAGCCAGCTGGGAAAAGAGGAAGAGCCAGAGAAGGA

GATTGGGAAAGAGCACCCAGGGAGTCA

GGAGGAGAACTGAGAAAAGGGCCTTTCAGAGGCCAAGGAACACAAGTGTTGCA

GAAGGAAGCCATGATCAACTGTGTGTG

GCGCTGGCAAAAGGCGGAGAAATATGGAAATTGAGACTTGACTATTTGATTTGA

CAGAGTGGATGTTGTTGGTGATCTTG

ACAAAGAGGAACATTAGAGAAAATAGCATGATGGAAGTGGGTTCAAGAGAAAAT

GGGATAAAAGGAAGTTTAAGCAGCAG

GGATTCTCAATTCTTTCAGTGACTTTTGCTGTACACTGTAAGGCAGTAGAGAAAG

AGGGCAGAATCTAGAGGAAGTGGAA

GATGCTTTTTAGGCAAGAAATCTATCTAGCTTGTGTGCACCTGCAGGTCATCTAG

AGTAAAGGGAACGTGGACTGGAATC

AGTGACAGGGCACTGGAGTCTCATGCCAAAAGGAGGGGTTGGCCTTAGGAGTG

CAGAAGGTCCACCCATCATAACCTGAG

GGGAGGCAGACTGCATACAGAGTACTTACGGGTGGGGAGAGCCTGGCAAGGA

GAGTAGAGAGAGTGGTTCCTTCTGTTTT

CTCAGTGAATGGGAAGCCAGGTTCCTTGGCAATGAGAGAGTAGAAAGCTGTCC CTTGGTATTGCTCATGACACCCCTGGT

GCATAATAAATATAGGCAGCGCCAATATTCTACAAGGTCATGGACCATCATCCTG CTCAGTCACATCCCTGGTGCTGAGT

GTGTTGCCTGGCACATAGGTGCTCTGTGATACCACAGAATCCAAAAGTAGTGCT TGTTGCCCTGGCCTAGGAATTTGTGA

TAGCCACAGGCAGGACCAGCTGCAAAACTGGTGGGCCCCTTGTTCAAAAATAAA GAATTTTAAGACAGCACAGCAGACTA CTGAACCAAGTTGCAGGCCCAGCTGAACACAAAGCCCTGTGGGAGTGCACAGG

CTGCACCTCCAGGAGACTGGCCCTAGC

CACAGGTACTTGTTAAATCTGATCAAACCCCAGTGATGCAAGAATCACAGTCAG

CAGACCCGTGGCAGTGGCCAGAGATA

AAACTCCCTGCAGAGACAAATTTTGCTTATTTTCCTCTTTCAGATGTTTAAGACTG

AGTGCAAGCAGCAGTCACCATCTT

CACAGATTAGCAGAGATGGAGGTTTAATGATTCTTGTAAGAAAATGAACAGAAAG

ATGCATCAGAGATATTCAGAAACTT

TTAGGAGCAAAATGTTCACTAGTGGTATCTCTTAAATGACGATACCACAAATCCC

TGCTGTAGGAAGAGAAAGGTGCCGA

ATGGTGGGACAGAGGGAGCTTCAGCAGTGTTGGAGGTTGCAGCCCATTGTGAG

TATGAGGTGGCATTAATGACCATTCAA

GAAAAAAACATTTTCAGACAAAACAAAATAACATAAGAAAACCTCCCCATTCATTC

CCAAAACTGCAAAGGTAAAGAGTA

GTATGGGTAGATTAGATCCAGTGCAAAGGTAAGATGACCTGTTTTATAGCTACTT

CAAATTGAGTTGTTTCAAAGATTGT

TTTTAAAAAACTATTATATAGAGACCTTTTTAATGGAGTTTTTCACTGGGCATGGT

GGCTCATGCCTATAATCCCAGCAC

TTCTGGTGGCCCAGGCAGGCAGATCGCTTGATCCCAGGAGTTTGAGACTAGCC

TGGCAACGTGGCGGAACCACATCTGTA

TAAAAATTACAAAAAATTTAAAAATTAGCTGGGGATGGTGGTGCTCACTTATAGA

CTCAGCTACATGGGAGGCTAAGGTG

GAAGGATCACCTAAGCCCAGGAGGTCAAGGCTGCAGTGAACTGTGATTATGCC

ACAGTATTGCAGCCTAGGTGACAGAGT

GAGACCCTGTCTCCAAAGAAAAAAAAAGCAAAACAAAGTTTTCCAACATTGTTCT

CCTTTTATCGGAAAAAAAGCAAGTC

AGCTGAATGAATGTGTGCACATGTATACATTCCATGTTGTGAGCATTTGGTTTCA

GTGGCATTCAGCCACATGAGAATTC

AAGCGTGAAGTTTAGGCCACCACTGGAATTGCTTTAGACATTTTAAGAATAAAAG

GAATTTTGGTATTCACTCTGCGGAT

ATCTGACTGTAGAGGGGGAACCTTGGCCAGCCCTTCCCTCTGACTGCCTTACA

GGAGAGGAGAATTCATTTTATGCAATG

TTGTCTAGAGGATTAGAGGTGGGTTTGTTAGAAATATCTGTCATATTCAGCTATG

GGAGG-TCACTTTAAGTCAGAGAAAA

TCTAAGTGTTCATTGTAAAAGGCCTACATATCCTTTTTTCATTCACCTTTTCACCT

GAATTTCCTGGTTTTCTTACATAG

TAAACAGCATGGCTTGACAATGGTAGGGTCTTCGAATTTTTCTTTTCTCAGAAAC

TCAGCTTAGCTCATTTTCTGGTGTC

ACTGGTATGCTTGTCTGCTCAAGTAAGTTGAGTTTGTGTCCCCTAGATTCAGCT

GACTACAAAGAGCCTGGTAAACAAGT

ACGCCCAGGGACCCAGACCTGGGACAGCAGACTTGGTGCCTGCACCTGGCCAT

TATGTTAGAGATGAAATCAAAAAATCA

CTCACAAGATGTATAGTTCTGGTGCGTGTATCAAACCAAGCGGAATTCATAGCC

TGTCAAGCATTTCTGTGTGCTGCTAT

GACAGCAGAGTGGAATGGTTGCAACAGAGACCGTGTGGCCCAGAAAGCCTAAA ATATTTACTGTATGACTCTTGACAGGA

AAAATTTACCGAGCCTTGCTCTAGAGTATAGGAACTCTCCCCTCCACACACACAC AAAAAAGGAGAATTAAATGTTTATA GTGTTGGTAAGTTAGTCTCCGTGCCCCATCATATACCTGTGACCTGTACCAACA

CCGTCCAATGGGTTAGTTTCAGTGCC

TTTGACTTTCACAGTGCAGCTGGTGTAGAGAAGTTCTGGGTAATTTTGATTATAT

TTCTTTGTTTACCTTGTCCATCCCT

GGAGTAAGGGTCAATCCCACTCACTTGCCTTAAAGTAATTCAACTCAGCTTCACC

AGTGTTTTCCCCCATAGGAAAACTA

ATTTCAAAATCATAACCAAAAATATGCCCAATGGATGTATATGATTCTTTTGGCCA

ATGTCATCAAATTTGATGCATCTG

CTTCCCTATGGGGAATGCCACGGAAGAACTATAATATCCTTGCCTTCTTGACTTA

GATTTCAACCTCTCTACATTCTGTC

TTCCTGATCTGGCCTTGGTATACCATCTATTAAGATGACTTTTGCATATACTTATG

TTGGTTTTGAGAGTCTCAAAGTTC

AGGGGTGCCTTAGTGTCAGTTGTTGTCTGACATCTACTTCGTGCTCATGTGTAC

AATGTGCTTTAAGGCTTGAGTCTTGA

ACATGATGGAATCTACATTTAGGGAAGTGATATGCTCTGTTATTTTTATCTGAGT

CTCCCATAATGGCAAATAGAATATA

ATTATTGATTAGCCTGTCTCATGTTCACTAAATTCAGATAATCTTTCTAAGGATTT

CATGAAAAGCTCTGCACTTTGTGT

TGTAAGCTATATTGTCTTAGCAGACAGATTCAGGGTCTTGCCAGAGATCGTAGC

TACCCTGGCCTCAGTCCAATGTCATT

GTACATGTGGTGAGAGCTTCCTCCCGGCTAGGAGTTCAATTACCTACTTCTGTT

TAGGGAGAGTAGTCTTTCACCTCACT

TAGACTTCAATATGAGTGAGGAGGGCCTCCACCCTCCCAAAGCACGGAGAAAG

GAGCCTGCAGGAATTGTGTATTCTACT

TCCTTTGCAACCACTACTGGCATATGTAGTCCCTGGGGTGTAAAGCTAGATCAG

AAATCCCCTACTACCCTTAGAATAAA

TGTGAACACTTATCTCTAAATCTTCAAGGAGCCAACCAGGCCTTCATGTCGTGCA

GCAAGACTCCATAAAAATAAAATAC

CTCTGTGAAGGAAAGCATAATTTAAGGAAATATCAGGCTGGGAGAAGTATCAGT

AGATATGAAAAAAGGGTTAAAACTAT

TTTGTATAAATCAATAAAGGAATATTTAAAAACTGGCAAAGGATTTGGACTGTTCA

CAGAGGGAATATAATTTTTAAGCA

AATATATAAGCATCTTTAATATATGCAAATTAAAAATGATGCACTGTTCTGTGTAT

GATAAATAGCAATGACTTTAAAAT

GATGACAGCCAATGTATATATGGTTATGGTAAAAGAGACACAATTTAAATTGATA

CAGTCCTTTGAAAACTATTTGGCAA

TATGTATAAGAAAACAGCGTATTAAGGAAGGCCTGAGAAATAAAACCACATAGGT

AATTTGGGCTAGATATAGTAGGCAA

TAAACCCTGATGGGTTTTGAGGGTTGGAGTTGCCTGAGATCTTTTATATAGATTA ATATGAAGGTAACTAATCTACTTAA

GAGAGAGAGAGACAAAGAACTAATCATTATGAGGCTTTGGTAAGTGTTGTACAT CCACAGGTAATAGAATTTTAAAGCTA

AATAATCTAGGCTGTCGTCAATTTGATAAACAGAGTGGGAACTGAGGAAAAAAT GGAAGTAACTTCCCCAAGGCTCAAGG

AGGAGTTACTAGCTCAGGGGAACTAAATGGACTCTTGCGAGTTCCAGTTCTTTG TTTTTCTTTTTAATATTGAAGAATTT

ATGGAAATCTGTGGTATTTACAGATGTAAAAATTTAATTATCTTATGTGGATTTTA ATATTTTATGACTTAATATATAAA CATTAACTTTTAAATGAGCTCTTAATGATAGCTATTTCTCATTTATAAATATTTTTG CTGTCTTGTAGAAATAATTAACC

TTTATAGCTTTAATAAATACTAGACTGAAGAAACATTTTTCTAATCATCAATTTTTT AATGTTTGCCTTGCACTCTAGAC

ACTTGCCATGCTCTCAGTGTGCAAACATGAATACCACTGTGTTCTGTTTTCCCAG AGTGGGAATGATGGACATGTGCTCA

AAATAATTTCATCAAATTATTTATTATCCACCACAAATGCAAATAATTGCAAAAATG TTATAATCCTATAATAGAGATAA

GAGTACATAATTACAAAGAAGGGACCAGAAGTTAGAGAATTCACAAAGATTCTTG AGTTTGATCTTCAGAGGCTAATAGG

AATTTCTCAGACAGCGGAGAGGGTAAGGAGTTGAGAAAAGGAGACAAGGGCCA GGTGCATTGGCTCATGCCTGTAATCCT

AGTGCTTTGGGAGGCTGAGGCAGGAGGACTCTTTGAGGCCAGGAATTCAAGAC CAGCTTGGGCAACATAGCAAGACCCCC

AGTCCTAGCTGCTTGAGAGCTTGAG

AGATATATATTGCAGGTCCCTTGAACCAAGGAATTTGAGGCTGCAGTGAGCTAT

GGTTGTACCACTACACTTCATCCTCA

GTGACAGAGAAAGGTCCTGTCTCTAAAMCTAAAAAAAAAAAAAAAAAAAAAAAA

ATGTGTGAAGCCATGAAATAGCATG

GCACTTTCAGAGAAATGAGATAATTAAGTTTGTCTATCTGAAGTAGACTATGAAG

AGAGGGTAGTTAGAGATAAGGTTGT

TGAAGGATGACTCGGTGACAACACTTTTTTCAGTTAGCTAACATCGTAGAGTGCT

AACGACATGTGCAGCTCCAAGTACC

TTTACATGTATGTTAGTTTTATTTTACCCCATTTATTCCCGTTTTACAAATGAAAAA

ACTGAGTCATGTAAAGGCTAAAT

AACGAGCAAGCAATACTACTAGTAAGTACTACAGACAAGAAGCAAAATGTGGCA

AGCCAATACATACCTAAGGAAAAAAA

AAACCAAATATCCCCAGATAAAAACTAGAAGGAAGCAAACGGTCAAACTCTAGGT

GATGGCAGTGCAGAAAATAGGTGGA

AGACGAGCCTATCGATCCTGGAAATAGCCTGTTGCAAGAATTAAGGCAAGATAT

AATGAGAGCCTCAACTTAGAGCAAAA

GTTTTTAGGAGTAAAAACCACAGAACTTAGTCTTAATTTGATGGAGACACTAAGG

AGTTTCTGGATTGAAAAAGTGGATA

GTTACTGGTACCATTAATTCAACTTGTTGATAGGGAGGATTCCTTTTGGTACAAG

TTTAATTATGGTGTCCATGGAATAC

TTGGGTAGAGAGGTCTAGAAATAGTGGGATACTCAGGTCAGGAGATCAAAAGGT

AAAGCTTTAGAATTTATCAGCTTGGA

GGGCATCAATTAAATTCCTCCTACAAATATTACCAAGCACTAAGAGCCTACAGTG

GGATAAGAAGGCCATGGAAGAAACT

CCAGTGCACTGCTGCTCAAAATATAGTCCATCAACTGGCTGCCTGTCCACTAAC TGTTTATTATTAGTGCAAGATAACAA

AGGATTTGCTTCAAAATGTAATCGACCATGTTACTAAGTAATCAATGACGTTACA TAGTGTTTTCTTCAGCTGACTATAT

TTTTAATATTAGCAAGACTTTTAGATGAAGGAAACATTGTGTTGATTCATATCCTG ATGCAAAATCTGTATCTTGTCCCA

GATCAGCACTTTGAATAACAGGGATGTAGAAAACACCAATATTCACACTTTGGTC AGAGCAGAAGTTCTCCAAAGAGACT TO

GATAAGGGATAGTGTAAAGCAGAAGTAAGGGAAGTCAAGATATTCTGAATTGGA

CATGATTTAAAGGAGCAAGTGGTCAG

CACAGCTGCAGAGGGGTCAAATAATATTGAAGACTAAGACAATAATTATTGGATT

TGGCCATTAGAGGGTCATCAGTAAC

AGTTTAGGAGTTTTACCCCCTGTTGTTTCACTCTTCTGCCTCCTTGAATCTGTAT

TATTGCCATGTAAACCAAATGCCTC

TTGACTCTAATCTAAAGTACTCAGATTCAGTCTTTATTTTACCTGTTTCTTGGGTG

ATCTGAAATTATTGATCACTCTCT

CCTTCCTGACATACTTTTTCATCTGCCATACATTGACTTTGGTTTTCCTCTTCCAA

CCTCCCCTGTCTTTCTGTGCCCAA

CCCTTGTACACACACTGCATTTCTTTAACAACACTCATTTCCTTAATTACATATGG

TGTCAGTTTCCAATCCTAAACCCA

GCCCATATTTCTTTATGGAGCTTCAGAGTAGTCTAGCAGCCTACTGGACATCTCT

ACTTGGATGCCCACAGCCCCTTCAA

ATTCAATATGTCCAAAGCGGAACTGAATTCTCACCTATACTCTTCCCTGTAATGC

CTTCTTCCTCCTAAGTCCCGTACCT

TGTATTACAATCTTCACCCAGTTGTGAAACCACTGACATGGGCATGATTTTTTAC

TGCATCTTTCTTCCTTATCCACTTA

GTTATTGATTTTACCTCCTAATGACACTTCAGATCTCCTTATGCCCGGCACCTGC

ATCACCATCCCTCACCTGGATCACT

GAATTATCCTCCTGGCCGGTCTCCCATTCTCAGTCTCCTCTCCAGCACCCTGCC

ACGTGGAGTGTGAGCCCCCACTCCGT

TGTTGAATTCTTTACCTGCCACTCCAGCCTCATCTCTGTGTTCTTTTTCTGTAGA

CTCAGGTCAGATTGGATTTTTATTT

TCATTTCTTAAATGTGACATGCGCTCTCGCACGCGCTTGCGCTTGTTCACTCGC

TTGCTCTCGTTCACTCGCTCTCATTC

TCTCTCTCTCGCTCGCTCACTCTCTCTCTCTCGCTCGCTCACTCTCTCTCTCTCT

CCCACTTTACCTTCACACATACTTA

TGCCCCTGTCTGGAACACAGTCATATTCCCTCTTCACTGGTGCATTTGTCCTCT

GTCTTCAGGAAGCTTCTGTAAGCCTG

CAAATCTGAGTTATGTGGACTTCCTCTGTGTTCTCCTAGCATTCTGCCTTTCCTG

TGTTGTGACCTTTCCACATTACTTT

GTAATTTCTCA I I I I I GTCCACAAGTCAATGCCTTCCTCTGAATTGTAAGCTCCG

AGAAGQCAGGAACCATGTCTGTTTG

CTACATCATTATATTTCATGCTCCTGGTACAGTGCCTGCATGGTGTGAACGTGAC

ATCCACTCCAAGTTTGTTAAAATCA

ATGAATGCCCTCCAAAGTATTTTCACATCCAGTATCTCAGTGGGGGTAAACTTGA

ACAATTTGGAGAGGGTTTTAGTGAA

ACATGAGCTGTCAGTGGATTAGAAGATTAAATGATTTGAATATTTGAGTTGTTTT

GCACTAGGAGGAGGAAGGAGAAGTT

AGAGTCTGGAAAGCTGGTTAAGAGAATAGAAGCATTTAATTCCACCTCATAGAAA

AGGAAGGGATTTTGAAAAATAACTA

GCTTGCATCACTTTTATTTTCATCTTCCTCCAGAAGTGGAGGCCTGCAAGGTAC GGGTGTGGGTGTATTTTAATATTTAA

AGGTGTCAGGTTGTCTATCTTTTAGGGGTTTTTTTCCCTTTACCTGGAAAAACTT TAGATAACATGATTTAAAACTTTCA

CTTTCTACTTTGCAGMTGMTTTATTTTCTTTTGGAAAGAGGCTTACAAAAGTAA CTCACTTCATTATAGCTAGTCTGT TTCCAAAAAAAGTGATAATAATAAAGACAGAATATATTTTAAAAGATTGAGTTTTT

AAAGTGGGGATAAACATTAGGAAA

TAAAGGAATGAGCCATCAATAGGAGGAAATCTAGGCTAACAAAATCTAGGAAAG

ATAAACGACTTTTACGACAAAATTAT

ACTGTCGGTAAGTCAAGGAAAAAGTTGTAACATGCTAACTTACATTGCTCGATGT

CTTTTAGCTTCTCTTCAAATACTTA

CTTCCAAAAGCCAGAGAGCTCCCAATTCAAGAGAGACTTTGTAGTAAAGTACTT

GAAGTGCAGGTTGAATTAGATGGTGA

CTGTACTGGTTGTAAAATGAATGCTTGGTATCGAAACATATTAGAGATTGATGTG

TGTTTTGTCTCAGATGGTGAATATC

TGGTTCTGCATGGAGACGTACATTGCAGAATATTTTTTAAAAAGGAAGGCCAAGA

CTTTTTCCTCAAAGAACAGCTTTGA

ATCCACTTGACTACTTAAAATTCTACTCATTTGGAGCTGCTGATTCTCTTTAAAAC

ATGGAAGAGAGATGCTCAGTTTGG

AACATCTTAAGGAGAGGATGCCCTAGTTATCTGCCTCTGTAATAAAAGACTATTA

ATGTAGATTTAGAGAATGATTAACT

GACAAGCCAGCATTATGTTGTAATGGTTCTGTGAGATGAGTCAGCTTAAAATTGC

ATCCCAAATAAACTAACCCATGGGG

TGGAGTTGATTTGGACTTTCTTATTGTCATTAATAAGCTTACAGGTCATGATGGT

GGTTTCTGGGTGTGAGAATCACATG

ACCAGTTTAGATGAGGAAGTTTGCCATCTTGTTATAGAAGAAGTCTCTTGCTTGT

TTTCTATAAGTAATAGGAATCTATT

TAGGTTTTAAGGTTAAATAAGAGGTATAGAGCTGTCTCCTGTGATTTAGAAACAG

AAATTTATTCTGTCAAAGCCTTTCC

TATCTACTCTGACTCAAGGCTTTGGAGAAGCACTTAAGCGTGGAGGTAATTCTG

AGACCATGCAGTTTGACACGTGGATG

GCCTTATTGGTATAGTTGAGGCTGATAGTGTGTTAATAAAGGCTACCAAGCCTTC

AGTTGTTCTCTTCAAACAGTGGTTT

TCTGTTTAGTGAAATAGACCAACCTTAACAACATCGTAGGGAAAATAATCATTCC

TCATCTTAGATGGAGAGATTCCAAA

CTAAATCTGGTACAGAAATCTGGAAAAGAGTTTGTGGGGTGAGCCAGCTTTTTG

GACGTCTGGAATTTGGGAGAAGAGGA

GATATTCTGATGTATGTATTGGATAGACTAAAAAGTACAAAATTGGTGTGAGTAA

ATGGCJ-I I I I GTTCAGTCTGAACAC

AAGATAAGCATCTTCCCTCGCTCCACAATCACTGCCCGACTCTTCCTTTGAGCCT

TTTGGGGGTTGTTCTTTATTTTGTG

GTTTCTATGTAATTATTTTAGTGGCTTCTCTGAGTTACCCAGATGAGTAATTTTAA

TGTGCTGCCTGGTTTTGGATGTTT

GGTTCAAAGGGACTAGGACCGAGGAAGAGCCGGTGATGGACAGTGTGTGGAGT

ACGGGTATTGTCCATGTATCTGCCTCT

TTATCTTGTCACCCGCTTGTTCTCACATAAAATGAGGGAGAAAATGGGGAATGTA

AAATTTTCAAAGAAAAATCTACCTG

ATGACTCAGTTTTCCCTGCCCCAGAGAGCTGGTTTTTTCTTTTCACCTCTTTGCA CGCACCAGAACCTTTTTGTAGCTGA

GAGAGCCACAGCTGAGGCAAATGTGTGAGCCTCTCAGTATGGCCAGGTGCTGT GACTGGAATGAATGAGAGAAGCGGGGA

AAGGCATGGGGAGCGTTCTCTAATCACAGAGAACCACATGGAAGATGCACGCA ACCTTGTACAACCTGTTGCCTCTCACC TTCAATTAAATTTTAATTGTTCCTCATCACACTTTCTTTCAAAACAAATCTTGTAAT

CAGATATCTTATAAATTATTTAC

CAGCGCAAGTAAAAAACAATGTAGGATTTCAGCAAAATGAAATGTGCTTTACTAT

TGAAACTCCCCTTGTGAATTAAAAC

AAGGCTAATCTTGGCACCATGGTGATGGAAATAAAAAATGAAAGTCCTTCCTATA

TTCCTGCAAAGTACAGCACAATCCA

CAGCCCATCAACACCCATTAAAAATTCAAATTCTGATCAATTGCTTCAAGGTTCA

AATGGGAAATCACGTGGACGGGGCC

AAAAAAACAATAATCCTTCACCTCCCCCAAATTCTGATCTTGAGGTACGTCATAC

CCAACACCCAAGTGAATAAAAAGGT

GTCTTAATAGCTAACTGGTGGTTTTACATACCTGCTACGTAAACAGCAGTGGTTA

AGGTATCAAATGCTTTTGTTACTGC

TTTCTTTTTTATATTAACAATAGATAAAGTAATATTTTCTCTCCTTTATTAACTATA

GGTTAAGCAATAATTTG I I I I I I

CTTCCCTATGAACTATATGGCAAAGGGTCTGTTTCTCTCTAAAGAAATATAGTAA

ATTATGCAATTCTACTTGAACAAAA

TTCTGTTATGCTGATCTGTATCATTGCCATTTTAATTAAGTGTCATACTAAAAATA

CTTGGTAAAATCCACTTGTTAAAA

GGCACAAAAAAAATAGAATGAGTGAGACCTACTATTTGATAGCACAACAGGGTG

ACTATAGCCAATAATAACTTAATTGT

ACATTTTCAAATAACTAAAAGAGCGTACCTGGATTGTTTTTAACACAAAGGACAA

ATGCTTGAAGGGATAGAGACCCCAT

TCTCCATGATGTGCTTATTTCGCATTGCATGCCTATATCAAAACATCTCATGTAC

CCCATAATTATATACACCTAGTATC

CACAAAAATTAAAAATACAAAAAATAAATAAAATCCACTTTTTGTTGTTTTGAAGAT

GGAGTCTTGCTTTGTCACCCAGC

GTAGAGTGCAATGGCGTGATCTCGGCTCACTGCAACCTCTGCCTCCCGGTTCA

AGCGATTCTTCTGCCCCAGCCTCCCGA

GTAGCTGGGACTACAGGCACCAAGCCCGGCTAATTTTTGTATTTTTAGTARAGA

CGTCATTTCACCATGTTGGCCAGGAT

GGTCTCCATCTCCTGACCTCATGGTCCACCCACCTCGGCCTCCCAAAGTGCTG

GATTACAGGCACGAGCCACCACGCCCA

GCCATAAAATCTACTTTGAAATGAAAGACACACTTATATATATTGAGAAAGGAAAA

CCTGGAATGTTCTAAACTGAAATA

GTAATCATCTAATGAGACATATTTTGGGGGAGTTCAAAAGCAGGAAGGATTAAGT

AAGAACTTAAAGAGTGGTAGATTTC

ATAGCCTTGAATCCACTGCAACTAAGTTTCTGCTTTTTGCCAGAGTGAAATCTGA

TGGTTGGAAAACATGATTATTTTGG

CTGAAAAAATGAAACTTTTAGCAGAATCTATGTATATGAATATGTACTGGTGTGA

GTGGGAATAGGAAGAAGTAAAACAG

GCTTGATAGGCTTTTTGTGGTATCCCAAAGACCTTTTTGAAATGAATTCCTTCAG TTCCCATTTTAAATACTTCAAGAGT

ATGTCTAAGAGCATGAGTGATGTGTATGCTTATTTTGTTAAATATCTATACCCTAA AGACTTAAAAAACCTGGCAAGAAC

AATGTGTATCCCATCCCTTTGATAACAGCTCAATAGGTAACATAAGGTGAATTTA TTATGTAAGGTTTTAGTCTTTGGAT

GCAATTTTAATACTTGGCTTCCTAAGAGTAAAACATAAGATTCATTATTTTAGTGC TTCTCAATGAACAGAGATCAGAGG TAAGGAAAAGATAAATGATATATGATTAGTTGCTCATCTCTATCTCAAGTTATAAC

CAGACTAGCTATAACTCATTTTTT

TCATTTATTCAGAGCTGAGACCGAAATTCTATTACAGAATTAGAAAGGACATAGA

TTATCTCTTTCCTATATTTATATTG

ACATTACAAATGTAATTATCCTCTTGCACCTCATCCAAAGTTATATCTGGAGAAAA

GTTAGGAGATACTCTTTGAAACAG

TTGTGTTTTTTTTGAAATAATAAATTCAGATTTTTCATTCATCTTCCGTTTCAAGAT

GACTTTTTCGTGTTGAAGTTAAC

TGAATAACAGCTTTCTCAGCCAAGGCAAAGACACATTGATTTGGTTCTTCCTTTG

TAGAGAGTGTTCATCTGGGACTTGG

ATGAGACAATCATTGTTTTCCACTCCTTGCTTACTGGGTCCTACGCCAACAGATA

TGGGAGGGTGAGTACCAAGAGGCAT

CAGTTTTTAATTTCTTTGGAGTAAATATACCTCTAATTTGTTTATTATCCAAATGAA

TGTAATTTAACTTTAACAAATTT

AAAATATAAATGGAGAAGAACTATATAGTCAAATATATATATGTAAGGTAAAAAAT

ATGTAAAAGAGCCAAAGAAGATAT

TATTTTTAGGTCTTTGGCAATAAGATACACTTTGTTCAATTAAAGTCATGCTTATT

CAGACCCCAGTGAATAAAAGTGAT

TGAATTTTATCCTAGGATGAAATATTAAAATACATAAGTGACATAGAAAGATGTTT

CGTGTTCTCTTTTACTTTTCCTGT

CTTTTTATTCCCTAACATTTAATGGAGCAAATATTTCACTTTCTGTTGTTCTCAGT

GATTAGAAGCTTTGGTATTTTATC

TTCAAAACAACTGCTACAAATGTCTCTATATGTCCCTTTGCTACTTGGGTTAAAT

GTATTTGAATGGTATGTAAGATTGA

TCCTCTGAAAGGCAGTGGGACCATGTTAGAGTTGTACAGTACTTATATTGGTATT

ATAGATAAAGAATAATATTATAGAC

CACCTTCTGTGAGTGAAACTTTGGTTTCCATGTTGTACATTTGGGTATCCCAAAA

GTTTGGAATTTCTTTTTTGGCATAT

TGATGATTTCATAAGTCTTTCCCAGGCAAAAATGTTATTTAAAGTAGCTATAGGTA

ATTTCTCAAGTTATCCTGCTAATA

TTTCATTTCTCTTGTTTTCATCTGTAAAGAAATAATAAGTCTTAGGTTTTTTTAAGT

TATTTGGAGCATTAGAGACTGAA

TTCTTATTATGTCCCAGTGACTTCAGCAGTTTATCATAATTAAGTGTTAGAATTAA

TACCATTAAAATGCACATACTGTT

ATTACAAATTTGACCGGGTGGTTGTTCTTTCAACAAATATTTATTTTAACAAATAC

ACACTGAGTATCTGCTATGGATAT

GCAGTCTGGGGGATTTAGCTTCACAGCCCAGATTTCCTTTTCATCATCTCAAAG GTACACCTAATCAAAAATAGGCTGAA

GCAATAATATCCCACCATTTTAATTCCTGTTCCGAGATAATATATCGGTAAAACAC

AGTATGTCTCATCTTATTGTTAGG

CAAATTCTCTATAAGAAGACAACAAAAATGCAGATTATAAAATATAAGGCCATAGA

ATCAATCAGCCAAATGGTCTCTAC

TGCGTGATTAGCACATAGTAGGAATTCAAGAAACATTAGCTGTGCTTTTTCTTGT

ATTATTTCTTCTGTCAATACTATCA

CTGTTCCTATTTCTAGAAAAACTACTACTTAAAAACTAAAGATTAATATCTGAAAT ATCAACATGTAGTTAGTAGGCAGA

ACCTTGAAGATCTCCTTAACTGATCCATTATAAAATCCCTACACCTGTCTTCTAAG GAGACCACTGGTGCCTGTAACTCA ?

CACAAGAGTGTTGAAAAGAGTAGGCACTTAGAAAATGAGCATTTGAAAACCACC

CAGAAAGTCTCCTATAAGCTCTCGCT

AAGGGCCAAATTGACGCAAAAGAGAGTTATCTAAGAGTATTCTATTAGCCAATGA

AAGAGACCAGCTTAGAGTTAAGGAG

TTAGGCATCCCATGACCTACTGAACTTTCAATCTTTGGCCATAAATTATTGGCTT

ACACATGGAACTTACACTTAACCTA

GAAATGCCTGGCTTCATACAATTTTTCATTCATGTCTAAACCATTCTCTAAATAAT

GTATTACAAATACCATGAGTTTAA

TTTTTTAATGTGTCAATCATTAGAACTTTGAGGCTTCTTCAAGTAGTAATATATTT

GTTTGTTTGTTTAGTTTAGTTTTC

TCATTCCAGGTCATGTGATAAAAAGTTTTATCTCGGCCGGGCGCGGTGGCTCAT

GCCTGTAATCCCAGCACTTTGGGAGG

CCAAGGCGGGTGGATCACGAGGTCAGGAGATCGAGACCATCCTGACTAACACA

GTGAAACCCCGTCTCTACTAAAAATAC

AAAAAATTAGCCAGGCGTGGTGGCAGGCGCCTGTAGTCCCAGCTACTCAGGAA

GCTGAGGCAGGAGAACGGCGTGAACCT

GGGAGGCGGAGCTTGCAGTGAGCAGAGATCGCGCCACTGTGCTCCAGCCTGG

GCAACAGAGCAAGACTCCATCTCAAAAA

AAACAAAAGTTTTATCTCATATAATTTCCAAAAGCCAAATTTATATACTCTCTGAA

GTACAGGCTATTTGAGTTTCTTAA

ATATAAGTGTATCCTTCATTGATAAGTTTATGGGGCTGTGGAAGAACTTCAGAAA

GCCAGGTTTCTGAGGAAGATCTTCC

TGCATGTGACTTTGGGTCTGAGGCCTGGAGTTACTGTATGGTCATCAGGAGAAA

GGGTGTTGTAAGCAGAAGAAACAGTA

AGTGCAGAGGCAAGAAAGGCTTTGCTGTTTGATGGACAGAGGCCAGTGAGACC

AAGCTTAGTGATGGAGATGGAGAGTCA

ATGAGATGTGGTCAGGGATGAAGGCAGGTTCCAAAGGGCCTCCCAGCCACACA

CAGTCAACAGGGCTAATCTCACTGCAT

GTGCCATGGGAAGTCATCGGGGGACCACAATGGAGGAGATGAGAAGATCTGAT

TTACATTTTACTTTAGCTAAGTGTGAA

GAATGAATGGTAGTGGAAGAAAAATGGGGGTCCAAATTGGAGGTTGTAGCAGT

GATCTGAGGCACCAGCAGTAGAGATGG

AAAGAATTATTTGGACTCTGGATTACATTGCAGGTAGAGAACTGGATTTAGAAAT

GATTTGAATTGATTGGAGTGGTGGG

TAGTAAACAAGACAGAGAAGTGAAAGATGATTCCTGTGTTGTCCTGGGCAACTG

AGAAAATAGTCGTATATTTGTTGAAA

TAGTAAAGAATTCAATTTGGAGGGAAGGGAGAGTTCAGTGTTGGACAAGTTTCA

GATGCTTATTAGACAACTGCTTGGAC

CAGGCAAGGAAACCACTGGAAGGTGAGCCCAGGGCACTGGGGGGACTAGTCA

GTACAGGAGATGGCAGATTCACAGTCAA

CATTGCACAGATTAGGGTTAGAGGCTCCTTGGAGTCTCTTAGCTTGATTTCTTCC TAAACATI 1 1 I I GAGCACCTTCCTT

GTACTGAACACTTGCGGTCAGCCTGTTGTCTCATTGCCTGGAGGTGTCTTTTCC TGGCCAGGTGACTTTTGAAGATGCAT

GGTTTAGGATGTTGAATTGTAGCTGGAAGCATGCAAGGAAGTCTACTTAATGATT TC CTC ATTAGAAATC ATTAC AGTTG

ATAAGAATTGGCTCTCAAATCTGATCATCAAGCTTTGTAAAAATGTGATTCAACAA GGATTCTTCCAAACAATAATAATA IS

AGCAAGCTGATCAGAGTGTCTTGCAGTCCTTTTGTTTCCCCCAGGGCACTGAGG

AGAAAAGTCTTAAAGGAGTTCAGTGA

CAAGACTTCTTTTTAGGAGCACAGATTTGGTGGTGTTCCTGGGCATTGTCAAGG

GGAGCATGTGGCTGAGCCTTACTCTA

CATGTATATAAAGAATGAATGCCTGACAATGTGATCGCTGTTCTGTCTCTAAGGG

AACCAGATACTTGAGAAGTAGCTTA

GTCACAGTGGCTTAGGCAACTCAATGAGTAGGCCATTCAGTAAGAGGACACAG

GATCTGGTTGATACAATGCAAAATTAG

GGCATGTTACAGCAATGTGTACCAAAAAAAAGTTTGAGAATGTCCCACCTTCCGT

GTAATCCTAGCTGTGGGAAGAAGGC

ACGTTGGGTGTTATAAGAAAGTCTTGTTTTATAACAAAGGTGATAGGGACAAAAG

AGGGTGCTGGGCACAGTCAGCCATG

GCTGAGGAAAACAAGTAAGATGTTAGGAAATTGAAGAGCTTGTGAGTGCTACGT

AAACTAGGATGTTTTAAATAGTATCT

GACCTAGCTGTTTTTGTTTAATGCA I I I I I AAAAGTTGCTTTAAAACTAATAACAA

ATTCCAGAATGTCATTGAAGAAAA

CTATTCTTGGGTTA I I T I I CATTGAATGGTCTCCTTATAATAAACAATGTAATTCA

ATTATTGTTATCAAAGTGCACACC

CAAAACTTGTATTTCAAATCAAAATTGTGCTGATGTCTCAGGCATGGTCCCCCTC

CTGTGGGAGGATTACCCAAGATATC

TACACTCAACGCCTTGATTGTTTCAACACTATCTTTTTATTGTTTAATGTCTGTGT

CTCACAGTGATTTGGGCTAGAATG

GATCTTAAAAATATGGATATTCACTTTTCATTTTCAACAAAAAACAAAACATGAAC

ACTTAAGATAGTGTAAAGCTTAGT

ATGTTTAATCATAGGCCCTATTAAGAACTTTCTATATGCCCAGCATTCATTTAATT

CAACATTAATGCTCCAAAGGTGCT

GTTATCAAGCCTATTTTATAGATGAGAAAATTGAAACGGGAGAGGTTAGAGACC

CAATGTAATAAATACTCAGCACCATG

TCTAGCATAAAAAAAAATTTCAACAAATACCAGTTATATTTGAAAGTAGGTGAAGC

TTCACTATCAAAAAAAAACAAAAC

AAAAACAAAAACTTGAATTCCTGCATAGTGGCCAGTGAGTAACATGGTGGCAGT

GTC CTC AAATTTATG AATTAC AAGTA

TACGCTATGCCTTAGAGTGTGATAGAAAGAGGATCGGATTTGAATTCAAGTAGA

TCTAGACTCAAATCCAAGCTCTGACT

TTTCACCTCTTTGACCTTAGGCAAATTATTTATTCTTACTGAGCCTCAATTTTCTT

ATCCATAAAGCCAGCTACGTGACT

TGCGGAATTGTTTTAAAAATTAAATTGTATCATGAATTTGGCCCAGCATGGTGGT

TTATGCGTGTAATCCCAGCACTTTG

GGAGGCTGAGGCGGGTAGATCACTTGAGCTCAGGAGTTCAAGACAAGCCTGGG TAACATGGCAAGAGTCCATCGCTACAA

AAAAAAAATACAAAAATTAGCTGGGTGTGGTGGCACATGCGTGTAGTCCTAGCT GCTTGGGAGGGCTGAGGTAGGAGGAT

CGCTTGAGCCTGGGAGGTCAAGGCTGCAGTGAACTATGATTGCACTACTGCATT CCAGCCTGGGTGACAGAGTAAGATCC

TGTCTCAAAAAAAAAAAAAAAATTGAATTGCTCACGAATGTAAAAGTCCATGCAC AGAGTCTTATTAATGGCAACAACCG

ATAGTAAGAAAGGAAAGCATGTGTCACCTATTGAACTTGTAGTTGCTCTCTAAAG TATTTGTTTTATAACTCACTAGGCT T6

TCTAAAAGGATCTCACTTGTCATAGTGAGTTTAGATAAACAGCCTCCCTTGTGGG

TGTGTTTCTGTGATCATTCAGTACC

TTGAGGAAAGAAAAACTGAAAGCAGTTTATATCGCCCATTTCATCAGAACAGCTC

AGTTCGCCAGTAGAGATTGTTGTTC

CTACTCTGCTTTAGGGGACAACACAACTCCAAGCTCTTTTGGTGGAGGATTTTCT

TCCATGGTGGTTGATGAAAAAGCTA

AACTCTTAGCATGGCACCAGAGCAGTAGGACACCAGTGTCCTCACTGGGTTGCA

GGAGATCTGGTTACTTAACCAGGATG

ATCTCCCTTTTGGAACCTCCAGTCCAAGGGTTGGCAAACATTTTCTGTAAAGCAA

CAGATAGTAAATATTTCAGGCTTTG

CGGGCCATATGCTTGCCATAACTACACAGTTCTGCCTTAGTAGTGCAAAAGCAA

CCAATGAATAAATGTGGCTGTGTTCC

AATGAAACTTGATTGAATGCTCCCAAATTTGAATTTTATATAATGTTCATGTCTTA

TGAAATATTTTTCTTTTGGTTTTA

AAAACTCTAGACCGAGGATTGACAGACCATACCCCATAGGTCAAATCTGGTTTAT

GGTCTGCTTCTGTCAGGCCTTTAAG

AATGTTGTTTAAGTTTTTAAAGTGTAATTTAAAAAAATAAAAGCACAAGGAAGAAT

GTGTGTCAGAGATGATATGGCCTG

CAAAGATAGAAATACTCCCTGTCCCTTTAGAGAGAAAGGCTGTCTGCCTGCCTC

TGCTGTAGGTGATGCTCTGAGCTTTG

CACTGGTTCATTTTCCTCCTGCCTTCCGGGACAATTCAATGACAGCAACAATGT

GACTAGACCGTGAACTCAATGCAGGC

AGTGCATACATGCGCCTGGGGCCCCAAGGAAACACAGACCCTCACGTGAATTC

ATTAGGGTAGGTGAAATGGCTGTTACA

AACAGATCTAAAATATAATGGCACAAACAAAAGAAATCTGTCTCTTGTTTAGTTGT

CTAGAGTGGGGATGTTCCAGGTTG

CTTGGTTTGGGCATGGTTTGGTGGTACAAATAATAGTAGTGCCATTTTCATCATG

CAGCTTTTGAAGCTTCTGGGATCAT

TAGCATTCAATCCCCAGGGGAGGGGGTGGAGGCAGGGGCAGTGTGAATGGGA

GGCTTTGTAAGCTAGGCTGAGACAGGCA

CACACCTCTGCCTGTGCCATTGACCTCAGTCAAATGACCCAGTGCAGGGGAGG

CTGGGCGATTCAXCCTTCCTGTGTGCC

CAGGAAGAAACAGAGAATGGGTTTTAGTGAACAGGTAGCACTCACAGCCCAGTT

CTCAACTATTTGACCAACGAATGAAA

CCCAAAGCACACTTTCAACCCACCTTGCAGCTCTAGCCACACTGACAGAGCCAA

GCCCCAGGCTATGTGAGTGGTGGCTC

TGGAGTGATGATGGCCAATCTAATCCCAAAGGACAGAGCCCTGATTTTATTTTCT

TCCTTGTATATCAATGCCAAATTAG

ATAAAAAGGATGAAATAATATATGTGTCTATATATGAAACCTCTTTATGCTGTAGA

GTGACATAGAATCTAAACTCTCAA

AACAAACAGAAGCACATTTTTTCAAATGCCAGCCAAACAGGCATGCTTCATCTCT

CTTCTGGCCATATCTTGAAGGTTTC

TCTCTTTTCCTTTAACAGCCAGCCCCTCTGCCTCACTACCAGTAGTATACAGCG

GAAATGTACATTCTTGCTGTTTAATG

GAACACAGCCTTACTGTTCTTGTTCATGAATATAAAAGGGAATGCAGTGAATGAA

CAGGAATCATCACAGCTCCTTAACC

ACTGCACTGGCCTGCCACCTGCAACTTTGGACCTCCCTTGCTCTGTGGCCTTGG AAAAAGCAGCCTATAAAATGGGAGCA ATTACAGGGCTGTTAGGGGAACAATGTGAACCACCTAGAAAAATACTCATAAGTA

CTTCATGAACAAGAGCTATTATTTA

TTGCTTACTTTGGGGGTTCAGAGGAGGAAGCAGGAAACCATGCAGACTTTGTTA

TTCCAGGCAGCAAAGAGGGAGTGGGA

AACAGAGAATTACAAGTGGCAGAAATCATAAGCAAAAAGGGTAAATAGTGATAAA

GTATATTTAGGGGATTAGACTTGAA

GAAGAATAGTAGCAGAAAATGTGTTGGATCAGTAATAAAAAGACTGCATCTTTAT

TCAACTTTTATGGTTTACACTGCTA

TCTCACACTTATTATCCAATGAACTCCTTCTTTTTGCCAATAAGAAAAGTGTAGCT

CAGAAAAATTAAATTATCTGTCCA

AAAGGCAGATCCATCAACTGGCAACACCAGTCTTGTAATTGACCCAGGCTTAGA

GAGAGCATTGAGTGCCAGGATTTCGT

TCTGTGGGCATGG " AATGCCATGAATTAGGGACATGATGGAAGAACATGATGG

CAATGGAGGGACATGCTGGAAGTGTAC

ATTTGGAAGACCGATTTAATAGCAAGGAAAACTATTGTGCAGTCCTCTGGTAGAA

AATAATAAAGGCCTAAAAAGTGGCT

GGAACACTCTGTAGGAAGGAGGGAGGTAGGAGAGTCATTACAAATGAAATCCT

GATAGGTCTTTCGACTGGATTTAGCTG

TTTAGCCTTTCCCTTAGCACCCCATGGGCCCCTTGGTGTTGAAGCCAATTTGGC

CAGGTCCATGCTGGCTTTATTCTTCT

TCCTCCCTCTTTGTACCACTTCTTGGCAGTTGGCCCATGTTTGAGATTCACTGG

GTTTTCTCCACAGTCAAAATTAGTCT

TTGGAATGTTGAAGTTCTTTCTCTTATTCTCTACTTGAGTATTATTTTCATGGTGT

TGAAAGGGAGTCTCTTAAGTAAGG

AGAAAATTAACACAATCGAGTGTCACGTATCTAATCAGTAAAAATGACCTGATTA

GCCCCACCACAAGTTTGAGGGTTTT

GGGGGTGTTTTTCCAGATCATTCAGGCAGTAGTTTAACCATGGTTTATTTTTTAT

GTGTATACAGTCTCTCTTGGTAATC

TGTATTATTTCTTAATTGGCAAGAGAATCACATCCAACTCCCAAAATCTGATATTT

CATATCTAAGTAGCCCAGTGATAG

GGCCTTAAACCTTGAAAAAAGTTAATATTTTATTGATGATATTGCTCCCTTTAGAC

AGGAGAAAACACAATAATGGAACC

TTTGGGTCTCGTAAGTTGTACAGATCATTTGGATAACACTTTTGGTTATGTGTTT

ATGTG N I I I I A I I I I I I A I I I I I I

AAAAAAACAATGTTTCAGCATAGTTTTTCCTTTCAAGATAGGAAGCTAATTTTCAA

AGCTTAACCTTAATCCTTTCCTTG

TGGGCAGCCAGCATGGGGTCTTAAAAATAAACCACTCATGTATTCAGAGTGCTC

ATTCCTG Π I i ΓCAGCTTATTTTCCC

CCAAAACAAAAGATCTTGGATGGGGAGAATTTTTAAATGACATTCAGGTCCTGTT TGTGGCTGTAATATTAGCATTCTAA

GTAAGGAATTGTTTAAGGTGCACTGAATGAGTATTTTTCTTTCTTTTGATTGATAC TGCCCAAAGATTTGTCCTTGATTT

GAGCATACTTAACAAGATTTTCACATGGACGAATAAATCTCATAAACCTGCTCAC ATTGTTAGCAACACACCTCACTGGG

CTACCACAACTCCCAGTAATTGCTTGCCAGGGAGGCTTAGCTGCCCATGTAGGA TTAGTACAGAGAGAGTTTAGGAACCT

TATTTTCTTTTATTCCAGAAATTCAAAATGTAGGTAGCATATCCTGAGGAACTAGT CATGCTTTCTGAAATAGTTGCACT GTCTTCTGATAACTC CTATCTCAAGATTAATTATTTAAATAG C ACTTTTTTAAAGAT

AATATATTCTTTGTGATTCAAAG

TAGAATAGCAATTGCTAGGAGTTTGTGTAGAGTATCATCTCATGGATATAAGTAG

GTACATATTCAGAGAGGAGAGATAC

AAAAGACATACATTATGTATTTAATGTACTTATAAATTGAAAATAATAGGATAAGA

ATTAAAAGGCAAAATAATTCCCTT

TCCTTTTTATTTTTAGTAAGAACACCATAAGTACTTTGTAGAGACCAAGTCAGAAT

GCTGAAGCTACTTAAAATTGGAAC

AATGACTTAGCCATTAAAATAGTTCTTAAATGTTGACTTAAAGCATTTAAATTAGA

CAATTTATATTATGTGTTTACTTA

TTTTGTTTTACTGTTAAAT I I I I I GTTTTCAATTTAGAAAAATAGCATGGCCAAAG

AAAATAAAGCTAAATGTGCAAGGG

TGCATAAATAAAAACTAGAGCTACATTAAAAATAGGAACAATTTTTCTTTTGGTGC

TTTACAGGTGTATCTAGCAATAAA

GAGAACCTGCTTCCTCATTAATATTTGATGCCATTTTTAGTGGTTCTTATAATAAA

ACTAAAGTGACTTATTCTTAGATG

ACATCAGATCTAATGTAATACTTGTCACTTGTGGCATTATTAAATCAACAATAAAA

ATCTTGGGAAGTATCTTTTAAAAC

ATAGTTACATAAAAAGGAGAGATACTTTCATGGTTAGAAAATGGTTACCTTTAAC

GTTTCTAGAGAAAGGAGAAGAGATA

TTTTGTTTTTATACTCAGAAATATATTCTGGATCCTTCTAAAAGCCCCAAAAGAGA

TAAACAACAGGTTTCCCTTCAACT

TTCTATCTGTTTTGAGTCTCCAAAGACCGTGAACTATCTGAACTGAAAGTAAATA

AAAACGGGTCAAGAAGATCATTTCT

GAATCAGCCTCACTTCTGTGAGGGGTTGGTGTTTGCCACTAAAAATAGACTACA

TTGTCATTTGAAGCCAGGAAGTGAGA

AGTGTTCAl I I T I TCTTGCCTCTTTCTGATCATTTTAGTTCAACGCATAACTCTAC

CTTTCTCCAGCAGGCTGAATTGTG

CGACTTTCATGAGTCTTACGTGTTTCTTTTATGCATTTTGCCAGAATACTGTGCT

TAAATGGTATGGGGCCAGTAGAGAG

CTCCAGGAGTGGGAACTCACTGAACTGGTAAATGACAGCAAGAGAAGTTAGGAC

AGATACCAGTGTGTCCACAGGTGACT

CTTGGTGCTCTTTGGCATCTGTGTAATAAGGTAACATGAACTGTGGTGGGCGGG

CATCTCCAGTCTGCTGTGCGGGAGAG

GGAGGATTGATTGTAGAAAGTCCTTAGTGGTACCAAGAGAATGTGATACGGCTC

TGGCTTGGTTGACCATAGACAGAGTT

CAGGGTAAAAAAGGTATTTTGTTGTTGTAGGGAGAAAGCTGCTGGATTTATAGG AAAAATGATGTGTGCTTACCCAATAC

TATCAGTAAAATAGTGTCTGGCTGCTTTACATTTTTCTTTAAAAAACACTATAAAT TCCACATTGCAATCCTTTTCTCAA

GCACTCTGAATGGGGTCACTAAGCAACTTTGTGACACAATTAGCTTCAGCTGTC CAGTTGGTCAAATTAGATTTTACCTC

TTCTCCCTTCCTCAATGTTTGTAAGCTGCCTCACGAACATGACTACAACATAAAA CATTTAACTTTCCAAAGAGGTCAAG

AGAATTGAGGATAAATACATACCGCCTTCAAAACAGTTTTCACAGTCCTTATTTA CAAGATAGTTAAGATCAAATATCAT

GTACAAACACTAGAATTAGCAGTGCAAATGTAACTATAATAGTTCTCCACATAAAT GGGCAACAAAATAAAATGAGCAGC ATAAAGTTTAACCTAGTCACTAGAATAAAGCAAAATGGCATGTGAGAATGTACTC

CCTTCCACTCCTTCCTGGGGACCTA

GAAAAATTCCTGGTGGTTTCTGTAGTGGGTAAGGTCCCCTGCTAATACCATGTG

GTCTCCATGTTAGGACTACCCAAAGT

TTAAGACTCCACCATAGAAGTATCAGGTGGTACTCAGAACGTTAGGCTTCTAGC

AGAGATGAGTTTCCCTGTGGATAAAT

GATCATTAAAAACATCCATGAAGTAATTCAGAAAATTGTACCACCAAATGGCACC

CTTCTCAAGTTGCACACGCAAAACC

TCCAGGGGTATATGGACTCCAGTACCCAGAACACATGCCTGGCTGCACACCTCT

CTCCTGCCTGAGTCACCATTTGTTTC

CTGGCCTGACATTAATGAGTCATTCAGAAAGTCATTCTTTTATGATTTATAGAATA

CATAAACAGTAATGTTGTAGGTGC

CTAGCCAAAATTTCTGCCTTCAAGGAGCTTTTAAATTAACAGAGGAGGCTGTTTG

GTAGGGGATGGATGAATCTTCAATA

ATAGTCTGTCATTAGGGTAAAACTATTCTAAACTAATGCCACTCTAAATCCACTAA

GGGTAAAACTGCAGAATCTCCAAA

GAAATAACAGTGCTAAGAAGGCAACAAGTCTCTCTGTATTATTCATGAGAGCCAT

TCTCTAGTAGCTACATCATTTAACT

AGCAAGAAATAGCCAGTAATAGGATGTCAGATGATAACACTAGCATTGTGAATCA

GAAAAAAAAAAATCAGGATCCACAG

GAGACATACAACCAAAAAATAAATGCCAGGAGCCTTAAATGAAATGTTTCTAGGG

TTACTGCTCTTCTTAAATTACACCG

GGATTTCTCCTTTGCAGCTGATCAGCACTGCAATTCAACACCGCTATTGCTAAAT

GTATGAGATGATCTTGTGGCTTCAA

AAATGTAGGAGTTCCTCTGGGTTCCTGGAAAAGAACATCCCATATGGTAAAAAT

GAATAGAGTGAATAGAAGCCAAGCAT

CGTCTGACATACATTCTTGTGTATATTCCAAATCCTCACTTATATCTGGGTACAG

ATTACCCAAGTATGCCCTAGCACCT

TCTTTTCAATAAAAGGTGATATGCTGAGGAACTTTGGCAGGCTTTTGCATTATGG

CCTTTAAAATGTTTTTGGCATTTTA

AAAGTATGGCTTGCACGTGTTTTA TTTTCAGAATAAATATTTTTAATCCATAGT

TTCACCACCAAAAACAGCCAATCT

TGTCTTAGTGAATA I I I I I ATA I I I I I I I I AACTTTTCATTATAGAAACTTGCAAAC

ATAGAAAAGTAGATAGAATAATA

TGTATAATGAAACCCAATGTAACCACTCCAGCTTTAACAATATCAAGTCCTAGCC

AATCTTGTTTATGTATATCTTCATC

GTATCCCTCTGCTTCCTGGTCCTGTCCAAGGATGATTTTTCAAAACAAATCCCAG

ATATCACATTATTTAATTTGTATAG

AGTTGAGTATGTATTTAGGAATTCATTAAAAGAATAGAATAGAAGTAATAAATAGT

ATTCAGCACCTTAGCCATCTAGTG

AAATTAAAAATAATTCTGTAATATCATTAAATGTCTAACCAATAGTTATATTTAAAA TTA I I I I I TATTTTAI I I I I I I I

TTGAGATGGAGTTTAGTTCTTGTTGCCCAGGCTGGAGTGCAGTGGAGTGATCTC AGCTCACTGCAACCTCCGTCTCCCGG

GTTCAGGCGATTCTCCTACCTCAGCCTCCCATGTAGCTGGGATTAGAGGCATGC GCCACCATGCCCAGCTAATTTTGTAT

TTTTAGTAGAGACAGGGTTTCACCACGTTGGTCAGGCTGGTCTTGAACTCCTGA CCTCAGGTGATCCACCCACCTTGGCC TCCCAAAGTGTTGGGATTATAGGCATGAGCCACCGTGCCTGGCCAATTTTTATT

TTTAATTTTGATATACACATAAAAAA

TTTTACCTTCTTAACCATTTTAAGTATACAGTTCAGTAGTTTTAAGTACCTTCACA

TTGTTGTAAAACCAATCTCCAGAA

GTTTTTTATCTCTCAAAATTGAAAGTCTAAACCCATTAAACAACTCCCCATTCTCC

CCTCCCCCCAATTCCTGGCAACAT

CTATTCTTTCTGTCTCTGTGTATTGCAGGTACCCTCATATAAGTGGAATCATATA

GTACTTCACTTTTTGTGACTGGCTG

GTTTCACTCAGGACAAGGTCCTCAAGGTTCATCCGTGTTATAACATGTCAGAATT

TC CTC C CTTTTTAAGGTTAC ATAAT

ATTCTGTTGTATGTATATGCCACATTTTATTTTTTTGATATATCTATCAATGGTCAT

TTGAGTTGCTTCCACCTATGACT

GTTTTAAATAATGCTGCTATGAATATGAGTATGCAAAATGTCTTCCCTGCTTTTAG

TATTTTTTGGGTATATAATCAGAA

GGGGAATTGTGGGATCATATAGTAATTATATACTTAATTTTTTGAGAAACTGCTG

TACTGTTTATCTTAGCAGCTGCAGC

ATTTTATGTTCCCACCAGCTGTGCACAGGGATTCCAATTTCTCCACATCCTCATC

AACACTTATTATTTTCTGTTTT I I G

TTTTCTGATAGTAGCCATTCTAATGGCTGTGAGATAATAACTCACAGTGGTTTGG

GTTCACATTTCCCTCATGAATAATG

ATGATGAGCATATTTCATGTGC I I I I I GACCATTTGTATAACTTCTTTAGAGATGA

TCCTTTTCCCATTTTTTAAATCAG

GGTTTTTTTTATTGAGTCATAGGAGGTCTTTACATATTCTGGATATTGACCCCTTA

TTAAATATATGATTTTCAAATATT

TTCTCCCATTCCGTGGCCTGCCTTTTCACTGTTGTTTCCTTTGATTATACACATTT

TTCATTTTGATGTTTTGAATTTAT

TTTCTTGGTTATTGCCTTTGCTTTTGGTGTCATATCCAAAAAGTCATTGACAAATC

CAATGTCATGAAGCTTTTCCTATA

CCATTTTATATTTTAGGTCTTTCATTTAGGTTTTTGATCTATTTTGAGTTAATTTTT

GCATATGGTGTAAGGTAACGATT

TACCTTGAA I I I I I I GCATGTGGATATCCAGTTTCCCCAGCACCGTTTGTTGAAA

AGACTATTATTTCCTCATTGAATGG

TCTTAGCACCCTTATTGAAAATCATCTTATCATACATGTGAGGGCTTATTTCTGG

TCTCTCTCTATATTCCATTGGTCTG

TACATCTGTA I I I I I GCCAGAACCACATTGTTTTGATTACTGTGTCTTTGTAATCA

GTTTTGAAATTGGCAAGTGTAACA

CTTCC GTTTGTTATTTTTTAAAATTGTTTTGGATCTTCAAGGTCCCTTGAGATT

TCATATAAACTTCAAAATGGAGTT

TTCTGTTTCTGCAGAAAAATACTATTGGGGATTTGATAGGGATTGCATTAGATCT GTACATCACTTTGGATAGTATTGAC

ATCTTAACACTATATCTTTCATCCATGAACATGGGATGTCTTTCCATTTATTTGTA TCTTCTTTAATG 1 I I I TAGCAATA

ATTTTTTAAAATTTTTCAGCGTACMGTGTTTTCCCTCCCTATTTAAGTTTATTCCT AAGTATTTGATTTCTTTCATTTC

TGCTATTGTAAATGGAATCATTTTCTTAATTTATTTCCTGGCTTAGTCGTTGTTAG TGTATAGAAACACAACTGATTTTT

GTGTGTTGTTTTTTCATCCTGCAACTGTGCTGAATGTATTCATTAGTTCTAACAG ACTCTGTGTGTGTGGTGGGGGGCGG SI GGTGGGGGGAGTTTTTCGGGGCTTTCTACACACAAAATTATGTCATTGTAAACA GATATCATTTTACTTCTTCTTTTACA

ACTTTGACGCCTTTTATTTGTTATTCTTGCCTAATACCTCTGGCTAGAACTTCTAT GTTTTGTTACACAGAAGTGGCAAA

AGCAGCCTGGTTGTCTTGTTCCTGATCTTAGAGGAAAAACTTTCAGACATTTACC ATTGAGTATGATGTTCTTTGTGGGC

TTTTCATATATGAGCTTTATTTTGCTGGGTAATTTCCTTTTCTTTCTAGTTTATAAA GTGTTTTTCTCCAGTAGTTTTGT

TTATTTACTTTACAGTTTATTATATGTCTCCTAAGTGTCTATAATCTATAGATTCTC ACTCAATCCCTTTTTATAAACCT

TGTGTATTTCTGTTGAAGTAACCAGACTGTCTGTCCTACAGAGTTCCTGGGAGT CTGGGTTTTCTTGACAGCAACCCTAT

TGTATTATTTAACATGTTTCTTTGTCCTCTGTATTTCCTACAATCTGATAGCTAGA TCTAGGAGCTTGCTTAGTTAGATT

CAGTTTCCTTTGCTTTTT I I I I I TTGTATTTTTGGTTGGCTAACACTTTATAGATG GGGTATATCCTTGAGCATCTATTT

GTTGTTTTTAAAACTTCAGTGCTCCAGTTGGAGAAGCTCCTTCAGCAGTCCATTA CAGAATGTTTTTACTTATGCTGTAC

ATTCTTTCGTTTTAGCTCTAAGGAGGTTGACTAAGAAGAAGAAAACTCAAGAGTT TGGAAGTTTTATGAACCACAGATTC

TTAATTGATCTGAATCAAATATATGTAGCCTTGCTGCCAGAAAAAAAGACTGACA GTTCCTGTTGCAGATAATTAGCTCA

GAGCTATTTTTGCTGAGGTGAAGTTTTGAGATGAAACTAAACCACTTGCTGAATT TTGATATTTTCTCAAGTATCAACTC

TTCTTGAATAGCTCTACAGTATTATTGCATATTTTTGATTAAGGCTTCCTCGTTAC CTTTAGTGGACAAGTAGGTGTCTT

GAGTGACTCATGCTGAGTCACCTGGCAGGTGCCACATTCCTTTGTTATCTTTTC AATAGTGTGGTGAAGTTCAATTTACC

ACCCTTAGTTTCAAGTGAGAGAACTAACTGATAGGATCAGTGAATTAACATGGTT AGTCTATAGCTATGCTTGGTGGCTA

ACTTTAAAGGCAGATTCCAGCTATCCTACTTGAGGACATTTGCTCTACGGGTAGT TTCTTTGTCTCAGTGTAAGAATAAA

TATTTATTAGAGATTTAGAGATTTATATACAATCTAACATGCTAATCTTGACTGGG AAGTAATCAACTATTTACATTACA

GAAATATGCTATACTCACATCAGAGAGCCAAAGAATAGACCACCTTTCCCCTATA TAATCAGTTTGATGATTCAACCAAA

CTTTCTATTTGTGG G AG AAGG ATC CTTTTTTATTTTTTAAATAATAC CCC C AAATT TTAAACTCTTTATCTTCTTCCTGG

AGTGGGCACAGTCCAAGTCACATGCATTTTACCCTGAGCGGACAGAATACCAGG TACTTGATACAGATACTCCAGAACTG

ATACTGGAAGACTCTACTTCTCTTTAGTATTATGGTAGCTGATATTTTATGTTAAT TTGATATTCTTAAAGTGCTTTCAC

AAATTTATCCTTCGACTTGACCACAGACAAAACCTTTCTAAAATATTATGTCATTC CTGAAATGCTAAAGTATTGGATCA

TTTTATGATCACTGAAAAATTTCAGGGACTTTACATTGAAGGCACTAAATTTGGA CTCTATGTCAATAATTCCTGTTTAT

AACCATGTATTACTGGCATACATTTGTAGATAAACTTGGTCAGGGTCAAGATGTT GCCAAGACCTATTCCCCCCAGTCAT υ TTGTTAATTGATTGGATTTGGGCTAAGCTATGTTCACAAATTGTCACTGTTAAAT GATTAAATGGTATAAGCAGAAAGGT

ATATGTGACTAACTTAAAGCAATCACATGACATGGCTATGCAAGTAATTGATTTTT TTGAAGAATAGTTCTAGGTTTCAG

TTGAACTGGTGCATTGCTCAGATCCCATCTGAATGATCCTACCTTCCTCTTAGTT CCTAGATCTTCAGAGTATAGGACAC

CTTTTGCCAGCTTCCTAAAGTGGGGTCATCTTTCCTGGTGATAAAAGCTTGGTT GACATCTTTGGGAAAAACCAGTGATG

GCCCCTTCTGGGATTGCTCATGTTGCATCGTCTATTTTAACTTCCACCTAAATTA GCTTTACTGATGACTGAGCTCTTAA

AAATATGGTCACCTCAAAAATAATTGGTATGAGGAATTGTATGAACATTCTATCG AAAGTGCCTTGAGTAGTAAGTACAG

CGAAAGTCTAGTTTGAATGTATGACTCCTTTATGGTAGTGCTGCAGAGATTGAGT GTGTTAGTCCATTTTCCATTGTTGT

AAAGGAATACCTTTACTTTTTAGTAGGCACAATACATATAGCACTTCAGAAATTTG AATTTACCAAGATTCTCACATACA

CAGATGAAGGCATTGTTGGAAGCAAATGACTTAGCCCTGCAAAAACAACAATGC CTTCATCTGGTGTGTGTGAGAGTCTT

GGTAAATTAAAGAATCAGAAGTGCTATATGTATTGTGCCTACTAAAAAGTCCATT TTTAATGATACTTGAAGTTTATCAC

ATGCCTTAGCTCACTGATGAAACAAAGCAGGGCGCCAAGAGGAAATATCCCAGT CCTCTGATGATTTAGTACACACATGC

ATTTATATACCCTTTTCTTTTATTTACTTCAAAC C C AATTAC AGATTTTCAAC ATTT GAATTCAAAGCTGACAGACATTA

ATCTCTCTAGAGACTGTATTTAATGGCATTTCAGTATTTCTGTGCACTGCTTTTGT CCAGAATATTTTTCC I I I I IAACA

TTTTAATCAAGATCTACTGTGATAGCTGTGATGAGGATTAAAAAAAATCAATAAAC AATTCAAGGTAGTGGACCAGGGTT

GATGCTGGTGATTTGGGTACATGATGAAAATTTTAGTCTCTGCGAGCAGTTCTG TGTTGTAGAAATAAGAGAAAGAGATC

TTGAGGCAATGGGATAATTAAAGACCACTTGACAGAGGATGAAGGCAAAACAAT TGTTTTATTCCTGGTTCTACAGACAC

TCAGCCAATTTCATCATTATTTACTTTTTCTACATAATGTTACTTTGAATTAGTAGA CATAATTΓGCCTTTGATGTACTG AAAACAGCCATGGGTAGCCTAGGATGACAACAAGTTCCTTCACTTCACTTTAGT

GACTTTAAGTAGACATTTGCAATTTA

ATATCATATCCAAATAATTTCGGAGCAGGGTTAGATTTCATTCCTGTGTGTGGGT

GTGAAGTTAGATGTTGTGTATTGAG

TTTTGTTTGGGTGAAGAACAACTTAAGAAGGCTTCCACTAGAAGAATTTCCTGTA CATAAGAACTATAGTAAATGTACAA

TTTCTTCGTTAAACAAGAAAATTGATAAAACAGTTGACTGTATTTTTTTCCTTTTG AGCCTCTTCACTGTACTATTCAAT

TTCTACCAGAGAAGTAAATTTCATGGCACATAAGAACATATTTTACAAAATGGTG TAATACATACCACTACTGAGTTTAC

AAAATCAGTGTTATGCTGACCTTATGGTAATATTATCCACTTCATTAAATAGAAAA GTAAGGCTTCTTATGCAGCTATTC

TAAGAATGGGCTCTTCCTTAATCAGGTAGATAGCATTTACTAGTGAACACGTTTA ATGTTTAATACTATCTTATGTAGAT Ϋ3

CAAATGAGATTTTCAGATTCAAGTGCCCATTTTTTCCCTAGGCTAGATCATTAGG

AATTGTTTTTTCACTGACAACACTA

ACAGCTAATGAAAGGTAAAATTTAGTATTTTCTTCTTTTCTTCACTGGAATTATTT

GAAATATTCTCTATTTATTGCTTT

TCATTCATTTATTCCTCCACCAAAGTTTGGGAAAGCCTTTATGACATGCATTGTG

TTGAGTACCAAGGATATACAGATAA

GGATATAGGGATAGGAAAAAACAAGGAATCATAGCCTAAAGCTTAAAATGTTGTT

TAAAGGCTGTAAAGAGAGAGAAAAA

TCTAATTGAACAAAACATTCTACACGAAATAGAAACAGAGCCTCCCACTATGTTT

TTTCAGAGGATTGATACCCTCTTTT

TGATTCTGCAAATAACAGTTTCATACTAAAACTAAAAAGAAAGAAAACCATACTCT

GAATCTAAAAAACAGGAGGAGGAA

GATACAGGGATTAAACCCCTGGCCTGCCGAGACAAGAACACATAGAGCTGTGG

TACCAGCAACTGTGGAGCCCAGCCAGC

AAGGATCAGGGAGGTGGAGCTGCGGAACTGGGAAAGGGGCCCAGCGGGGAG

CGCTGCCTGTAGGGATGGAGATGTGAGTG

TACAGGCTCCATCTGAGACCACGGTTCATGAACCTGTGTGGACAGATTCTTTGC

CCCAGTTTAGGCTTCTGCCTAGTTGT

GTTTTGAGGAAAGGATTTCATGATCAAGACAACAGTTTGAAAAATCAGAAACAGG

CCATCTTGAATATGTAAGCAAAAGT

TAGACATATACGGGTCTTTTGTACTTGTTAAATGTTTGAATGTTTTTTGAAAGAGA

TGAGAGGCTGGAATGAAGATAGTG

ACAACCTAGGAATAGACCCCAGTGAAGGAGACCATACCAAATATCCACAAAAGA

CACAAGGCTAAGGGGAAAAAAAAAAA

ACAAGAAAGCAAAGAACAATGGCAATGACAATAAGGGAGGCTGAAATTTATGGT

CCGAGTGACAGAGTGTTGAAATTGAT

TCAATATGGGGCTAAAAAGAGGTGGCTAGGAAAATGCCAAGGTCTGGGTTGGTA

GGATGCCATTCCTCTAAAGAATGAAT

ACAGTAGGTGTCCAGACGAAATGTGATGACTCCAACTTGCCACACATTGAATTT

CAGGTGCTTTATGGGACATCTAGGTG

GAAATGACCAATAAGTAATTGATATATGAGCCTCCAGTTGAGATCTAAGATCTGA

TAGAGGGGAGTGGGTGGTGGTTGAA

GCCATCAGTGTGCTTGAGTCCACAAGGTGATCTGAACAGAATGAGAGATGGAAG

GATAAGAATAAAGTCCTAGGAACCAA

CGTCATTGATATGGTTTTGCTGTGTCTCCACCCAAATCTCATTTTGAATTGTAGC

TCCCATAATTCCCATGTGTCATGGG

AGGGACCCAGTGGGAGATAATTGAATCATGGGGATGGTTACCTCCATGATGTTC

TCATGATAGTGAGTTCTCATGAGATC

TGATGGTGTTATAAGGGGCTTTTCCCTGCATTTGCTCTGCACTTCTCCTTGCTG

CCACCATGTGAAGAAGAATATGTTTG

CTTCCCCTTCCTCCATGATTGTAGGTTTCCTGAGGCCTCCCCAGCCATGCTGAA

CTGTGAGTCAGTTAAACCTCTTTTCT

TTATAAATTACCCAGTCTTGGGTATGTCTTTATTAGAAGCATGAATGGAATAATAC AGACATTTAACAAGCAGATGATGT

ATACATAGATTCTAGGAAAATGCCCATAAAAGATTGCTGTCATAGAGGAGAAATG TCAAGAAGAGAGTAAACAGCAGTGG

CATATGCCACAGAAAGGCCACGTTTTGAAAAGAATGAGAACATTTGGCAATGAG ATTGACTTAAAATGAGATGTTACTTT GAAGTGGAGGCAAAGCTCTAAATAATTTTTTGAAAGTGATATAACAAAAAATGGT

GTTTTAAAAGAAGTCCTTTTATAAC

TGCAGCTCTGGAATTTGTTATTGCAAGAAAATTATGTATTATAAAAATAGTAAGAT

TAAACTAAAGACCTGGTGATTTTA

ACATCTCAGTTTTGGTTTAAGAACGTGTTTTTTTCCTTGTTCTTAATATGCCAAAA

TAAGCCATCAAGCCAGAGTAATTT

ATTAACTGTTCAATGTTAGC I I I I I I CACATTTGACTTGTACTCGTATGAGACTTT

AGAAATTTTAACAGTGTTAACGTA

TTAGCTATTATAATAGGAAAATAAAATCTAACCTTGATATGAGAACTCCAGTTGCT

AGAATATGTAAGCAATTCCATGAG

CAGAATTATCTATCTGTCCTTCTGCTAGTTTCCTCTTGGGTAAAACACTCAGGGT

GAGATGGCTTTGCACTCTGCTTGCT

GGGTCATCACGCACAAATCACAACCCTCTAGGAACTCCGTGCTCTGTTTCTAAA

GTCAAAGAGTATGTGTTTTCTAGAGC

TCTGCCTTTCAAACTGTAAATTGAGACAGGGCTGTGAAATGGACAGTGTCAGAG

TATATCCACACAGTGAGCAAAATCTT

GCTTTGGCAAATATATTTCAGCTGTGTGTGTGCACATAAGAGAGATTCTTAGATC

C CAGTGTTAATTGTG I I I I I I ACTG

AGCATTGTGGTTCAAAGATGTCTGAAAAATACTGCTCTCAACTAGAACCCCCTTC

CTATTCTAAAATTGTCTCATTCCAG

TTAGCTGCCAGACTGTTCTGTTGGTGTAGTAAATGTTTCAAAACAATATCTTGGA

GTGGCAGGTATAAAAACTGTTGAGG

AATGACAGTTAATAACAAAAGAGATTAACATTAAGAGGTAAATAAGACCAACATC

TGTGTTGTATTTTACCACTAGACTG

AACAAGCCTTTAAAATTTATTAGTATTCTGACTAGCAAGTCATTACTGTAATTTAT

GAAGAAAAGTTAAAGTATGCTATA

TATTAACGAACAATAGAGTAATTCTTTCAATGGTCATATAAATAAGCAACCGTATC

AAGGAGAMTAATACTTTTTCAAA

AGTAACAATACTATGGCACATAAATTTTAAAATTGGCAATTAAATAAATGTGGTGA

GGCTCCATCCTTGCCACATAAAAA

GATTCATTGACATGGTTATGAGACAAGTCAGGGTATTTTTATTGGCCTGAGCTAG

AGGGAATAAACACCTTAGTCAGGTG

TTTTGTTTTAGACACATCTGTTTGAGCTTCCCCTACAGGTTAATTCAGCTGTAGG

GTCTATTTGAGCATTTGC I I I I I CC

AGCATGCCTACTGATACTTAAACTCCTTGTGAATGAGCAATTTCTTATAAATCCAA

GAGTAAATGAGTTAG I I I I I CTTT

ACAGTAATTTGTAAACTGTAATTCTCCTAAAACCTATGAAATTTCATAGAATTTCA

TAGATTTCAGGTGTATTAAAAAGG

GACAATTTCAGAATGCTAAGGAGTATGAATCATCATACACAAAATTTGAGGGAAT CGTTTTCCTTTGTCCCTCCCAGCAG

AACTCTTAGACACTCAGCCTCTGCATCTTTATTTCCTCTCTGCCTTCTTATCAACC CTGGGGTATTTGCCAATCTAACCC

CTGACGTTTGGCAATACTATGTCCTCTGCTTCCCTTCCACTGTCAGTCAGCTGG CAGAAAAGGCACAGGCAGGAGAGGAA

CTGAGGTGAGTGAATTGCACCATCCTGCCATATTTCACGTACTCTGTTCCTCCTC TAGGGCTGCTGGAAGGTAGAACTTG

GGCCACACTGGAGAGGTGGGGGCAGTGCTTAAGGGGGTGGGATGTACTGTAX GGATGTGGGTTATGCTGGAAGGGTGTGG GTAGTGCTAAGGGGGTGAGAGTGCTAGGAGGGAAGTGAGCTGTGCCAAGAGG

GTGGGAGGTGCTGGAGGGATGTGGGAGG

TGCTGGAGGGATGTGGGCGGTGCCAAGACAGTAGGAAGTATTAGAGAGATGTG

GGAGGTGCTGGAGGGATGTGGGTAGTG

CCAAGAGGGTGAGAAGTTCTGGAGGGATGTGGGAGGTGCTGGAAGGGTGTGG

GTGGTGCCAAGAGGGTGGGGAGTGCTGG

AGGGGTGTGGGCAGTGCTAGGGGTGGGCAGTGCTGGAGGAATAGCCAGGTTG

GGTTGGGTTGACCTCCAGGCTACAGCAG

ATGGGCCCCTGGCAGGTGCCCAGCAAGGCTCCGCTCCCATCTCCCACCCTTTC

CAGCCCCTGCTCTCTCCTAACTGTACA

AACATGTACGATTCTGACAAATGAAAGACAAGATTTTTTTTATTAGTCTATGTGAG

AATATTGTGTTTGCCATCTTGCTC

TCAGATTTATTAGTGAACAAACCAGGCTCGATGAGAGGATGACACCTAAGTGGG

AAAGCAAAGGCAGGGTCTGGCCTACA

GTCTAGGCCTCCTCTCTCACCACTTCTTGCCACTTCCCAAACTTAACAGGCATG

GCAGATGTTAAAGAGTAGGGGTAACA

CACGGGCATGTCTAGATATTGGAGATAAAGTTAGAGTGTTCAGAAAACAAGGTA

GATAGTGTACAAAGTTGACATTGGGC

AGTATCGAATCAAAGTCTTTTCTTTGGATTGTATGTTCTTGACTCTAAGACTCAAA

GGCACATGACACACATATCATGAC

ACATGGCATACCTGCCCAATGGGGCCTACCTGAAGGCAGTTTCTCTGCTTACAA

TCCCCCTGGTTACATAATGATGCAAG

GAGATTCCATACTTATTCCAAAGGTTTGCCGTGACAATAAAATAGTATATCATAAA

TTATTGTTCTTTGGAAGAATTGAA

AATGGCATACATACGAAAGTTGGTATTATTAATAGAATGCCAATAAACCCTTAAAA

AATTCATTTTATAGCCTTGTATAC

TTCCTGGAAATGTTATCTTTCATTACTTCCTATACAGGATCTTCAGCCATGAACC

ATGTTGCTTCTTGGATTTGATACAC

AGTAAATACAGGATAAAACTGCAAATACCTGCATATCCCCCAAGTTCATTTCAGT

GCCAGTAAATGTCATCAGGGTGGCT

TGTAGGGAGAGGCTGACTACTCACTGCTAATGTCCAACACTTGAATTTTTAGATA

GAAACCACGGTGCACTGTGGAATGT

AGCACTGCTCTCTGAGTATGGCATGGGCTGTTTGATGCCCGTACAGTGTCATGT

GAATCTTAAGATCAGCTCCCTGAGCT

GGACAGTGCCTGGTGGGGTCAGAGTGGCCATCACTTCCTGCTTTGAGTACACT

TAAAATTGACAGGTTTAATGAATGCAA

ATAATGATAGAGAGTTATCTAGATTAAAATTACTATGTACTACCCTTCTGAAATTA

ACCTATAGTGCAGGGCTGTCCAAT

CTTTTGACTTCCCTGGGCCACACTGGAAGAAGAAGAATTGTTTGAGGCCACACG

TAAAATACACTAACACTAATGTTAGC

TGATGAGCTAAAAAAAAAAAAAATCATCACTTAGGCCGGGCGCAGTGGCTCATG

CCTGTAATCCCAGCACTTTGGGAGGC

TGAGGCAGGCGGATCATGAGGTCAGATCAAGACCATCCTGGCCAACATGGTGA

AACTCCGTCTCTACTAAAAATACAAAA

AAAAATTAGCTGGTCGTGTTGGTGCGCGCCTGTAGTCCCAGCTACTCGGGAGG CTGAGGCAGGAGAATTGCTTGAACCTG

GGACATGGAGGCTGCAGTGAGCTGAGATCACGCCAATGTACTCCAGCCTGGGT GACAGAGCGAGACTCTGTCTCAAAAAA AAAAAATCATCACTTAAAACATAGTGTTATAAGAAAAGTTTATGAATTTGTGTTGG

GCTGCATTCAAAGCCGTACTGGGC

TGCACGCAGCCTGCAGGCTGCAGGTTGGGCAAGCTTGCTATAGTGTATCTATTT

TCTGATATTTGTGACAGTGCACTTAG

ATGCTCTTAATGGATTGTGGCGTAACATAACCAGTGGCTACTTGATTCACTGAAT

TTTTTGCTCCTGAAATAGACATAAA

AAGTTGAATTTCCTTTATTTTATATGTCTAAGTAACCCTAGATTTTAAATTTTAAAA

CAGTGTGTAAAGCACCATCAACATTTGGGGCTCTTTTTATAAAATAAGTCCCTGA

AGTTTGCACACAGAATAAGTCATGC

ACACTAAAATAAGCAGCTGTTGCCCAAGTCTCTCTCTGTGTCTCTTGCTTTCAGC

CTCTCCCAATGCCTGCTTCCTCTTA

ATGAGACACTCTCTTTCATGTAACCTGAAGTATACATGTTCTTCACCTGTCATATT

CTTATTTTAGGATCCACCCACTTC

AGTTTCCCTTGGACTGCGAATGGAAGAAATGATTTTCAACTTGGCAGACACACAT

GTTTTAATTTTTAATACATA I I I I I GCATGTCTGAAAATAGGTGATTGGTTTTAAAA

TAAAGATAGGATATTA I M I M A

ATGTATAATTGGAATAGTGAGACAGAGGTTTGTTGGTAATATGGCAATGTGTTTT

GCCTTTATGGGATTTTTATTTTGCT

TTACTTTGGCTGTTGATTTTCAGAGTTAGATCCTACATGACCAGTAGTAAAGATT

AAAATTTGATTTCAAGAATTTTAAA

CTTTGTTGATTATCAGAAGCCACATA I I I I I CAGTTACATAATCACCACTAAGATA

TGTCCAACATAATTTGTAACACTT

GCATATGTTATGTCAAATTC I I I I I I I I I I I GAGACTGTTATGTCAAACTCTTACT

GTATTAGGACCGATGCTTTACTTT

CTGGCTGATCCAATTGGTAGGAAGATGTTTTAGTCTGGCCTCTGCTGTTTGGGT

TGCCAGGACTGCTTATGTCACACTGC

TGTTAATTTTATTGGTCCATTATGATTTCAGGGCAGACTCTTGTAAGATGGCTAG

CTGAATTGTTACAGGCACTGACAAT

CACAATGGTCTTGATTAGCAGCATTCTTAAGAGTAGAGAATATTGTTTTTCATATC

TC C ATTATG AG AAAATATGACTAT

AACTACATTTCGTTCTCTTCTGAATAGAGACAGTTGTTTATTTAAATTCCGCTGTT

AAGCAAGCTTGTGGTAGGGCTGCC

AACAGGCATTAATCATCAAGTTGTTGTTTTGTTCGATTTATAAATGTATGTTATTT

CAGACAGGAGAAGAATCTGAAAGA

TTCAAATAGAACTTTTACTGATCAAAGTTAATTGGCAGCAATTGGCCTTTAAATAT

TTCCTGATGTTTCCCTTTTCAAAA

TCAAGCAAAATGCACCTTATTTAATAGATGAGCCAAAATCATTTGAAAAATGCCA

GTGTGTGACAGAACTTAAATGCGGT

TTGTTCAACATCTGTCCTGTATTTTGATTTCTTTTCATCTGCAATGAAACTACTCA AAGACGAATTGGCAACTTCCAGTG

CAATAAAGTTATTAAATATGCTTTATTTTTTCTCTTCCTTAGTGAAAAATTTATTTT ACACTTAAGGGTAAAATACAAAA

TTTTTTTGC TTTAAAGTGTTTTCCTCAAGAAGAAATTACATGTAAGGCATTACT ATTTTTTTAAGGTAGAAATATTCC

CATTGAGTGACAAGTTTAGAATTAATTGCCTTTAGAAACACATAATTATCATACTT ATTTTCAAAAGCTGTTTCTGATTG ATAATTGTTTTCACTTAGAGAGCAGCACAAAAAATTCCACATTTTACTGGTATGTT

AAAAAATGCCTTTCCTAAGTTTCC

ATTTGTTTCTAAAG ATAC AG AGTAG ACTG ATTTTTTGTC CTG G GTAC AATCTTGTT

ACCTTTATTGATGTTAATAGGTGT

TAATAACAAACATCAAGAAGAAATTTGTAGTAATTTCCTATTGTTTTAAAACTCTG

TACCTTCTTAAAAATGCCAAAACT

TGGCTGGGCGCAGTGGCTCATGCCTGTAATCCCAGCGCTTTGGGAGGCAGAG

GCAGCTGGATCACCTGAGGTTGGGAGTT

CGAGACCATCCCGACCAACATGGAGAAACCCCCGTCTCTATTAAAAATACAAAAT

TAGCTGGGTGTGGTGGCCAATGCTT

GTAATCCCAGCTACTCAGGGGGCTGAGGCAGGAGAATCACTTGAGCCCGGGAG

GCAGAGGTTACGATGAGCCAGAGATCA

CGCCATTGCACTCCAGCCTGGGCAACAAGAGCAAAACTCTGTCTCAAAAAAAAA

AAAAAAAAAAAGTGCCAAAACTTGCT

TATACAGTGCCAAAATTAGGTGGTGAAAAGTGAGATGTTAGGCAGTCATAACTT

GTTATAATATAGACCAAAAAACTTCC

AAACTCAATTTCAATGTAGTAT TCAGATTTTTGTTTATTGACGTTATTTACACT

GATTATATAGAGACTGAAATACTA

CTCAAATATATTAAAAGCTCTAGG I I I I IAGGGTATAGGAGATGTATTTTTTTCTG

TAAACCCAAATTTCAAAAGAAAAA

GAAAAAGCAGTCTATGAAGTTACTGAAATATCAGATTCTATTTTTGGCATGAGGA

CTATTCCTTAGTAGAGGTAAAGTAG

AAAAGAGAATGAAGGTATAATTCAACCAAATATATAAAACAATCATGGGAAAATG

TTTAAGCTTGTAGCCTTACAAAAGT

TGATTTTATGTTAGGGATATTAACAAAAGTATAAATGAAAGATTCATTTAGTCTGT

CTTTGGAAATTGTTTACTTGCCCT

AGATTTGTACAATATTTTAAAAAATAAATTGGTTTTTTACTTATTTCAATATTTTTTA

TTTTATTTTA M i l l GAGATAG

GGTCTTGCTCTGTCATCCAGGCTGGAGTGCAGTGGCACCATCTCAGCTCACTG

CACCCTCTGCCTCCCAGGTTCAAGCAA

TTCTGCCTCAGCATCCCAAGTAGCTGGGATTACAGATACACACCACCATGCGTG

GCTAA I I I I I GTACTTTTAGTAGAAA

CGTGTTCTTCATGTTGGCCAGGCTAGTCTCAAACTCCTGACCTCAGGTGAGCCA

CCTGCeTCGGCCACCCACCCAAGGCA

CTGGGATTACAGGCATGAGTCACTGCTCCCAGGCCAATCTTTTATTTTATATTTT

TGAAATTCAACAAACCTGGGACTGG

TCATTGCAAATATGTTTTCTTCTAAATAAAAAGTTAAATTATTTTTTGGCAAAAAAA

AAAAAATGTCTATTGGCATAATG

CTCCTCAGAACACCAGAATTATCTCATCAGGGAGAAGTGATGTGGTAATGCCTG

ATTTCACACTCCAACTAGCAATGGAT

AGTGGTAGGGGACATGAATATTCTAATATATTTAAGTTACATGTTTTTAGCTTTGT AAGGTCTATAGCATAAGTAATTTC

AAGGAGCTTAATAGTCTCTTGGTTAGCTTGAAGTATGATGCCTGAACTCTAATTG TTGACTTTTGGTCTTATTTTCTTCT

AAATCTTAGCTTTTGCCAGCTATCTCTTGGTATAATTTGGTTAGTTCACTCCTTGC TTAATAAGACATTCTGACTAGTGA

AATACACCCCCTCTGTGAGGGGCTTTCAGTAACATTTGTCATATTATGAATCAGT GTACATGCTTAAAATGGTTATTAGG ATATGATTTAGAAAATTTAAGGTATAATTTTTGAAGTACTTTAGAAATATCTTAATC

GAACTCTAAGAACTAAGTTAATT

CATTTTAGTTTCATAAGTCTTGTTTGTAATCTCACTTAGTGACTTTTTTAATTTAAA

GAAAATCACAATTTATATCAATT

CTGTTGACACACCAGGAAAAACAAAAAGGAGAAAAAAATGTTTTCTGTACCATAA

AAAATGTTTGACGTATGAAGGAACA

TGAGTGACAGGGAGACATAATTATCTTAGGAAATAATATTTTCCCCCCAAGAGAA

GGGTTTCGACTTTAGTTCCGTAGTG

TACGTCTGGCTTTCCTATTTCTAAGTCTGACTAACGTCCTGCATATCATTTTACAA

AAAAACTCTCCATGTGCAATACGA

TTCAGCTTCTTTGGTAAAGTATGAC

TCTTTCTGACTGGTTGGAGAAAGAGTTCTTTCTGTTAGCTTTTTATTGAAAGGCA

ACCTTCAAACGGAGTGAGGAAAGGC

CTTCAGCATACGATAATATTTCAGTTTAACCTGCTGCTGGTCAGTTCTCCTGCTG

GATTTTTCCACAGTTAGAAGAAAAT

ATTTTATGAAGATGGATACAGGTCTAAATAAAAATAATGCTTTAAGTTCATGTTGA

ATTTTCTTCTTCTGTCTGGTTAAG

GAATATGATTTCACTGGCATTATGGTTGCCATCCTAACCCATGTAGACAGAGTGA

CCTTTAAGAGTCAGCAGGAACCAAA

TCTCTGGTCTCGGGTCACCCGTTACCCCTGAAGAGTAAGGATTGGGAAGAGTG

GGAACCCTCCTCTCGTGTCTGTGAGAT

GGTGGCACATGAAACTATTGCACATACCACGCTCACCCCGTTAGTAGCAAAGCT

GTTCTAAACTCCCCTCTCCACTCTTT

CAGTGCATTGTTTAGTTGGAGCAGACCAGAGCATTCTTTGCATCAAGATTTACAA

CATGAATATTCCTTGGGGCCTTCAA

TATGTAGACTCCTAGCCCGGGTCTGATATTTGAACACCTAGACTTGTTGCCCCTT

GTTTTCACCCTTTCTATTAAAAAAA

AAAAAAAAGGCCTGGGGCAGAAAGTTCTGGTCCCACTAGAGTATGAGTTTGATG

AGTCCAGGGCCAAGGAAAGGGGCTTG

TGGATGGTCTTTAGGTAATAGCATTATGTAAAAGGAGAAAGCTGCAATGATTTTT

CTTGTAAAGCAAAGAGAAGATTATC

CCTTGGTTTAATTCAGTCTTTAAAGTTGCAATAATATTTTCTTACTTTAAAACTTAG

GTTATGATCTATAAATTATTTTA

ATATGTGTTATTTATGTTTAI I T I lAGCTAGGAAAAAATTCTTTCTGTAAAATATCA

GTTACGCCAATGTTAAGAAACTT

CGATTTCAAGTAAAGCACTTGGTACTGACATACTGTTTGTGCTTCCAAAAAGTGT

GAAATATTTAATCCGATTCAGAAGT

TGATTTCTCAAGTTGGGAAAATATAACTCACCTAGAAAGTCCTTTATTCTCTATAA

ATAAAGAAGAAACTCTGATGCTCT

ATGTTCAGCTTTTGTGTGGATGCTAGAGCTCTGGTTCTCTGCTGCCAAAGGATC TCACAGGTGCTTGCAAGGAAGACACT

GTAGTTCTTTGCTGCTTCCAAACCCCTCCACAAGCCAAGAGTAGCCATATGAAA GGGTGTGAGCATATGGATAATTACAG

TCCAAACCTAAGGGTCCCTATGTATATGTACACACATATGGAATGCCACTTGTCA TTTTCAACCCTGTACACAGACTGGT

ATACTTGGTGATTTTCTCCTGACAAATTCAGCAAACATAAAGGACCACATTTTTTA AATCCTAGTAAACATCCCTTATAA CACTCAGAATGTTGCCTTATATATGTTGACCATGAAATGGATATCCTTTAGACTG

AATTGTTATGTGTTGTCTTAGACTA

G AATAC C G ATTTTTGTTTC CTTCTTC ATAG AAG ATAATAG AGTTG ACTTTG C CTTC

CTGTGTTTTCAGGTAGGTTGTTCA

GCCTTGTGCATTTCCTTAGTTGACAAGAGTAACATCAAAACCCTCAGTTTAGGGT

GGGGAGGACAAAATAAGTTTTTTAA

GCAAGAAATGATTATTAGTCATAATAAATGCGTATTGAGTTTAGCTGCATTGTAA

CTAATGGCCAAAAAGAAACAATGCC

CATATTTTATTATTACTAATGAATAATTTTTATTGCTCTTCTTGTTTCTTCATCATTT

CTTATAAGATTGTCATCTTTAT

TTTATCTGAAGTTAAAAATAGAAATATAAAAGTATATTTAAAGATTTCCTTAACTCT

GATTTTCATCAGAACAGTCTTAA

AATATAGCTATTTAACACAGTAAAAGATAATATATTATAAATAAATTTATACTCTGA

ATACTTTATATTCTACTGATAGC

AAACATTGTACTTAGCAGTAAAATACTGGAAGCAAACTGTCATTACAAGTAAAAA

CAAGATAAGAATGCCTTCTATCAGC

ACTATTATTCAGCAGTATTCTAGGAATTTTGGACAGTACAATAAGACACAAGCTT

TACTTAATGAAATGAAAGAGATTCA

AATTATTTTTGTAGATAATTTGATATGTAGAAGACTGAAGAAAATCAACTAGAATA

TTCTTAGAATTTACAACAGCCTTC

AGAAAAATGAAGAAATATAGAATTCAAAAATCAAATTGATTTGAAATTTTAATTATA

ATTTAAAATAGCGGTAGTTACTA

AAACACAAAAACAAAAAATATCATAAAAGGAAATTTAGGAAGAAACCTTATAAGAA

GTATTTGTGAAGAATATATGAATA

AACCTGTGACATCTTACTGAGAGATATAAAGGAAGGTTTCAATAAATGAGATTCT

TTCTTTCTCATTTTTGTAATATGAA

AAAATTGTTCTAAGTTCTATTTGAAAGGATAAATCAGCAAAAATACCTAGCAATTT

TTGACAAAACATGGGAAATGAATG

TCTGACTGAACCAATTATTAAAATGTTTTATAATTCTACAATAGCGTGAAGAATTA

TGCGGCCTAATGTTGACATTGGAC

TAAGTATGAGAGAAGAAACAAACTGCTCAGGCACAGACCCAACTTAAAGAAAAAT

TGTGTATGTTTTAAATAAAACCAAG

CAAATTAATAGTGAAGTAATAGATTATTCAGCTTCTATGCTAAAATAGGTAAACAG

TTAAAAAATGAAAGAATGAAACAC

AAGGAGAATATATAGGTAAATGAAAAGACTAAATAAGTCAAAATCTTCAGATGGC

CATTTGGAAATAAGTATTGAGATCC

TATAAAAGGTTAATACCATTTGTACCAATTATACATCTAGGACTTTTTACTAATCA

GAGATAAAGGCAAGAATTTATAAC

AATAAACATTTCTAACTTATGAAATCCATCATATTTTGTTTCTTCATCTGGGTGTT

AGTTATACCCATGCATTTAGTTTA

TAAAAATTCATTGAGCTGTAATATTTGTTTAATAAAAAGTTAATTTTTAAAAACTAA TATTTGAGGAATATACACACATA

TTGTTGGCTCACAATTCTATTATAAAATGTATACCCTATGTATAATATATTCACTG CTAATTTCTCTAGCACAGGACCTG

GCCCATAGGAGGACCCAGTAAATATATATTGCATAAATAATGGAAATATATGTTG ACTGTCTTATGGAAATATAAATCGT

AGAAGTTTAACATTGATTGTTTATGGAATTTGTGGTTTTATTATTTTTTTATTTATG CTTTTCTGTATGTCCCAAATTCT GTAAAGCATATGCTTATTTCTTCTGTAGTCAGAGACAAATACTATTTTTAAAAGCC

ACAGATAATATTAAAGACACAACT

GAGCCAAATATAACCAATCAAAGAACTAAGATTACTGCAATTCATTAAGAAATAG

GTAAAGAAATAGCTCCACAGAAGAA

ATGAGCAAAGACCATGAACAGATAATTCACAAGAGAAAAGCATTCAAATTATCAA

GCTACAGACGAAAAATGTTGGATTT

CACTTXTAATAATCAATCAGGGCGGATCACGAGGTCAGGAGTTTGAGACCAGCX

TGGCCATCATGGTGAGACCCCATCTT

TACTAAAAATACCAAAATTAGTTGGGCATGGTAGTGTGAGCCTGTAATCCCAGTT

ATTTTGGAGGCTGAGGCAGGAGAAT

CCCTTGAACCCGGGAGGCGGAGGTTGCAGTAAGCCGAGATCATGCCACTGCAC

TCCAGCCTGGTGACGGAGTGAGACTCC

ATCTCAAAAAAAAAAAAGAAAAGAAATGTCAAGTCAAACAGCCTCTTGCACATGA

AATAGGCAAAGGTTTAAAATAAATA

ACATGATCTACAATGCAGTAAGATGAAAACTTGTGTACATTGCTGGTAAGGATAT

AAAGGGATACAAGTTTTCAGAAAAG

CAGTTTCATAATATGTATTCAGAACTTTGATGTGCTGATCCTTTTACTCAAAATTC

CACTTTTAGCATCACTTCTAAGAA

AATAGAATTACAGATAATTAAGATAATAGCAATAAGCATTTGTCAAACACTTGTCC

TATATCTTTTACATACATTATTCC

ATTTAACAGCATGATATCTCTACTATAGTAACACTATTAATATCTTAATTTTATGGA

GGAGCCAGGTGAAGCTTAGGATA

GGTAAAAGAAAACTTGTCTAAGGTCACACAACTGTAGAGTGGCAGATTCTAGCT

CAGGTCTGACACTTTCCAGAAATCCA

CTCAACCCCATAATATACCCACCCCCCATGGTTTTTTTACCACTACAATAGAGAA

ATATTTTAAATATCCAATAACAGAT

TAAAAATGAAATAAATTAAAATACATCCTCATAAAACTTGTTTTAAGACAATTTATT

GTATATAGGGAAGGTATTCATGG

TCGATTTCAAGTTTTAAAAATTAGCTCTAAGATACAATCTAGCCTATTCAACATGT

CATACCTCTTCAACATGTATGGCA

TATTTAAAAAGTGTAAAATTCGGCCAGGTGCTGTGGCTCATGCCTTAATCTCAGC

ACTTAGGGAGTCTGGGGCAGGCAGA

TCACTTGGGATCGGGAGTTTGAAACCCGTCTGACCAACACGGTGAAACCCCATC

TCTACTAAAAATACAAAAATTAGCTG

GGCGTGATGGCGTGTACCTGTAATCCCAGCTACTCGGGATGCTGAGGCAGGAG

AATCAAACCCGGAGGTGGAGGTTGCAG

TGAGCCAAGATTGTGCCACTGCACTCCAGCCTGAGTGACAGAGTGAGACTATTT

CAAAAAAATAAAATAAAGAGTAAAAT

TTGTATTCAACACGTATGGCATACTAAAGAAAAACTGGAAGGGAATACATTTTTT

AAAAGCCACGACGATATATTTTCAA

GTTTAAAAGTGATCAGTCTACAAAATGAATGATAGTATATAATCCTGATTTAATGT CTATGACATATTAAAGGTAATATT

TACAAGTTGCCAGTGGTTATTTCTGGGTGTTGGGGCTATAAATGCTATATTTCTG TGATGTTTCAGAACCGACATGCTAT

CGCATCCAGTAGTCTTTTTAGACTTTGTCATTTCTTTCCGTCATTCAATATTCAAT ACCTATTTATTGAATAACCATTAA

GTACCTAGGCACAGTTCTAACTACTGGGGATACAGCAATGACTTTATTAAGGAC ATTCTCCTTATGGAGTTAGTGCTGTG GAAGAGAAGTAGGAATAAACAGTATGTAAACAAAACACTTGCAGGCAGAGATAA

GTGCTATGAAGCAAATCAAGCAGTAT

TGGCTAAGGGATGAATTGGAGTGGGGCATTGTTAGCTGAGATGATCAGGAAGA

GATGTCTCTGAGGAGGTAACATTTGTC

AAGAAGGGAGAAGCTGGGAGGGAAGCAACTGCAAGGATATGGTAGGAAGTCAT

TTTGGGAGGACAATAGTAGGAATGAGT

TTCACTCCTTCAGGAAGTGAAAGATGCCCAGTGTGGCTGAGACAGAGTAACTAG

GGAAGAGCGGCAGGGTCCAGATGATG

AAGGGTCTTGTAGACCATGGTAAAGAATATTTTTTTTCTAATTGCAGTGCGAAGC

CATTGGAGAGTTTTAAGCATGGACA

TGATAGAATAGTGTTTACACTTTTAAAACATCAGTCTGGTTATCAAATGGAATCG

GGGCAAGAGTGAAACCAATGAGACC

AATTAAGAGACTGTCTCAGCCAGCCTAGGAGGACATTAGCTTAGGGCTGAGGAT

ATAGCAGTCTGGATGTGGTGAGAAGT

CACAGGACTTACAGTATCCTTTTATGGAAATATGGGCAGAATTTGCTAATGGATC

AGACGTAGGTGGGGAGGGAAAGCAG

AATCAAGGATGAGCACTAGGTTTTGCCTTGAGTACTGGAAAAGTGATCACACTTT

TGAAGAAGAGTGTGTTTGGGGGGTG

GCTGGGGAGGAGTTGGATTCAAGCATTCTGTATTGAGTGTATTAAGTATGAAAT

ACATATTCAACATCCAAGTCAAGAGA

TGACATATAAGCAGGTAGACATACAGGTTTGGATCTCAGAAGAGAGAGGCAAAT

TTGAAGGTATAGTTTTGGGAGTCCCG

ACGCCATAAAAATAGATGGCACAGGGGAAGGAATGAAAAATTGAGAAGGGGGC

CTAAGATTGAGCATTTTGGTACCAACA

ATTAGAAATAGGGCCAAGAAGAAGAGCTTTGAAGAGAGAATTGTATGGTAGAGA

GAATTCCACAAGGTAGGTGGAAAAGT

ATGAGCTATCACAGAAACCTGGAGAATAGTGTTTCAAAAAGGAGGGAGTGGTAT

ACTCCTGTGTCAAATGTGGCCAAAGT

CAAGTAAGATGAGGACAGAGAAGTGAGCGACTGTTAGATTGGGCAAGAATGTTG

GCTGTTAATGACCCTGATAAGCACCA

TGTCAATGAAGTGGAAGACATCAGCCAGATGGGAATGGGTTGAGAAGAGGATG

AGGGCTGACAGTGAAGACAGAAAGGAT

CAAGAATTCCCACAGAGACAGACACAAAGAGATGGGCTATGGTGTGTGGAAGG

CATCATGGAATCAAGGGAAGGTGGTTT

CATTTTTAAATAAAAGATATTTAGGCATGTCTGGATGTCAGCAGGAATGATTCCA

TAGGAAGGAAAATACATGTGGTATG

GCAAGGAGAATGACTACAGGAAAGAAGTCCTTGGATAGCAAGACAGAATGAAAT

CCGGAGCACAGGTTACATGGTGGGTC

TTAAATAAGAAGGCAGGTAAAGTCTGCAGGTACAGTGCAGTCCAGGAGTGGGG

ATTTGGTCATGATCAAAGGAGCTGGTT

CTTGCTATAAAAATATTTACTGCACTTTATTAAGATTTTCTGCATACCTCGGTCTA

CCCCCCACTGGGAAATCCCTTGAG

GCCAGGAATCAGGATTTGTCCACTTAAATCCTGAGCTCCTGACACAAGATTTGG TACATGGAAGGCTCTCATATGTTTTC

TTTAATTACTTATTTTCAAATTTTTCTAATAGTAGTCAATGATGAGCATTACCTTTA TTATTTAGAAAAGTTAACTTTTA

AAAGTAAATGAAGATTAATCTCAATCTTAACAATTAAAACACCCAAGATATTAGAC TTTCATATTACGTTAAGTCAGATT 9^

TTCAAAATCCTTAATGTCAAGACACAATATACATAGAAGAGGTATGTTATAAAATG

ATATGCAGCGTGGTTCCAGTTTGG

GGATTAAAAAACAAAGGTATGTATATCAAAAGAAAACAAAGGCTTTCTTTTTTTTC

TTTTTTTTTTTTTTTTGAGACGGA

GTCTCACTCTGTCGCCCAGGATGGAGTGCAGTGGCACAATCTCGGCTCACTGC

AAGCTCTGCCTCCCAGGTTCATGCCAT

TCTCCTGCCTCAGCCTCCTGAGTAGCTGGGAATACAGGCGCCCACCACCACGC

CCAACTAATTTTTTGTG I I I I I AGTAG

AGACGGGGTTTCACTGTGTTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGAT

CTGCCCGCCTTGGCCTCCCAAAGTGC

TGGGATTACAGGCATGAGCCACCGCACCCGGCAGAAAACAAAGGCTTTCTATAT

TTAAAATGATACAGAAATAATTTCAA

CAATAAACACTGATTTATCTCTAGATAGTAGGATAAGAGAAATTTTCTGTTTTTAC

TTTCTATATGTAATGTTTCTTATA

ATTAGGAAAATGCTGAATGTAATCATGGCTCTCTTATTCCAGCCTGACTCAAAGC

CCAGTGTTGTTTATGTGGTTTGTTA

GCTTGCAGTTCTAGAGACACCAGAGGTGGAACTGGTGAATTCATTCATTCGTGA

GGGCCCCCTTTATGCCTCAATTTCCC

ATCTGTAAAACTGGATGATGATATACCTCATCGGGTCAGTGTGAGAATTGCGTG

AGTTAAAATCTGTTAAGTGCATACAA

CTAGCACTGGCTCGCAGGAAGCACTAAGGAAGTCTATGCTGCTGTTATTGTCCA

TTGAGTACTTCTACATGCAGAATGCT

GGGTCAAGCCCTGTAAGGCTGTAGGGACGAAGGGAAAGTGACTTAAGGAGGGA

CGAGGGAATGAAGGGCATGAGAAAATA

AGTGGTTTGCTGCCAAATTCTCTTGTAGAATTCCAACAGAAATCACACCAGACAC

ACCAAAAAAAAAAAAGAAAAGAAAA

AAGAAAAAACATCTGTCAGTGGCAAATGCCCCTGCTCCAGGTAGATCACAGAAT

ACTTCTTTGGGTCCATCCTTATTCAT

GGGCTCCTCCCACCCCCAGTTGCATGCCGGGAGGTGTGCTGTTGGCAGTTAAG

TAGCTACTGTATCTGGTTCCCGTCACA

GTGTGCTGTCCCTCATAGAAAATGAGGATGCTGGCTTCGTGTAAAAAGACACGT

TTCCTTGGTTGTGCTGATAAATGTCC

TTTTACTCGTTGTCTTCCTACCTCATGTTTTGAAGGATGGCTCATGGAACTTGCT

GGTGGTGTCACGGCATGCAGCGGAG

CCTGGCTGGGTGTGAAGTCACTGTGGAGCAGAGCCTCCCCTTCTCAGGCCGCT

TGGACTGACAGTGCAGGCTGCTGGGCG

TCCTTAGTGAAGTAGGAGGCAGGGGGCAGCAGTCCTTTGGGAAACTTTTCTCAT

GTCAGTACAAAAGCACTGACTCCATC

AACACTGTGAATTCAATCTTATTATCTGCAGTTTACACATGTGGGAAATTGAAGG

GAGGTGGTTGTTGCCCAAAGCCACT

TGGTTAGGTGATGGTAAAGCTGAGGTTGAAACTCAGGTTTTCTTTTGCAAACATC ATTGCACAAAACCTTTCTAGCCAAG

TCAGAGAACCTGGCTATTCCTTTTTAGCAAAGGAAAGGAAGTCAATAACACCTTG CTGCTGTCACACAAGGATGCTATGG

AGCGGGGCCTAATAACTGGATGGGGCTTTCTTGACTGTATTCTTTGAAAGCTTT CTTTCTCACTGTGTCTTACAAACCTG

CAAGCCATAGTTGCCCAGGCAACTTAAAATCTATTTTGGAAATGTTGAGAGGTAG GTCAATAGATAGAGAGTGGACAGAA AG AC ATGTTGTTGTAATGAAC AAC G ATAAAATAATATTG G AG C AGTC C CTAC AC C

ACAGACTAAAAAGAGCTACTTTGCG

CTGAATGTCCTCATGCTCACTCTATTCTGGACACTAGCTAGGCACTGAGGATAA

AATATGAAACACATTCTTTGCTGTCA

AGGAGCCCAGATACTGGGTAGAGACAGAAAAGTGAAAGAATAATTCCATCACAG

CAATGATGGAGATGTCTGTGGTCATT

AGCTAGAGTCTGAGAGATGGGATGGCTATCCCAGATTGGGGAGAGGTGGCAAG

GCAGAGTGTTGGAGCAGTGACTTCCCC

AGGAAGAAACATTGAGCTTGAGAAAGCATGACATTTAGGAACAGAGCCTGTGTA

GGTATAAAGCACAGAACACACAGAGA

GTGCTTGTGAGATGAGAAAATGGAAAGAAAGACAGAAGCCATGTCATCAAAGGT

TTTGCACACTTTGCTAAGGGAATTGG

ATTTTACAATGGATGCCGAGGGTAACCAGTAAAGAAATTATAGCACTACCAAGGA

GATGAGTTGTATTGTAAAGTCAAAG

GGGTCAATAATGATGAGAAATCAGTGGCATTTGTTTGTGGAGACAGAGAACAGG

GGCTGATGTGAAAGCTACTGAGAAAG

TAGTTTGGGTAGGATTTGCAGATATATTCAGTGTAACTATTCAGTGAGCCAGTCT

TTTAGTTTTTGGCTTGAGCAATAAG

GTAGAGATAGAAAACCCAGGAGAATGGTCTGGCTTTGGAAGGAAGATGCTGAAT

TCAGATTTGAGATATTGAACCTGAAG

TGTGTGTTGGACACACAAGTGGAGTCCAGTGAGCAGGTGTATGCACAGATCTG

CAGCTCAGAAAACAGAAGCTGGAAGCC

ATCATTTCTGACATCAGTTAAGATTTCCCAGGAAGAATATATTGAATGAAAAAGA

AGAGTTATGTCTTAGTTTAGAAACA

CAGAATCCAAATATGTATTGAAAATTCCAGATTCAGAAAATTAAATGATTTATGTG

AGGCAACTGTGGCCATTTGGAGCA

GCAGTTATTCCAACAGGCTGTTAATTAACCTGTTCATGAAACTGCTTTCCTAGTT

TCGCCATAGTTTCTCTATTCTGTTG

GGTGAAGCTACCAGGAACAGTGTGGTCTGAAGGGACAGTAAACACTTCAGAGG

TGGAGAGACCTGGGTTCAGTCCTAGCT

TCCTGCTAATGTATGGCCTCCACACAGTCAACCCTGACTGGCTTCAGAGAGGGT

GACAGGTAGGTGTGGAGTTAAGGGAG

ACAACAGAATCTGGAGCTCCTGGCCGGTGCCTGCAGCATAGTGGTGCCTCAGC

GAATAA€AGAGTGTCTTCCTTTCTTGT

TGAATGGAAATACCACTTCAGCACTCACACAGTCTGCTGGATTGCTTCAGTTTAT

TTTAAAGTCGTGTGAAATGGACCTG

TCAGTGTCACCTATGACAGGCTTTTGGTTTAGATCTTGGATTTTGACAGGTGCTT

GCTAATCTTCAATCTGGGAGAGAGT

CACTGCTTTGTAGAGCCAGATTTTCCTCAGTTACCTTCATAGGAAGTTTGCTTAG

TAAAGAAAGGAATTCAAGGTGCCCT

TAGACCTTT ΓT I I I I TATTTTTTAAGGTTGAATACTGCAAGAGAAGGCCTGTATGC

CTGTAACTTCAACTTCCCCAAAAT

GTGTTTAAGTTGCAGTCCCTGGAGACTTCTCCCATTTCAGTGCAGCTACAATAG GGCCTCTATTTGGGCTCAGTTCAGTA

TTCTAGCAGCATTTCTAAAGAAAGGCTTTCTAAATGCTGGATCCTTCTCACTGCT AATTGTGTGCTACTCCTTTGTAAGG

CAGGTGGACGGTCGGGGTATGGGGAGAGCATGTTGCTTTTGATCAACATTATTT TATAATCTTTTGCCTCTTCAGAAATA ATCAACTAAGAATTAACTTCTGGAATTGGTATTTTGTCAGAAAGTTAAAGGGAAC TCTTCTGTCCAAGATATCATCCTTT

TCAATTGGTAGGAAACAGCTTGGACTAGAAGAATGTTACAGTGGTTACTTCTGA GGGATGGAATTGGAGATGAATTCTTT

CAAAATTATTTATTTCCACAAAAC

TAACTTCAATGACAAAATGCACTAACATTATCTATTAACTGATTGGGTCATCAATT

CTTTCATAATCAAATCCTGTCAAT

CAGATAACCTAGAAGGTTTCAGTCCATGATATCTATGTGTTTGGTCCAGCGTTAG

AAAATAAGTAGGAAAAAAAGACACG

TGAAAGATGGTCTTTTCTTCAAAGAGTTTCTTATCTGGTAGTGTACACAGAAGTT

GTCTGTGTAAGTCTTTGCTTATTCA

TCAAATGCAGAGTTTTCAGGAGAATTGGAAAGAACGTGAAATACACATGACACAA

AGACTGTAGTCTGTCAATAATGGTA

GTTATTATTGAGACTGTGGGTTGCAGAGAGTATGTCAGGGATGGAGAAGGGAAA

GAAACCAATAAAAACTTAATGGAGGA

GCACTGCCTTGAAGGAGAGAGAAGGCATTCCAGCAAGCAGTAAACACAGCTATT

GTCTGGTGCAGGGAGGCAGGCTGTTC

ATGATAGAATGTGAGGGATGGGCACATTGGTAAGGTTAGGGCTCATTATAGAGG

CCCTTGAATGCTCAATTGCATAGAAA

TATATTAAGTGTTTCCAATTTAA I I I I I CTAATACATCTATTGAGGGAGATGGACT

AAAACAGAAGTAAAACATATTCCC

TGTCTCCCCTCCCGCTAGATGCTTTTGCTCTTTGTTGGAGACTGCCTTGCCTGG

AACAGCAAAACCTACTTTCAGAAAAA

TGTATCTGAACGTGCTGCCCAGGGTGGGTTGGAAGAGGAAGGGCTTTACAAGA

ATTTAGTACCTTCCCTGACAGAAAATC

ATAGTGACAGTCACTGTACCCTTTCTCTTTAGCCAGTCCCCATGTTTAAAACCGT

AAAGGCAAGATCTGCATCTCATTTA

TTTTTGTGTGTGTGGTGCCTGGAATAGACATAGGTGTTCAGTCCTTATTAGTTGA

ATGCATGAGAAAATGAATGAAAGAA

TCGAGAACATGTGGTTATGTATTTTCTAGTGACACTGCTACAGTTTAATATATAAC

ACACATAATACTCATGCTATTCTA

ATGATACATAATATTTTCCTGAATAACATATAAACAACCAAAGCATGATTATTTAT

AGCTCTAAATTATACTTCTTGAGA

AGAGGGATGTTTTTATAGGGAGTGAAATTATGAGTATAGACAAAACTTTCCCTTT

TTTTTTTCCTTTCGGGCAGCCAACT

CCATTTCCCCTTTTAACACCCAATGATTTGGGGGCAGGCACACCAGGGCTCCCC

ATGCTGGAAGAAATTTATCTCATAGT

GCAGTTCACACCAAGGCAATGGAGCTTAAATAGTTCACTCTTACCTGAAGTCTTT

GTCTTACAAAAGAAGATTCAGAAGG

GATGATTTTGTGTGGTGGAATTTCAGACCCACGGATACTTAGAGGAGAGAATTT

TTTGAGTAGGAAAGTGCCCCTCCAAG

GTCCTCTCCTCGCCGCCAGGGTCTGACAGTGAGGGAGGTGATGATGGAACCCT

TCCTGCAGCTCACAGGGAACTGCTTTT

TAAAAAGTGCCTATGTCCGGTTCTGCATATTTTTCTTTCTGGAGTCAGTAATAATT ACATGGCAGAGAAAATCAGCTTGG

AAAAATCTTTATTAACTTATTAACTAAGGATATGATTGGGCCTGATTTATTTTGAG CTGTATGTTCTTTTATGCAAAATT GAGTGTTTCTTACTATTAGAAAATTATCTAGCAGTATAAGTTAACTGAAGTTCATA

AATTAGCCTCTTTTAAACTTCAAA

ATTTCAACCCCACTCTCACCCCTGCAAAAGAAAAAGACATTTAAAAAGGTTAACT

ACAAGAAAATATTTCTTTACAAAAT

GCATTGACATATAGTTTGTAGTTAAAGATGAATCTATAGACATGTAATGGCATTA

AACAGAAACTAATATTTGTTGCAAA

TGATTTTAGGAGGAGTGGGAAATTGCTCCAGAAAAAAAAATAATGTATGACCTAT

TTAATATTGGAGCTTATAGTTCATT

GTTTTATATGAGCGAAACAAATATTTTTGTGCAAATGAAAGCTCTAGTTAAATGCT

TTGTGAATAAATTGGTATGCGATT

CACTTTCTGCAAATGGCCATGAAAGTTAAAAGAAAGCCGTTGCAGAAAAGGATG

AAAGTTCTCTCGGTCTGAATATTTTT

CCATGCTTCTGGCAGCAAGTTTGCAATAAAAAGAATGATTTTTATGACATTGTGA

TTATTCACAGCAAGCCTAGAGTTGC

TGTCACTATGAAAAATTCTTCTTTAGTAGATTCTCTCAGCAGGAATAATAGATCTA

AAAGAAAGGAAACAGGAGTTACAA

AGAACACTGTTGCCAGTGACAAATTATGTTTGCAAAAATGATGAGCAAGCCCGTT

GAAAATTGAGAAATAAGTTCAGCAG

TAAATCAAAGGCTCATTCTTAAGAGTAGTTCTGAGTTGCTGTTCCAAAAAAAAAT

GATATTTATATTAGATGTGTGCTGA

CCATTTTAAATATGTGTAATATTTTAATGATAAAATTGTACAATATAATTTGGCATT

GTCAGCAAACACTAATGCATTGC

CCTGTGTGGTATATGAAGGTTTGCACTGGTTATGATACAAATTTGTAAAATTCTA

TGTTCTCTTAAATTAACTGAACCAT

AAATAAAGCTGTGCATTTGGACATAACCTTTTGTGTACAATGTCAGCGGTAGGG

ACATAGAATGCATTGAAATACCTCTT

ACTATTGCATAAAACATAACTTAATAAAAGACGGATCTTGTACGAGATAGTATTTA

TTGTATACTTTTAAAAAAGCAAAG

AAATTTACAAGTATTGATATCATTAGATCAAATGTAATATATGCTATTATCTTTTAA

TTATCCTTATATTAAAACTGTTC

AACACTGAGTCACTAAGGTAATGGATTAGTTAATGGATTAATCCATTACCTTTAG

AGACTGAGTGTTGAACAGTTTTAAA

TAAGCTCATTTTGTTTAAAAGTTTATTTTCAATTCTTTCTGGCTTTCATTAAGTGA

GACTTTCTTTTCCTCACCCTAAGG

TTGAGAGTGTGAATAAAGACAAATATATTTAATATCATGTGGTAAAGAAGGCATT

CATGGTGACAAATATATTTAATATC

ATGTGGTAAAGAAGGCATTCATGGTGAGAAAGAAGGAACTCCACGCACAGTGAA

ACTGACATATCATATTTATTAACATT

GTACTCACTAAGAAGGAGCTATCTTCATGTGATACCTTTGGGATTCTGTAAGCCA CACGCTAAAGTGATATAATGATGTC

TTATTTTTTTTAATTAGCACATTTTAAAAGGGATTAATATTATAAGAAAATATCAAA ACTTTGAAAAACATGTGTTCATG

CAATCTTCTAGTTATTAATAAGTTTGGGCATCCTGGAAGCCTTTACTTAGTGATC TCAAATATTTACCATAATTTTATGC

ATATACCAGCACATAGCTTAAGCACTTCAATTTATTTTTTACCTTAACATACCATC TTCAGGAACTTTCAGTAAAGACTT 36

TGTTCTCTAAATATTTTATGAATATTAATAAATAGAATCAGATCAAAATAGCTAATA

CCTTACCTTTGTTGAAACTTCTG

TAATTCAAATCTTGTGTTTCTAATACAAGCACTTAGGTCTCCTAGCTAATATAAAC

ATAATTCTAGTAAAATCTCTCTCA

CTTAGATAATCTCTAAAGGATATTATAACATTAAGTATATTAAGACACTGTTTTTG

TTTGTAAATGCCATATGTGTGTCC

ACAAGTTGCATCTTATTGCCACCTCTCGAAAATAGATGCTGAAATTCCTCTTTTT

GGTTTCTGAAATTTGAAATTGTTTT

CTTTTCCCATCGGCTTGTATAGTTGTGTCATCCTATTCCTACCATAACACATATA

GATATTTAAATATTGACTTAAAATA

TCGGTTGGCACCAATAGTATGCTGAGTAGATAGTTTAGAGTGGGAATTAAAAGA

TATATTTGTCCAGGTGTGTCTTAATT

CCTTGACTTGCACTTCCCAAAACTGCCAAACAGAATGGGGCAAATTTCAAAACTC

CTTTTGTTTTTTATCCCCTGTGCCT

TAGCCACAGGTATCTGCACCA I I I I I I TCTTTTTCTGCAAGGAAGCATCATGGTG

TGAAGGCCAGGGAAATAGGAGGTCA

CCAGGTGTACCCTAGACAGAGATGACAGGGTCTGCCAGCGATTGCCAGTGTGG

AGAGGCTACAAACCACATACCCTCCCC

ATCTCCAGGAGTCAAGGGCTTCCAGAGAGTCCATGATGCTGGTGAAGCTGGGC

AGAAGAAAGCCTAGCAGCGCTTGAGCT

CTTCATGAGGTCTATTTGAAAGCACAGTGAGGGCTTGGACGTGCCCTTAGAAAG

CACAATGGAATCATGCTGTTCTGCAT

CAGTAAGTATAAATACACAGTACATCAGTTGGGCAGTCTCCTCTTTACAGAGAAA

AACACGCAAGTAAAAACATGGTTCC

TGCAAATTTACTAATTCATGGACAGAGGGTCTTTATCACGGAGCCTAGACTTGAT

GTACCAAATCAAATTGTCAGCTAGT

TGGAAGCCAGTTTCTTCAAATCTGTTCCATCAATTGGCTTCTTCCTTGACTACTC

CTCTCCCTCTGTAGAACTTCTAAAT

CATAGAGAAGTTTAGAAGATGTCCCATTTCACTTAGTTTTTCAAATTAGTTATGAA

AATATCTGGAGAGTTGAGAGTTAT

TTCGAAAAACCACCATGGAGAGCATTCCCTTAAGACCTTTGTCACTCAAACACTG

GTCACCCTGTAAGGCCATCTGTGTA

ATTTAGTTTTCTGTGAGAAAGATGGTATCTGGATAGATCCACAGACAGATATGCA

TTCTTTT CAAGTGCTGCTAATGGC

TTGACAAAATTAGCTTTCAAACAATTTATGTGAGTTTGTCACTAAAATACTAAAAC

TACCCATGTCCCATGTATAAATAA

ACATTTTCACATTTAGAAATAAGAGGAATACTTCTTTTAGTGACCTATAAATGTCA

AATATCAGGCAAGTATCTAAATTG

CTCAATAGGGAAGTCAGAGCCATGGATGTAATCACTTAATGACAGATAAGAGGG GACACAGAACCCAATGATCACTTCCT

CTGCCTTGGCCAGCTCCAGCTGGTCTCAATAGGCACATTGATGTAAAGACACAA TGAAAACAGCTAGTTCTCTGCTGACT

TTTATCCTTCCATTTTTGTTTTGTAAAAGGAAGTATTGAAAGTATTGAAACAATTA GTTGGCAGTATGGTGTTAGTTTTC

TCATGGTAAATTTGGAACATGGAGCATACTATAGCCTTGATCAGCAGGATTTGG ACTTGTGTAACTCAACTTCTCCAAAG

ACACATTGT CTTTTTCATATTATTCCCTTTTATAACATGCAAACTGCCTCACAG TCTTCCTAGAGATTTCCTTCTCTG TTTATTCAAGGACTTCAGGTTTACTTTGAGTTTGTGTTTTTAGTTTCATGTTCAGA

AGTATACAGCATCAGTGTCCTAGC

TATTACGTCACTGTTGGCACAGATAATAAGAATCATTGTAACTGTTTTAATTTTTT

TTATTAATTTTCCAAAAATACCTT

GAATTGTATGGTGTATTTTTACATAATTATGGTACAAGTGAAAGGCTTGGGGGGA

AAGTACAACTACTACAGTATCAAAG

CAGAATAAATATTTGCAGAAAAATTTTGCCATAAATTAATATTTTGCATATGAGCC

TTTGTACAAAATTTGAGTTATTCC

CGGGGATTGGTCCAAAAGAACATGTTTGTGGCCCAGCCCTAGAAGGCCAGAGA

TGATGATGGATCAGAAAAAGGTCAGCT

TGAGTCTTGATGCCAAGAATAATGACAGAAGGGACTAAAAAGGCAGAATAACAG

TGCAACCAGAGTTCCAAGAGCGAATG

TGCATTTGCCGCATGGCTGTGAGATCGTGCCCCATGTGGCTTTCTGCCCACTCC

CTATACCTACACCAGAACTGGGATTC

AGGACTTCATGTGTTGGCAGTTTCCCCAGCCACCCATGTGAGCATTTTATAATC

CAGCTCCATGAGTAGTTAGATGACTG

TTAGCTGAGGGCCAGCAGTAACTGAAGCTAACTTTATTTATTTTACTCCTCTGTG

AAGCACAGGTGGTGTCATATAAGGG

GAAGGCTGGCAAXGGTAAACCATGGTCATTTCAATTTTGGTATTTACTGAAAGAT

GCTTGTAGGATCCTGTTGTGGAAAA

GCAAAAGTTTGCAGGCTGGTAACTGTGGGCACAACACCGCACCTCTGACTGGT

GCCATCTGCCCTCTTTTCAXCTTCCCC

TGCCATGACGGTGGCCTAATCCGTTTTCCTGGTCCTCAGCTCTCCTACTTATGA

CACGCTGTGGCAGTGTGATATGCTAA

AACCTATGCTTTCACCACCTACAGTTGTACTCTCTACCCTCCACGCCTGGCAGA

GACCCGGTTACTACAGTGGCTCTCCT

TCTGGGAGCATTTTCCTCTGAAGTACCAGTCATTTAAAGGCCTGCTGATGACAT

GAAGCGTTGGGGCTTGTGCTGTAGGA

TGAAGGACCCACATCCATGCTCCAGGCAGGAAGAGGGGTCCATGACCACGGAC

ACAGAGCACAGGTCAGAGTGGGGCAGA

CAGAGCTGGAGTCCCCTAAACTCAAAACAACTGAGAAGCCTCTAAGAACCTAGG

TCGTGACCAGTGTCTAGAGGGAGCAG

TTGTCTACACTGTGTGAAATTGAGGAGCCTGGGAAGAAACGGCGAGTTGAGGG

TCAAAGACATCAGCATCGCAGCATTTA

TCTCACATTATTTAGCTTTTCTGAATCTTTATTTTTCATATCTGTAAAATGGGTTGT

AACATTAAATACTATTGCATTTT

CACCATAAATTCAACTTAAGAACTAGGAGTCTGTGTAAGTGAGAGAAACTGAGCA

CCTGCCTCTGCAGGAACTCCACAAT

AAGTGTAAGGAATGCCTTCTGCAGAAGCTATTGCTGTGGGGGAAGGAAGAATAT

CCTCTACGAGGGATACAAAGTGCCAA

GATGTGGTGCTTTGGTAGTCAGGTATCAAGGGATGATGAATCTCTGGTGTCTTG GGACTTCTGAAGAGAGAGACCAACCT

CAGACTGGGAACAGCGGGCTGGATGCGTTAGCAATACCCAAGTTTGGTAATTAA TTGTAAAATTTTTCTAGTGGAGGATA

ATCGCAACTTTGCCTTGCACTGGCCCTAGTTCTTCCACATAATCTATCTGTACTA TGTGACTGTTACCAGTGGCAAATAC

CTGAGTCACACGGCACCAAATATGTTACCAGTGGAAGGTATCCGAGTTACCAGC AGTGAATCCCTATGGTCTGCAGCAAC CTCAATTCTTACCTCCTCAGAAGAAAGAATTCAACTGGGGGGCATGAGGCAGAA

AGAGAGACTGAGGCAAATTTCAGAGC

AGGAGTGGAAGTTTTCTGAAAAAGGCTTTAGAACAAGAAAAAAGGAAACTGCAC

TTGGAAGAGACCCAAGCAGGCATCTT

GGTCAAGTGCGGTGTTTAACCTTGATCCTAGGACTTTATATGCTCGCCCCGTTC

CCATGCATCTTCCCTTAGGGTGGGCT

GCCGGCATGTGCATTGCCCCCCTTACCCTTGGGAACTGAGCCTGTGCAGTGTG

TTTAGGAAGCTGCACGCATGTCTATCT

GAGGCTTTCTTCCCTTTTCCAGTGGAGTGCCCCCAGAAGGTCATACTCTGCCAT

TAGGTCTCTTAATGTGCATGCCCAGG

GACCCTGGTGCCTGCATTCAGTTAACATTTAGTGCAACAGGTGTGGACCATCAG

GAAATGGCCTCTCCCTGGCATTGGCT

GCCAATTTATCACTTTTAGAGAGACAATGTGATAATTGCCAGACCATCACCCGAC

ATTCCTAGTGGGTGGCGAAGAGCCC

TCTCCTGCCCCGGCTCATGGCTAACTACCTGTATGTGAAAATTGGCTAAATAGA

AATGTTTATTATATATTCCTCTACTG

CTGACTTGTTTCTGAGCCCTTATGCAAACATGTTGAATTTTTCATCCCTTTGGGA

AAGCCATTAACAAAAAATTACTCTC

TTCCCACGTCTTGATCCCTTTCCAACTCCCATATCAGTTCTCACATCTGTGACTT

GGGGGTGAAAGAAACATGTCTTCAA

AGGCTTCCCCTCACCTCTCTGAGGCCTGGTCACTAAGCCCCCGGGGTTCCTTT

GTACCTTCCCCGCCCCATCCCACCCCA

CCTCACTGCCATTTGAAGGTAGAGGTCCAGCATGGATAGTCTGGGACCAATAAG

CTGTTTTGTTCCTGTCACCACAGTTC

AGTTGTTAGGAACCATCTCCAGTAGGCCACCATTTTATCGGTTTGATTGTTTCAA

TCACAAAAGCCACAAGGACAAGCGG

TGGACCAXGCTGTCCCTCCTGATGTACCAAAATATAATTTTAATTCTTCAGACCT

TTGCAACTCTCTCTGGCTAAATAGA

ATGTAGGACGATGTTCATTTGGATCCTAGGGAAGTTCTGCAGGATTTACCATGA

TTTTAATTATCACGTGTAGTTAGTTA

ACTTCTCTCTTTCCCCCATAACCATGTAAGTGCCTTGAGGGCAAGGACTGATAA

CTTGACCACTGCTTCCTTGGCAAAGC

CTGCTCCTTGCTGGGATCTGCAGGCACAGGGAACTCTTCTCTTCCTCTGCCCTC

TCCTGGCTCCTTCCTCCTACCTCCTT

ACACCAGTGTTCTCCTGCCTCAGGGCCTTTGAACTTGCTCTTTTATTTGCCTGGA

AAACTCTTTCCCTCACACCCACTTG

GCATTGCATTTCTCCCTCATCTACTTTTTTGTTTCCTTAGCACTTACCACTATCCA

GCATATGTACATTTTACATATTTT

CTCGTTTGTGACTAACTAGAATGTTAGTTTCAAGGCAGTAAGGACTTTTGTCTGT

TGTATTAAGCGCTGTATCATTAGCA

TCTGAAACACTACCTGGAACACAGCAGCCTCTCAATAATTTGACACGTACATGAA TGACCACCTTTCTAAGCTAAGATAC

CCATTAGTTTACTGTGAGTGCAAAATCTTTCAGGAGTCCTTGAAATCACTATCTC TGTCTTCTCTGAGGTCCCAGGCCAG

ATCCCCAAACACAGCAGGGATCCCCTGGGCATCTCAGACCCTTCCAXTACAGAT TAGCGCATAAAATATGCTGAACTGGA

GTGGCCCCTAGAAATGTGAATGCTGTCCCAAGATTTGGATTCAATAATGATTTCA TC AC CTTAAAAGTCTATC AC ATAAT 93 TTTGGTCGTGTATTTCAAAAGTACAGTTTCAGCACGTTTAGTCTGTTTTTGGAAT AAAAACTTATTTTCCCATAGAATTA

TGTATGTTCTGTACTCTACTGAACACCAAATTAGGGTTTTTAGGAATATATATTG GCACCTGGAAATATATTAAGCTTCC

ATTTTTGTCTTTGTCAGTAGATTCTTCCAGGAGAGCTTTAAACCAGGTTGCTGCT GTTGGTTCACGGTGCCCAGAGCCCC

TTCTGCCTAAGCCATGGACTCTGAAATCAGGCCCTGCACCCTCCCTCAGTTATC TCATTGTCTGGGTTTTAAACAAATTT

GGAAGCTAAAAACAACCTAGTCCAGAAGGCAAAAATGACAACCTAAAATTTGGCT CTTAGGAAGACAAAAAGCTAGATAG

ATGGTGTCTGAGAAATGGTAGAAGGAAAGTTTCTCCCCACAGAAGAGGCTGAGT TTCAGGATATCTGATGGGAGACTTCA

GTTTCTCATTTGAGAGCTGCTGGACTTACAACAGGTTAACACATTGTGCAGTTCT GCTGTGGAATATTGCAGTATCTTAA

TATTCCTCATTCTTTCATGAATTAAAATGTTAATAAGTAAGCATTTAGATAGCAAT ATTATAGTTTCTCCTTAAGATTTA

AAACAATTTTTTAAAAACATACCTCCCAAGGCATTTTATGTCATTATTTGTAAGTT CCTACAAAGACACAGATAATGAAC

CCTCAGTATGTATTTTATGTATTTGAAGTTTGTACAATATAGTATTTTTCTATTCAT TAAGTGAAATAAGCAACAATTAC

ATAGACACATTTTACATAGACATTTTAATGATT I T I I I TTCATTCCTGGAGGTGCT GATGAGGCCCCTTAAAGATTACTC

TTGATTTTTAAATCTCTTTTAATTAAAATCATTCTACAGAAATTCTGATGAGGAAT GAGGATTTCCTTGTCACTACTCCG

AACTTTGCTTTAATCAAGTCACTGTGAAGTGAGTTTGCTATCTGCAGGTAAAAGC TTGAAGGTGTTGACACTAAGAGCTA

AGCTAGAGATTGAACAATACTTCAAATGAAGTCCTCAGCCTGGGAACAAAACGTT TTAAATGAAGCTGGCAGGAGAAAAC

AAATACAGGATGTGGAAGAACAATAGCAACAATGGGGTCGTTCACAGCTGTTAG AGGATAACTGCCTTCTCTGTTAGTAA

TGAGGATTTACAGCTAAATTCGCTTCAGAAAAGCATCTTAACACCCTGCTTTTTC AAAAAACTCTTTCCACCCTTAGAGG

GAACCAGGGAATTCCAGGTCAGTTTTGATTCCTTGAATTAAATTCTTTTGAACAT ACATTTTCCTCTTCTACTAGTTCCT

CAACTTCCTCCTCCTTTTTGTTCATTCTCCCAAGAGAGAAAATTTCTCCATCATG GCGCTATGGCCTTCCCCTCTCCCCC

CACCCCCAACGCTAACCACACGCTCTAAAGAAGTTTTACAACATAAAAATTACAT TCCTGGTTAAATATCAGAAATTTCT

ACTCAGGATTTAGTATGTTGTCGAAAACACAAGCGATCATCATTCATCTTCTAAA ATAAGATTACATTAATAAATGAGAA

GTTCCCAACCACGTTTTCCAAGAATGACCTGTTTTTAAAATGTTAAATTCACAGT CAGCATGTGAGGGCTCCTAGACTTG

CTTTTCCTTTTCTCTTATCTGAGGTTATGGACCTCAGGAGAGCAGGGGAGGGTT ACAAGGTCAAAGTCTTTTCCTCCTTG

GAACTTGCTGCGTGCTGGGAGAGGGCTGCCTGGAAAATAGATGCTTTAGAGAA GAGGTTGGGGGTCACGATTTTAATTCT

GTTAAGGAACAGCTGGTTTGAGTTTCTTTCCACTTCTATCTCCTCCTCCCTTCCT CACAAATCCATGGAGAGAGAAGAGT TTGTTCAGAAGGGACCGGCAACATAAAAATATACCTTCTATTATTATAGTGCTTT

CTAAGTGTCATCTTTGTTATGAAGT

TGAGTCTAAGTGCTAGAGAGATGGGCTAGATTTTATAAAATTAACTCTATCTGAA

ACATGACCAGACCTTCAGTCAAAAA

GAACCTTTTAAAATACAGGATGTGCGCCTGGAAATTTCTTTTAATAATATTTTTAA

ACTAAAACTTTAATGGAAATAAGT

ACAATAAAGGCCTCTGGGGTATTTGGAGCCTGACTGCAAAGAGAGGAACATGAC

TGAAATATGAAAGCAGCCAAGTGGGG

GTGTTAAGTTTCTGGACTAGAGTCCTTGTCTGCCTCCAAATTAATATGCCTTCAT

TCATTCAACAAATGTTTATTGAGTG

CCTGTTCTTCCCACAATTCATCTGGGGATACGGTGGTTAAGATATATGGGTGCT

CCGATATATCTGCAAACAGGTGCAGA

AACGTTAACTGGGCCCAATAGAGCTTTAGCCTGTAAGCATGGCTTCCTATTCTAA

GTAGTTTACACAGTTTGGGATTTAA

AAATAAGATCTGTTCAAAATAACATTATGCCATAATAAACTCATAAATCCAGTGAA

TATGTAATAATAGTAAAACAGCTA

AAAATAACACAAAAAATGTAAAAAAAACTAAACAGCAAACAAGCACATGGTTTGA

CTTTTTATTTAGGTTTTAAAGTCTC

CCATGGTTTTTACCGTGCACATGTGTACACACACACACACACGTACACAGAGAG

AGAGAAAGAGGGAGGATAAAACTCAC

ATCTCTCAAATAAATGGAAACCAGTAACAGATTTTTTGTCTGGGCTCCTCAACAC

ACTGGCCTCAGGCTGTGTTGGTGTG

TGGCTCAACAGCAATCAGAGGTCATAATTTCATTGGCAGTTAGCATCAGGTTTAA

TTTTTCTAGCATGTTACATTTACCA

AAATGTCTGGTTTCAGTTATGCTTAGGCACTGACTGAATCAGCTCTGAGCAGCA

TGATGGCTTAAGGTACCTGAGGTTTA

TGGAACACATATGCT TTGTTTTCAATCACTTTCCTTTCTAAACACAAAAATGTG

TTTTCTGAAGCTGAATAACAGTAG

TCCTTAACGATGTTGCTTCTTTCTTACAGGAGAGGACCCTAAGTAGGTAAAAATG

TCAATTTTACAAGACTGCCACCTAC

TGATTGACATAGTTGAAAAATCCTCCAATTAAGGTGCTTTAAAAAATATGTATGGT

TTTTCCAAAACAAAACAAAAAAAT

TAGGTACTCTTAAAAAATTGACATGTATCATTTTAATTGCATTTTAGGAATGTGAC

CAAGTGCATATAGATGATGTTTCT

TCAGATGATAACGGACAGGACCTAAGGTGAGCACCCTTGAATGTGAAGTGACCA

TCTCCCTTTTATAGTTTAATTACTTA

CCTAATTAAGATGGACTTTCTGTTCGGCTAACTTTGGGCTTTTCTTGCATATAAA

TAGCACATATAACTTTGGAACAGAT

GGCTTTCCTGCTGCAGCAACCAGTGCTAACTTATGTTTGGCAACTGGTGTACGG GGCGGTGTGGACTGGATGAGAAAGTT

GGCCTTCCGCTACAGACGGGTAAAAGAGATCTACAACACCTACAAAAATAATGT TGGAGGCAAGTGATTACCACCAGTAC

CTCAGCAAGTTAAGACAGTCCAGATACAATTCCAAAAGTACTGTACACTTTTGTG TCACACTGCTTTGAATATAATAGCT

TCTATTTGGGCAGTTTCATCTCACTGGCCATTATTCTGCTGTGATTTAGTGACAG AAATATTGCACTGGAGCCAGCTGCC

CTGGCTATTCCAGTCTAGTCCTGTCTCTGTTACCTAGTGATGTGACCTGXGGCA AGTTTCCTGACCTCTCTGACATTCAG -λcA

TTGTCCTTTCAAGGAAGGATTAGCATCCTTCGAAGAATTAGCCACCTCTGCCCAA

ATCACAAGAATGGTTGTAAATCTCC

TCCTACAGGGTAAATACTTGGATTAGTTTGAATCTTTTGACTTTGGTTCATAATG

GCTTTAGCATGAAAAGGAAATACAG

TGACTTAATAATAAAAAGTTTAGGGGTGAGGTTGGTTTCCAGCATGGCTATATGC

AGATGCTGAAATGATGCTGTCGGGG

AAGCCTGTGCCTTCCTTTGGATAAAGGCCTTTCTAGGTCAGTCCCTCCCAAGAG

GTGGCAAAATGACCACCAGTAGATCC

AGCTTTATACTCTACCAGTAGTTTGGCACATCCATGAGGGGAGGGAATCTATCA

TTTCCTAATAGATTCAGCAAAAGTTC

CAAGACTGACTTCCACTGGCCAAGTGAATGCTGCACACCTACCCTGGGAGCCA

GAAAGTGGAGTCACCTCTGCCCAAACT

ACACTTAGAGGAATGTAXGAAAGTAGGTTCCCTAAAAAGAGATGACTTCTGTAAC

C AAAC C AAATAAATACTTGTC AAAT

GAATGGTACTATGTGAAAGATACTTTGTATACTATAAATCTGGAGGCTGGTATTC

ATAGTAC I I I I I AAATGTTAATTAC

TTGCAAAGCCGAAGAAATATGTTGGTCAGGAAGTAGTTCATCATGTGCTTTTAAA

TCCTCAGGTCTGCTTGGTCCAGCTA

AGAGGGAAGCCTGGCTGCAGTTGAGGGCCGAAATTGAAGCCCTGACCGACTCC

TGGTTGACACTGGCCCTGAAAGCACTC

TCGCTCATTCACTCCCGGTGAGGCTCTCTGCGGGGTCTTTGCCAGGAAATGGG

CAATGCTCGTGCTTGTTCATTTTGGAG

ATTTATTTTTCAACAGTCTTTGTTTTC I M M GTTCTCATAGGACAAACTGTGTGA

ATATTTTAGTAACAACTACTCAGC

TCATCCCAGCATTGGCGAAAGTCCTGCTGTATGGGTTAGGAATTGTATTTCCAAT

AGAAAATATTTACAGTGCAACTAAA

ATAGGTGAGCTGCTTTTCTTTTCTT ^"I I I AACAATCAGCTCTTTTCCTTCAGGATAA

TAAAAACGAAAGGCAATAAATGAA

ACTTTTTTTCCAGAATGCTACATAGGTCAAAAAAAAAATACAAGAATAATTTAACC

AATTTGCAAAACTGGATTTAGACG

TAAGAGAAACATAAAGCATAATACAGTAGCTGAAGTCACAAAATTTTACTTGACT

GGCTAACGTTAAGACCACGGCATTT

TAGGTAATATAACAGCATCGATTCTGGATTATTATCATATTTTTATCCTTTTAGGC

AACATTAACAAAAATATCTGATTT

TCTAAGCTAAAACAAACAGCAAGATAAAATTATAAAAAGTAGAAATGAAACAACTC

TAATAACAGATTTATTACTAAAAA

TATTTGTTTTGTTTTCCATTCAATCTGTATAATCAATTGAGTTTCCATTTTTGTTGT

GGAAATTTAAAAGCTTCTTGCAT

GAAAAAAACATAGTTGGGAGGAAAATACTTATGAAATTTAGTTAGAATATAAATCA AGGCAAAATCAACATAGGAATAAA

AAGACATGGCTCTTGTTCTCAGGGGAAGCAAACAGTTACATGACAATCCAATAT GATACAAGAGTATGCAAATGAGCATT

TTTTAGTTGTCTACTAAATGCCCTGAACTGTACTAAGCATTTTGCATTTAAAGTCA ATCTTTATAATCTTTCCATTTTAC

AAATGAGGAAACTAAAGTCCAGAGACTTAAGTAAATTGTTGCAATCAATAGTTCA CAAACACAAGGCTCAAGCCTCATGT

TTCTCTGATTCCAAGGTCCAGACCCCTTGCTGTATCCTACAGGGTCCTATTGCTA AGTGCTTGAATGTGCACATGACTGA -AOZ,

GGAAGCATTTACTGCCACCCAGGAAAAAGTAGAGGCCAAGTGTCTATGAAAGTG

TGAAAGCTAAAGGGAAGAAATGACAT

TTGATCTGGGTATAGAAGAGCAAGTAGGAGTTCAACAGGTAGATGGGCTAGTGA

GAACAATGCAAATGGACAGAGAAATT

AAAATGTGTTCCCTGCTGAGGGACTTGTGAGTGGCCCTGGGTAGCTAGTATGCA

GTGCTCATTAAAAGCCAAAGCTGAAA

AGGTATACTTGGGTCTAGGCATGAATGGCGTTGTATATCACCCTAAGGAXAGGA

ATGTTATTTTAGTCCAGGGGAAGGGG

TATGAGCATTCTAGCAGGTGGCAAAAGGGCTGTCTTTGGCTGCTCGACTCCATA

TAATAAAAAGGGGCAAAAATGGAATC

CCTGTAGGTCTAACTTCCGTTTGCCTGCACTTTTACACTTCCTGTGCTTCTCCTT

CAAAGCATTTACTAAATGATTACTA

AAAAATTACTGACTTTTATAATTATTTGTTCATAGTATGACTTCCCCGTCATACTG

TAATAATCTTGAGGACTGGGTTTG

TCTCTCTTGCTCATCATAGTAGCTGTAACACCCAGCACACAGTAGGTATCAATAA

ATGTAGAGATGAATTTGTTGAATGA

ATGGCTGAATAAATTACCATTTATTTTGAAGAAGGCCGACACAAGATTTCTTTTA

GGCACGTGCGTCAATAGCGGCACAA

TTAACTGATGGGGAAATCAGGTTTTATTTTTATCAGTGTTGCATTGGAAGAGACC

CAGTGGGGTCTTCAATCAATGGCAG

AAAGACACAGTCAGCAGGGTGCTGATGGGATTCATCCAGGCTAACCTTTCAAGT

TAGAAATACATATCTGGAGTTTGAGA

ATGACTGATGCTTCCCAAGAAGGGCCAAGAACAAATTGTTGGAAAATATCAAAAA

GCAAGAAATATATCAAATAGAGTAA

AATTCCTAGTAGCCCATTATAACCAGAACTTTTATGTGCTTTCATGTTCCATAAAA

ATAGCAAACAGTTTAAGTCACATG

TTAATTCTATTTTCATCTATATCAATTAAGAGGAATTATAATTATATTGTGTTGTAA

ACAGGGATTGTGGAAAAGGTTTA

CCAGCACATGAAAGTTGGAACAAATGCCGAAGCAGCCCATAGTACAGAAGCGG

GTAGAGCTTAGGTTTTCAGAAAGAAAG

C AGGAGTAGTGTATTACATAGC ATGTGCATTGAAGTTTAATC C CAAATAATTTG A

AAATGATAACAGGAAATTACAATCC

AGTAAGCATTGTTTTTTTAATGGGCATTTTGTTTAAAATATTAAGCTTTATTTTTTT

TTCAATACAGAAAAAAAATATGT

GGTGGTGGAAGTAGGATTTCACCATCAAATAAGGTATATGTGGGGAATCAATGC

AAAAAACCCAGCAA I I I I I I I I I I CT

TTTGACATGGAGTCTTGTTCTGTCACCTAGGCTGGAGTACAGTGGCACCATCTC

AGCCTGTGCCTCCTGGGTTCAAGTGA

TTCTCCTGCCTCAGCCTCCCCAGGTAGCTGGGATTACAGGCACACACCACCATG

CCCAGCTAATTTTTGTATTTTTAGTA

GAGACGGGGTTTCTCCATGTTAGCCAGGCTGGTCTCAGACTCCTGACCTCAGG TGATCTGCCTTGGCCTTCCAAAGTGCT

GGGATTACAGGTGTGAACCACTATGCCTGGCCCCAGTAATTTTTATTGTAGGCA AAATGCTAAAGTCATAGGAGGAGATG

ACTGGTATCTCAGTTGGCAGAGCTATGATGACCTTTCAGACAAAATGGACTCAT CAAAATGAATACTTCATATAAAACTA

ATAGTTAACTTCTAAAATCTAAGCTTCATTTTCTTAACCTCTATTGTGAAAAATTTC TACATGACCCCAAATTGGCAATG AGCCCAATAGGAGATAGATAGTATATAGGCAAGGATTTTTTTTGTTTACCACAAC

TTCATCTTTAAATTACATTGAATTT

ACCTTCCTTTTTCCACAAGAGGGAAAATTTGCAAAAACGCATAAGCATTTCCCCG

TAAGGAGAGAAAAAACTTTCATGAC

ATCGAAGTTTTTGGTTGTATATTTTTGCAGTGAAGACTATACTTTGCATTTGCTTC

TTAAAGTGATTTAATATTTCACAG

TTTAGTGGACAACTAAAGAGGCATTTAAATTGCATCTTTGAATAAGGCTAGCTCT

ATTTCATTGTGTCAGATGCAAACTA

GAGATTTTAGTGCAAAATACAAATATAAAGCAGACATTTGTTTAAGCAAACAAGC

AATTTGTTCATGTGTATATCTGATC

CTTATTTGAAAGAGGTGCAGGCTTCGCTCAGCCATTCACATTTGAAAACTATAAA

CTGTCCCATTGTCCATTAAAATGAC

CCTATTGGCAGTCCAAGAGTTGGAACACCAGTTTCTTCTCTGAGAAGAAACAAT

GCTTGCATTAACTGTGGGAATGTGCT

GACAGTGTAGCCACCACAGGAGCCCAAACTAAGTCATGACACGGGAAAGATAG

TAGACAGATAAGGGTATTCTATTACCT

TTATTCCAGTTTTCCTCCAGGAAAGTGCTTCATTGAACATTAGATGCCTGGGTAA

GGCAGGTACCATCTTGCTGCACTAC

CTCAAATTATCTGACATTTATTTGAGAAGGAGTTTTACAAGACTACATCAGACTTT

CAGGGTCTTTAATTCAACCTGAAA

ATG GG TAAGCTAATTCCGATGTTTGCGTTTTCTTGCTAGAACAAAAACAAG

TCCAGTTATTTTTTATCATTCTCAA

TAGTCTTTGGATCAGATATTTTCAAAGTTTGATATATGAAGAAAAAACTTTTTAAA

ATCAAGTTAAAATATGTTGCCAGA

ATTGCCTATGTTTGAGTTCAGCTTATATATAGGGGGAAAAAGAAGTCCCACTGCA

AAACAGTACTTTCCCCCACCACCCC

AATTGCAATGCTGTGATTGACTAGATTCCATTTTTTAATAAATGAGTCCTTGGGA

AGAGAGTATTTAACTATCAATTTCC

AAAGGACAGGACACCACACTGCTAATTAATTTACTTTGTAATTTATTGTGCTGTG

GCACATACAACCCAAGTGGCATAAA

AGCATTCCTCTTAGAGGTACTAGTGGGGCATTCGAATCAGAGCATGAAGAAAGC

ATTTTATATAAAAAGTTCAAGCACTG

TCACATTAGGTTACCCAAACTTCACATTTTCTATCATTTATTTTTGTTCACTAGGA

AAAGAAAGCTGTTTTGAGAGAATA

ATTCAAAGGTTTGGAAGAAAAGTGGTGTATGTTGTTATAGGAGATGGTGTAGAA

GAAGAACAAGGAGCAAAAAAGGTGAG

CATTCCTAGATGGTGCATCTTGTCTCATTTGTTTTTTCAGTCCTCAGTATTCTCAT

AACTGGATCATTTAAACAGGTTCC

ATTTATGTGAAACGTGATGAATATGTGCAGTACTCTAAGCACTGAACAGGGTAAA

GTAGAATTCAATAAATATGGAGTGA

TCTTTCTGAATAGTAGTGTGCACAAACTCAAGGGCCACAAGTGGTGTTCATAGA

AGGTAGAATTTGGGAGCAGAACTGAA

GTGTTGTTAGTGGTGTAGCTGTCATCCAGAGCAGAGATAACACATGTGCCAGCA GGACCTTTTCTGTACCCATGGCAGAC

ATGCCAGGTGGTTCAAACCTTGTTACCTGAGCCTGGACGCAGCCTAAGAACCTT TCAAACATCCTTCAGAAAGCCTCTAT

CAGTTGGTCAAAGCTGACTTGGAAGATAAAACTATCTGCTGCCCCAGAAGACTC GATGTCTTCTTCATATCATGAGGTCC TACCTGTCTCTCTCAGGGCAGGGGGAAGGGAGTGGACCCAACTATAAATCTCTT

GACTCCCGCTTTGCCCATTGCTGTTC

ATTATATTCTTCTGAATCTCTGCAGGTGTAATTCTATCATTTCTCTGAGCAAAAGC

TTTCTCATTGGGCCTCTCATTGCA

TTTTTAAAAGCCAAGGACCACCTTCCTTAGCCTGGCACTGAGGGTCTCCACAGT

CTGGTTACAGCCTGCCTTTCTGATCT

GTCTCCCTCCTGTACCTTACACTGTAGTTGTGTCAGTCCACTCAGCTTTCCTTGA

CCATGCCCCACAGCTTTTCCTTCCT

GTTGTGTCTCCTTGGAACCTTTTCCCTAATACCATTCCGTCATTGTCTTATTCATT

TGCAAAATTTAATTCATGTACTAC

CTTTTCTGTTATGCTTTTAAGAGATCAATTATAATCATGCTCCCTTATATTTTAACT

GAAGGAGCTGTAGTGATAAATAG

CTTGGATTCTTGAGCCAGACAGCATGAGTTTGAATTATGACCCTGCCACTTACC

CTTC CTGTG ACTTTG AAC AAGTTTTT

AACCCTCTGTTTCCCTTTCCTTGTCTATAAATGGGGATAGTAATAGCACTTAGCT

CATTGGGCTAGTTTGAGGATTAGAT

ATAGTAAAGATATATAATTCCCATAATATAGATATGTGTAAAGTGTACAACAAATG

GTAGCTATTGTTATTGTTTATGTG

CCTGTTTCAGCAAAGAAATGTGAGCTTCACAGATTCAGGGATCTTACTTTGCTTG

GTTTGTTGAGACTGTCTTCAGATTT

TTTTATAACTCCCTAAATCTTGTCATAGTTGTACTCTGTAAATGTTTCTTGAATTG

AAGAACAATTAAATATTTTATGGG

ATTAACCAACAGCATAATTTTCCATTCCCTTCCTTACACACTCCCTCATAGGAATC

CATGTATATTTCCATAAATGATAC

AATATCTCAGCCCTTCTGGAATATTTCAGCATATACCAGAAACAGCATGTAAGGT

CACCAGATTACAGATGCAGCACTGA

GCCTAAGTATGATACATGGCCAGCCTTCCTACAGGCTCTGAAGATGGCCTAGTT

TACCTTCAGTGCCTCTGAGTTCCACT

AAAATCATAATCACTGACCCTAATTTTAAACTAGATAATTTCATGAATACCCATAA

ATACAGAATGTATCCCAGTCACAA

ACCTGCGTGAGTCTGGTTGGTGCTGGTAGAAACCCACACGTGACTTCAGGTCT

CCTCATGCTCAATGTGAAGCAGCATTT

GCCATTTAAGCAGTAGTTCTAATCTTCCCACTTCACCGACGACCAACCACTAGTG

TCATTGGGTGCCTCTGTTACCTCTC

TGGGCfGTCTTTATAAAATAAAACCTTTCCCAGCCTAAGTCAGAATCCTCAAAAC

ACAATCCTGGAGAGTTGAAGAGAAT

GTTTTCTGACATCCAGACTCAAGTCTTCTTTCTAGGAAAGCCTCTGGCATTCCCA

AGCCATTCCTATTCAGGACCCTCCT

AGTGGCCAGCAGGGCAGGGATGGAACAGATTTGTACCTCTGACCAGTATGGCC CTAGGCCAAGGTGTGAAGCCATCTCCA

GGCTGGTCCTTCTCCCTCTGGTATCTACCAGAAAGTTTAATATCAAAAATTGCTA AAATGTGGCTTTCTAGCTTGTGGTA

GGCAGAGAGGGATGTTTATGAAAGTGATTACTTAAAATGCAGGGAACACAAGTG AAATC CTTTGGATCTTATCAAGGTTT

CTGTGAGTGAGTCCCTTCCTAAACCTTTCTGATAAGTACAATTCTTAGAAGTTTG ACTTCAGTGAACACAACATGGAAAA

AAACAAAGGAATTCTTGGTTTCAGGTTAGAATTGAAAGTATTGATAGGGCTAGAG TATTTGCTGTCTTTGAAGAGCAGTT -AoS

AAGATTTTTTTTTCTTTTTTTTATTATACTTTAAGTTCTAGGGTACATGTGCACAAT

GTGCAGGTTTGATACATAGGTAT

ACATGTGCCATGTTGGTTTGCTGCATCCGTCAACTCATCATTACATTAGGCAAAT

GCTGACACTTCCTGGTAGATTCTAA

TCCTGACAGCTTTGACAAATGGGTGCTCAATACATTTTGTTGGTAGAGCGGACA

TGAAAACTTTATGTATTTCTGATGTA

ATAAAAGGTTTTAGTTAAAAGATTTTAGTTAATTTTTATACTGGAAATAGGCTGAG

GTTCGTTTTTGGTTTTTTGTTTTA

TTTGAGAAAGAGTCTCCGTCACCCAGGCTGGAGTGCAGTAGCGCGATCTCGGC

TCACTGCAACCTCCTCCTCCTAGGTTC

AAGTGATTCTCCTGCCTCAGTCTCCTAAGTAGCTGGGATTACAGGCATGTGCCA

CGATGCCTGGCTAATTΠTGTATΠT

TAGTAGAGACGGGGTTTCACCAGTTAGCCAGACTGGTCTCAATCTCCTGACCCC

AAGTGATCTT

SEQ ID N°1-F : This sequence is contained in the subclone J4A10 derived from the

P1 10910 and contains exon H (Contig H) which is the last exon of eyal. Exon H spans from the nucleotide at position 362 of this sequence. The translation stop codon is localized at the nucleotide at position 440 of this sequence. The polynucleotide of SEQ ID N°1 -F is contained in the clone HSEYA1J4A10 deposited at the Collection de Cultures de Microorganismes under the accession number :

XXX. The nucleotide sequence of SEQ ID N°1-F is the following :

AATTATAAGAACAGTTTTAAATTTCGCXGAAAACCATGAXAGGGAACACTGCCAT

ATTTGGCAACGTTXTTTTATTTAAAGCTTGGAATTGGCATTCAAGTCACAGTGTA

GATCCAGTTAGGAAATATCCATCCCCTTCTTCAAAGAAACAGACTCTAGATTACA

TTTTACAACTCAGTCCAGTATTCTTAGGGGAGGATTGAGTTTGAATTTTTGTTTTA

AACAGATGTGATGTTAAGATGTTAGCTGGCATTTCAATGATACTGAAAATGGGG

GTCAGCATGGGAGTGGATTTCCAAAAAAGCTGGCAGGGCTGGCGCTGGGTGCT

CTGTCCTCACATGTATGTGTGCCTCTGCTGCAGCACGCGATGCCCTTCTGGAG

GATCTCCAGCCACTCGGACCTCATGGSCCTGCACCATGCCTTGGAACTGGAGT

ACCTGTAACAGCGCTCGGCACTTTGACAGCGCACAGCTGCTCTGTGACCAGGG

ACAGATCCAGCAGGCCCCAGTCTCGCATCAGCGCCGGCCTCCARAACTTAGCA

ATTTCCGCCTGGTGATGCGCAGTTGCTGTCAgTCTTGACCTCTGCCTTTGTGGT

GAATGGAGGACCACGTCTATTTCATCAXAACAGCTGTTGACTCTAGTACTGTGAA

TCCAGTGAAAATAAGCCATGAXAATGTTTTAGCACAGCGTTATGTGTCTGCCACA

TTAACTACACGGTTCAAACCTGTGAAAAAAGGACcTGCAAACGCTTCAGTTGTTA

GCATTTTCAATGTGATATAAACAGCTTCTCCAATACAGCAAACCTAATTGCACAA

CAGAXACTGAAATGTGTTTCCTGAATACCAGTGGAGGAATTTTCTTGTAAAGAAG

GTTTACTTTTTGGTGTCTCATACCCAGGGTAATCTGTACATCTCTACTTATTTATG

AACAGACTTTTTTTAAAAARATAAAAAAACAGCTTTATTGAGGTATAATTCACCCA

CCAGACTTTTTTAAACATCAAATAATTGAAGAGACAATAGCATTAGAAATAAGTGA

TTAAAGGCCTCTGCCTCACAACATGGCAAGTACAGTACTTTGAATTTTAGCACAT

TGCATAATAKTTTTAAGTATGTCTAATTTAAACGTATAATATGTACATCACTGAGA

CAATCATGTXCAGAAAGAATTTTTGGTGTAAATTTGTAATAATGGATAATTCTTTT

ACATATTGTTTAGGGAAATGATATTGAAXAGGTAGCAATGCCTGCATAGTGAAXC

ATGAGGCAGCACGTGCACAAATTCATGTGCCGTGCCTTATCTGAGTTTTCGGTA TAAATATGTA -Λ06

ANNEX 2

SEQ ID n° 2 :

5 -TCTCCTTTTTCTCTTTTGGTTAAAAGAGGGCATTGTCGTTCTCAGCCATG

TGCTCTGTATAATTAAGAGCTGACACTGAAGCAGAGTAACAACATCTTCT

AATTTTTTTACCCCTGATCACAGGTGCAAACATCTCAAGCCAGTTCAGAT

GTTGCTGTTTCCTCAAGTTGCAGTTAAAACAGAGCCAATGAGCAGCAGTG

AAACAGCTTCAACGACAGCCGACGGGTCTTTAAACAATTTCTCAGGTTCA

GCAATTGGGAGCAGTAGTTTCAGCCCACGACCAACTCACCAGTTCTCTCC

ACCACAGATTTACCCTTCCAACAGACCATACCCACATATTCTCCCTACCC

CTTCCTCACAAACTATGGCTGCATATGGGCAAACACAGTTTACCACAGGA

ATGCAACAAGCTACAGCCTATGCCACGTACCCACAGCCAGGACAGCCGTA

CGGCATTTCCTCATATGGTGCATTGTGGGCAGGCATCAAGACTGAAGGTG

GATTGTCACAGTCTCAGTCACCTGGACAGACAGGATTTCTCAGCTATGGC

ACAAGCTTCAGTACCCCTCAACCTGGACAGGCACCATACAGCTACCAGAT

GCAAGGTAGCAGTTTTACAACATCATCAGGAATATATACAGGAAATAATT

CACTCACAAATTCCTCTGGATTTAATAGTTCACAGCAGGACTATCCGTCT

TATCCCAGTTTTGGCCAGGGTCAGTACGCACAGTATTATAACAGCTCACC

GTATCCAGCACATTATATGACCAGCAGCAACACCAGCCCAACGACACCAT

CCACCAATGCCACTTACCAGCTTCAAGAACCGCCATCTGGCATCACCAGC

CAAGCAGTTACAGATCCCACAGCAGAGTACAGCACAATCCACAGCCCATC

AACACCCATTAAAGATTCAGATTCTGATCGATTGCGTCGAGGTTCAGATG

GGAAATCACGTGGACGGGGCCGAAGAAACAATAATCCTTCACCTCCCCCA

GATTCTGATCTTGAGAGAGTGTTCATCTGGGACTTGGATGAGACAATCAT

TGTTTTCCACTCCTTGCTTACTGGGTCCTACGCCAACAGATATGGGAGGG

ATCCACCCACTTCAGTTTCCCTTGGACTGCGAATGGAAGAAATGATTTTC

AACTTGGCAGACACACATTTATTTTTTAATGACTTAGAAGAATGTGACCA

AGTCCATATAGATGATGTTTCTTCAGATGATAACGGACAGGACCTAAGCA

CATATAACTTTGGAACAGATGGCTTTCCTGCTGCAGCAACCAGTGCTAAC

TTATGTTTGGCAACTGGTGTACGGGGCGGTGTGGACTGGATGAGAAAGTT

GGCCTTCCGCTACAGACGGGTAAAAGAGATTTACAACACCTACAAAAATA

ATGTTGGAGGTCTGCTTGGTCCAGCTAAGAGGGAAGCCTGGCTGCAGTTG

AGGGCeGAAATTGAAGCCCTGACCGACTCCTGGTTGACACTGGCCCTGAA

AGCACTCTCGCTCATTCACTCCCGGACAAACTGTGTGAATATTTTAGTAA

CAACTACTCAGCTCATCCCAGCATTGGCGAAAGTCCTGCTGTATGGGTTA

GGAATTGTATTTCCAATAGAAAATATTTACAGTGCAACTAAAATAGGAAA

AGAAAGCTGTTTTGAGAGAATAATTCAAAGGTTTGGAAGAAAAGTGGTGT

ATGTTGTTATAGGAGATGGTGTAGAAGAAGAACAAGGAGCAAAAAAGCAC

GCGATGCCCTTCTGGAGGATCTCCAGCCACTCGGACCTCATGGCCCTGCA

CCATGCCTTGGAACTGGAGTACCTGTAACAGCGCTCGGCACTTTGACAGC

GCACAGCTGCTCTGTGACCAGGGACAGATCCAGCAGGCCCAGTCTCGCAT

CAGCGCCGGCCTCCAGAACTTAGCAATTTCCGCCCTGGTGATGCGCAGTT

GCTGTCAGTCTTGACCTCTGCCTTTGTGGTGAATGGAGGACCACGTCTAT

TTCATCAGAACAGCTGTTGACTCTAGTACTGTGAATCCAGTGAAAATAAG

CCATGAGAATGTTTTAGCACAGCGTTATGTGTCTGCCACATTAACTACAC

GGTTCAAACCTGTGAAGAAAGGACCTGCAAACGCTTCAGTTGTTAGCATT

TTCAATGTGATATAAACAGCTTCTCCAATACAGCAAACCTAATTGCACAA CAGAGACTGAAATGTGTTTCCTGAATACCAGTGGAGGAATTTTCTTGTAA

AGAAGGTTTACTTTTTGGTGTCTCATACCCAGGGTAATCTGTACATCTCT

ACTTATTTATGAACAGACTTTTTTTAAAAAGATAAAAAAACAGCTTTATT

GAGGTATAATTCACCCACCAGACTTTTTTAAACATCAAATAATTGAGGAG

ACAATAGCATTAGAAATAAGTGATTAAAGGCCTCTGCCTCACAACATGGC

AAGTACAGTACTTTGAATTTTAGCACATTGCATAATAGTTTTAAGTATGT

CTAATTTAAACGTATAATATGTACATCACTGAGACAATCATGTACAGAAA

GAATTTTTGGTGTAAATTTGTAATAATGGATAATTCTTTTACATATTGTT

TAGGGAAATGATATTGAAAGGTAGCAATGCCTGGATAGTGAAGCATGAGG

CAGCACGTGCACAAATTCATGTGCCGTGCCTTATCTGAGTTTTCGGTATA

AATATGTAGATAATGGA I I I I I I I I TAGATAATGTTGTCAAGACCAAAAG

CATGGATGTCAAGTGTCAGTAAGGATTTTGTTTCTAAAATTTTTTCCTGC

ATCAGTTCTTCTGAGGGCCTTGATGAAATAACACAGCAGTTTCTTAAACA

ATTTGAAACAAAATGAGCTCTCCTACCACCTCACTTTTTCATTTCCACAC

TAATGTATTATATGTAACTACTTGGAAAAAATAATTATTCAAATGCTTCT

TCCCACAAAGAATATAGATGATAGTAGATATATTTTATTAATAAAATGGT

TCATGAATCGGAGACTAACAAAGTTTTCATGTGCTCAGAATTATTAATTA

TCGTGTCTGCATTTTCTTTCGATAAAGGAAGACACACGATGCTAATCCGG

AAATCAGCAAACTTTGCATTACTCCCTATGTGCGTATTTTCTCTTTCTTC

CTGTCACCCTGAGGAAGGTTCATTGCCATTGTCATCACCATGGAAACAAC

GTTCCTCTCCACCTGCATTATGTACTACATGACAGGCATCAATCTGGGGA

AATAATAAAATTATCACCTTTGTCAGACCATAAGAGTTTCTCCAAAAGTG

GTCAGTTTGGCTGGGCAATATTTTCTCTCATCTAACAAACACAATCCATT

GTCATGAAATTACCCTTAGGATGAGTCTTCTTTAATCAATCATATATTGG

GCGGAAAAAACACCAGCTTTGACCCGAAGTAGTTGAAGAGCTACTTCATT

CTTTTCTGAAGTTGTGTGTTGCTGCTAGAAATAGTCATTTGTGAATTATC

CAAATTGTTTAAATTCACAATTGAATTAGTTTTTTCTTCCTTTTGGCTTG

AAGCAAACAGTTGACCATTTTT CCTTTTCATTTTATGTTTTTGTACTC

TGCAGACTGAAAAGACAAAGTTTATCTTGGCCTTACTGTATAAAGGTATG

CTGTGTCCACCGTTGTGTACAGAATTTTTCTTCATTAATTTTGTGTTTAA

GTTAATAAAATTTATTTGTGATGTACTGTAA-3'

SEQ ID N° 3 :

AAACCAATAAGGTTAGGACAAGAGAATAGCTGTGGTTTGCGTTGCAAAAA

CCAAAAAAAAAAAAAAAAAAAAAAAGAAAGCCCCGAGGCTCCATGGGCAG

ACCTACAAGGCTGCGCAAACAAATCGAGGGATGAGATTCTGCTGTTTCTT

TGTCTAGGGTTCTCAGATGCTATCTGCCGCTGCTGTTTGGTGGGGAAGGA

GCGCTGGGCGCAAAGCTGTTACCAAACAGAACGGTGGGAGCTGATGGCTC

CGAGTTTGGGGCGAGGTAGAAACTCTCCAGTGCCACTTCCGACTTTAAGC

CTTCCTGTTGCCGTCCACTGTGGCGGGTTTCTTCCTGGGGAACACGTTTT

CGCTCAGTCGCTCGGCAGCCCGAGCCTGCGGCAGCGGCCAGGCGCCTGCC

CCCTGCGCCGAGCTTTCCCCTGCAGAGGCGCTCCACTCCCAGAAGCGCCG

CGGCTGCACCAGAGCGCCTGAGAGCCCCCGCGCGTACCCATCCAGGAGCA

AAACTATGTCAGGAATGGAGGTTTGCTAACCCAGAAAATTCGAAGGAACA

CATTAAACTGGTGGATGCAGCAGATGTAAGCGCTGTGCAAACATCTCAAG

CCAGTTCAGATGTTGCTGTTTCCTCAAGTTGCAGGTCTATGGAAATGCAG

GATCTAACCAGCCCGCATAGCCGTCTGAGTGGTAGTAGTGAATCCCCCAG

TGGCCCCAAACTCGGTAACTCTCATATAAATAGTAATTCCATGACTCCCA ATGGCACCGAAGTTAAAACAGAGCCAATGAGCAGCAGTGAAACAGCTTCA

ACGACAGCCGACGGGTCTTTAAACAATTTCTCAGGTTCAGCAATTGGGAG

CAGTAGTTTCAGCCCACGACCAACTCACCAGTTCTCTCCACCACAGATTT

ACCCTTCCAACAGACCATACCCACATATTCTCCCTACCCCTTCCTCACAA

ACTATGGCTGCATATGGGCAAACACAGTTTACCACAGGAATGCAACAAGC

TACAGCCTATGCCACGTACCCACAGCCAGGACAGCCGTACGGCATTTCCT

CATATGGTGCATTGTGGGCAGGCATCAAGACTGAAGGTGGATTGTCACAG

TCTCAGTCACCTGGACAGACAGGATTTCTCAGCTATGGCACAAGCTTCAG

TACCCCTCAACCTGGACAGGCACCATACAGCTACCAGATGCAAGGTAGCA

GTTTTACAACATCATCAGGAATATATACAGGAAATAATTCACTCACAAAT

TCCTCTGGATTTAATAGTTCACAGCAGGACTATCCGTCTTATCCCAGTTT

TGGCCAGGGTCAGTACGCACAGTATTATAACAGCTCACCGTATCCAGCAC

ATTATATGACCAGCAGCAACACCAGCCCAACGACACCATCCACCAATGCC

ACTTACCAGCTTCAAGAACCGCCATCTGGCATCACCAGCCAAGCAGTTAC

AGATCCCACAGCAGAGTACAGCACAATCCACAGCCCATCAACACCCATTA

AAGATTCAGATTCTGATCGATTGCGTCGAGGTTCAGATGGGAAATCACGT

GGACGGGGCCGAAGAAACAATAATCCTTCACCTCCCCCAGATTCTGATCT

TGAGAGAGTGTTCATCTGGGACTTGGATGAGACAATCATTGTTTTCCACT

CCTTGCTTACTGGGTCCTACGCCAACAGATATGGGAGGGATCCACCCACT

TCAGTTTCCCTTGGACTGCGAATGGAAGAAATGATTTTCAACTTGGCAGA

CACACATTTAI I I I I lAATGACTTAGAAGAATGTGACCAAGTCCATATAG

ATGATGTTTCTTCAGATGATAACGGACAGGACCTAAGCACATATAACTTT

GGAACAGATGGCTTTCCTGCTGCAGCAACCAGTGCTAACTTATGTTTGGC

AACTGGTGTACGGGGCGGTGTGGACTGGATGAGAAAGTTGGCCTTCCGCT

ACAGACGGGTAAAAGAGATTTACAACACCTACAAAAATAATGTTGGAGGT

CTGCTTGGTCCAGCTAAGAGGGAAGCCTGGCTGCAGTTGAGGGCCGAAAT

TGAAGCCCTGACCGACTCCTGGTTGACACTGGCCCTGAAAGCACTCTCGC

TCATTCACTCCCGGACAAACTGTGTGAATATTTTAGTAACAACTACTCAG

CTCATCCCAGCATTGGCGAAAGTCCTGCTGTATGGGTTAGGAATTGTATT

TCCAATAGAAAATATTTACAGTGCAACTAAAATAGGAAAAGAAAGCTGTT

TTGAGAGAATAATTCAAAGGTTTGGAAGAAAAGTGGTGTATGTTGTTATA

GGAGATGGTGTAGAAGAAGAACAAGGAGCAAAAAAGCACGCGATGCCCTT

CTGGAGGATCTCCAGCCACTCGGACCTCATGGCCCTGCACCATGCCTTGG

AACTGGAGTACCTGTAACAGCGCTCGGCACTTTGACAGCGCACAGCTGCT

CTGTGACCAGGGACAGATCCAGCAGGCCCCAGTCTCGCATCAGCGCCGGC

CTCCAGAACTTAGCAATTTCCGCCTGGTGATGCGCAGTTGCTGTCAGTCT

TGACCTCTGCCTTTGTGGTGAATGGAGGACCACGTCTATTTCATCAGAAC

AGCTGTTGACTCTAGTACTGTGAATCCAGTGAAAATAAGCCATGAGAATG

TTTTAGCACAGCGTTATGTGTCTGCCACATTAACTACACGGTTCAAACCT

GTGAAGAAAGGACCTGCAAACGCTTCAGTTGTTAGCATTTTCAATGTGAT

ATAAACAGCTTCTCCAATACAGCAAACCTAATTGCACAACAGAGACTGAA

ATGTGTTTCCTGAATACCAGTGGAGGAATTTTCTTGTAAAGAAGGTTTAC

TTTTTGGTGTCTCATACCCAGGGTAATCTGTACATCTCTACTTATTTATG

AACAGACTTTTTTTAAAAAGATAAAAAAACAGCTTTATTGAGGTATAATT

CACCCACCAGACTTTTTTAAACATCAAATAATTGAAGAGACAATAGCATT

AGAAATAAGTGATTAAAGGCCTCTGCCTCACAACATGGCAAGTACAGTAC

TTTGMTTTTAGCACATTGCATAATAGTTTTAAGTATGTCTAATTTAAAC

GTATAATATGTACATCACTGAGACAATCATGTACAGAAAGAATTTTTGGT -Hog

GTAAATTTGTAATAATGGATAATTCTTTTACATATTGTTTAGGGAAATGA

TATTGAAAGGTAGCAATGCCTGGATAGTGAAGCATGAGGCAGCACGTGCA

CAAATTCATGTGCCGTGCCTTATCTGAGTTTTCGGTATAAATATGTAGAT

AATGGAT I I I I I I TTAGATAATGTTGTCAAGACCAAAAGCATGGATGTCA

AGTGTCAGTAAGGATTTTGTTTTCTAAAATTTTTTCCTGCATCAGTTCTT

CTGAGGGCCTTGATGAAATAACACAGCAGTTTCTTAAACAATTTGAAACA

AAATGAGCTCTCCTACCACCTCACTTTTTCATTTCCACACTAATGTATTA

TATGTAACTACTTGGAAAAAATAATTATTCAAATGCTTCTTCCCACAAAG

AATATAGATGATAGTAGATATATTTTATTAATAAAATGGTTCATGAATCG

GAGACTAACAAAGTTTTCATGTGCTCAGAATTATTAATTATCGTGTCTGC

ATTTTCTTTCGATAAAGGAAGACACACGATGCTAATCCGGAAATCAGCAA

ACTTTGCATTACTCCCTATGTGCGTATTTTCTCTTTCTTCCTGTCACCCT

GAGGAAGGTTCATTGCCATTGTCATCACCATGGAAACAACGTTCCTCTCC

ACCTGCATTATGTACTACATGACAGGCATCAATCTGGGGAAATAATAAAA

TTATCACCTTTGTCAGACCATAAGAGTTTCTCCAAAAGTGGTCAGTTTGG

CTGGGCAATATTTTCTCTCATCTAACAAACACAATCCATTGTCATGAAAT

TACCCTTAGGATGAGTCTTCTTTAATCAATCATATATTGGGCGGAAAAAA

CACCAGCTTTGACCCGAAGTAGTTGAAGAGCTACTTCATTCTTTTCTGAA

GTTGTGTGTTGCTGCTAGAAATAGTCATTTGTGAATTATCCAAATTGTTT

AAATTCACAATTGAATTAG I I I I I I CTTCCTTTTGGCTTGAAGCAAACAG

TTGACCATTTTTAACCTTTTCATTTTATGTTTTTGTACTCTGCAGACTGA

AAAGACAAAGTTTATCTTGGCCTTACTGTATAAAGGTATGCTGTGTCCAC

CGTTGTGTACAGAA I I I I I CTTCATTAATTTTGTGTTTAAGTTAATAAAA

TTTATTTGTGATGTACTGTAA

SEQ ID N° 4 :

TCTCCTTTTTCTCTTTTGGTTAAAAGAGGGCATTGTCGTTCTCAGCCATG

TGCTCTGTATAATTAAGAGCTGACACTGAAGCAGAGTAACAACATCTTCT

AATTTTTTTACCCCTGATCACAGGTGCAAACATCTCAAGCCAGTTCAGAT

GTTGCTGTTTCCTCAAGTTGCAGGTCTATGGAAATGCAGGATCTAACCAG

CCCGCATAGCCGTCTGAGTGGTAGTAGTGAATCCCCCAGTGGCCCCAAAC

TCGGTAACTCTCATATAAATAGTAATTCCATGACTCCCAATGGCACCGAA

GTTAAAACAGAGCCAATGAGCAGCAGTGAAACAGCTTCAACGACAGCCGA

CGGGTeTTTAAACAATTTCTCAGGTTCAGCAATTGGGAGCAGTAGTTTCA

GCCCACGACCAACTCACCAGTTCTCTCCACCACAGATTTACCCTTCCAAC

AGACCATACCCACATATTCTCCCTACCCCTTCCTCACAAACTATGGCTGC

ATATGGGCAAACACAGTTTACCACAGGAATGCAACAAGCTACAGCCTATG

CCACGTACCCACAGCCAGGACAGCCGTACGGCATTTCCTCATATGGTGCA

TTGTGGGCAGGCATCAAGACTGAAGGTGGATTGTCACAGTCTCAGTCACC

TGGACAGACAGGATTTCTCAGCTATGGCACAAGCTTCAGTACCCCTCAAC

CTGGACAGGCACCATACAGCTACCAGATGCAAGGTAGCAGTTTTACAACA

TCATCAGGAATATATACAGGAAATAATTCACTCACAAATTCCTCTGGATT

TAATAGTTCACAGCAGGACTATCCGTCTTATCCCAGTTTTGGCCAGGGTC

AGTACGCACAGTATTATAACAGCTCACCGTATCCAGCACATTATATGACC

AGCAGCAACACCAGCCCAACGACACCATCCACCAATGCCACTTACCAGCT

TCAAGAACCGCCATCTGGCATCACCAGCCAAGCAGTTACAGATCCCACAG

CAGAGTACAGCACAATCCACAGCCCATCAACACCCATTAAAGATTCAGAT

TCTGATCGATTGCGTCGAGGTTCAGATGGGAAATCACGTGGACGGGGCCG A O

AAGAAACAATAATCCTTCACCTCCCCCAGATTCTGATCTTGAGAGAGTGT

TCATCTGGGACTTGGATGAGACAATCATTGTTTTCCACTCCTTGCTTACT

GGGTCCTACGCCAACAGATATGGGAGGGATCCACCCACTTCAGTTTCCCT

TGGACTGCGAATGGAAGAAATGATTTTCAACTTGGCAGACACACATTTAT

TTTTTAATGACTTAGAAGAATGTGACCAAGTCCATATAGATGATGTTTCT

TCAGATGATAACGGACAGGACCTAAGCACATATAACTTTGGAACAGATGG

CTTTCCTGCTGCAGCAACCAGTGCTAACTTATGTTTGGCAACTGGTGTAC

GGGGCGGTGTGGACTGGATGAGAAAGTTGGCCTTCCGCTACAGACGGGTA

AAAGAGATTTACAACACCTACAAAAATAATGTTGGAGGTCTGCTTGGTCC

AGCTAAGAGGGAAGCCTGGCTGCAGTTGAGGGCCGAAATTGAAGCCCTGA

CCGACTCCTGGTTGACACTGGCCCTGAAAGCACTCTCGCTCATTCACTCC

CGGACAAACTGTGTGAATATTTTAGTAACAACTACTCAGCTCATCCCAGC

ATTGGCGAAAGTCCTGCTGTATGGGTTAGGAATTGTATTTCCAATAGAAA

ATATTTACAGTGCAACTAAAATAGGAAAAGAAAGCTGTTTTGAGAGAATA

ATTCAAAGGTTTGGAAGAAAAGTGGTGTATGTTGTTATAGGAGATGGTGT

AGAAGAAGAACAAGGAGCAAAAAAGCACGCGATGCCCTTCTGGAGGATCT

CCAGCCACTCGGACCTCATGGCCCTGCACCATGCCTTGGAACTGGAGTAC

CTGTAACAGCGCTCGGCACTTTGACAGCGCACAGCTGCTCTGTGACCAGG

GACAGATCCAGCAGGCCCAGTCTCGCATCAGCGCCGGCCTCCAGAACTTA

GCAATTTCCGCCCTGGTGATGCGCAGTTGCTGTCAGTCTTGACCTCTGCC

TTTGTGGTGAATGGAGGACCACGTCTATTTCATCAGAACAGCTGTTGACT

CTAGTACTGTGAATCCAGTGAAAATAAGCCATGAGAATGTTTTAGCACAG

CGTTATGTGTCTGCCACATTAACTACACGGTTCAAACCTGTGAAGAAAGG

ACCTGCAAACGCTTCAGTTGTTAGCATTTTCAATGTGATATAAACAGCTT

CTCCAATACAGCAAACCTAATTGCACAACAGAGACTGAAATGTGTTTCCT

GAATACCAGTGGAGGAATTTTCTTGTAAAGAAGGTTTACTTTTTGGTGTC

TCATACCCAGGGTAATCTGTACATCTCTACTTATTTATGAACAGACTTTT

TTTAAAAAGATAAAAAAACAGCTTTATTGAGGTATAATTCACCCACCAGA

CTTTTTTAAACATCAAATAATTGAGGAGACAATAGCATTAGAAATAAGTG

ATTAAAGGCCTCTGCCTCACAACATGGCAAGTACAGTACTTTGAATTTTA

GCACATTGCATAATAGTTTTAAGTATGTCTAATTTAAACGTATAATATGT

ACATCACTGAGACAATCATGTACAGAAAGAATTTTTGGTGTAAATTTGTA

ATAATGGATAATTCTTTTACATATTGTTTAGGGAAATGATATTGAAAGGT

AGCAATGCCTGGATAGTGAAGCATGAGGCAGCACGTGCACAAATTCATGT

GCCGTGCCTTATCTGAGTTTTCGGTATAAATATGTAGATAATGGATTTTT

TTTTAGATAATGTTGTCAAGACCAAAAGCATGGATGTCAAGTGTCAGTAA

GGATTTTGTTTCTAAAA I I I I I I CCTGCATCAGTTCTTCTGAGGGCCTTG

ATGAAATAACACAGCAGTTTCTTAAACAATTTGAAACAAAATGAGCTCTC

CTACCACCTCACTTTTTCATTTCCACACTAATGTATTATATGTAACTACT

TGGAAAAAATAATTATTCAAATGCTTCTTCCCACAAAGAATATAGATGAT

AGTAGATATATTTTATTAATAAAATGGTTCATGAATCGGAGACTAACAAA

GTTTTCATGTGCTCAGAATTATTAATTATCGTGTCTGCATTTTCTTTCGA

TAAAGGAAGACACACGATGCTAATCCGGAAATCAGCAAACTTTGCATTAC

TCCCTATGTGCGTATTTTCTCTTTCTTCCTGTCACCCTGAGGAAGGTTCA

TTGCCATTGTCATCACCATGGAAACAACGTTCCTCTCCACCTGCATTATG

TACTACATGACAGGCATCAATCTGGGGAAATAATAAAATTATCACCTTTG

TCAGACCATAAGAGTTTCTCCAAAAGTGGTCAGTTTGGCTGGGCAATATT

TTCTCTCATCTAACAAACACAATCCATTGTCATGAAATTACCCTTAGGAT GAGTCTTCTTTAATCAATCATATATTGGGCGGAAAAAACACCAGCTTTGA

CCCGAAGTAGTTGAAGAGCTACTTCATTCTTTTCTGAAGTTGTGTGTTGC

TGCTAGAAATAGTCATTTGTGAATTATCCAAATTGTTTAAATTCACAATT

GAATTAGTTTTTTCTTCCTTTTGGCTTGAAGCAAACAGTTGACCATTTTT

AACCTTTTCATTTTATGTTTTTGTACTCTGCAGACTGAAAAGACAAAGTT

TATCTTGGCCTTACTGTATAAAGGTATGCTGTGTCCACCGTTGTGTACAG

AATTTTTCTTCATTAATTTTGTGTTTAAGTTAATAAAATTTATTTGTGAT

GTACTGTAA

SEQ ID N° 5 :

5'-GGAAATGGTAGAACTAGTGATCTCACCCAGCCTCACTGTAAACAGCGATT

GTCTGGATAAACTGAAGTTTAACCGTGCTGACGCTGCTGTGTGGACTCTG

AGTGACAGACAAGGCATCACCAAATCGGCCCCCCTGAGAGTGTCCCAGCT

CTTCTCCAGATCTTGCCCACGTGTCCTCCCCCGCCAGCCTTCCACAGCCA

TGGCAGCCTACGGCCAGACGCAGTACAGTGCGGGGATCCAGCAGGCTACC

CCCATTACAGCTTACCCACCTCCAGCACAAGCCTATGGAATCCCTTCCTA

CAGCATCAAGACAGAAGACAGCTTGAACCATTCCCCTGGCCAGAGTGGAT

TCCTCAGCTATGGCTCCAGCTTCAGCACCTCACCCACTGGACAGAGCCCA

TACACCTACCAGATGCACGGCACAACAGGGTTCTATCAAGGAGGAAATGG

ACTGGGCAACGCAGCCGGTTTCGGGAGTGTGCACCAGGACTATCCTTCCT

ACCCCGGCTTCCCCCAGAGCCAGTACCCCCAGTATTACGGCTCATCCTAC

AACCCTCCCTACGTCCCGGCCAGCAGCATCTGCCCTTCGCCCCTCTCCAC

GTCCACCTACGTCCTCCAGGAGGCATCTCACAACGTCCCCAACCAGAGTT

CCGAGTCACTTGCTGGTGAATACAACACACACAATGGACCTTCCACACCA

GCGAAAGAGGGAGACACAGACAGGCCGCACCGGGCCTCCGATGGGAAGCT

CCGAGGCCGGTCTAAGAGGAGCAGTGACCCGTCCCCGGCAGGGGACAATG

AGATTGAGCGTGTGTTCGTGTGGGACTTGGATGAGACAATAATTATTTTT

CACTCCTTACTCACGGGGACATTTGCATCCAGATACGGGAAGGACACCAC

GACGTCCGTGCGCATTGGCCTTATGATGGAAGAGATGATCTTCAACCTTG

CAGATACACATCTGTTCTTCAATGACCTGGAGGATTGTGACCAGATCCAC

GTTGATGACGTCTCATCAGATGACAATGGCCAAGATTTAAGCACATACAA

CTTCTCCGCTGACGGCTTCCACAGTTCGGCCCCAGGAGCCAACCTGTGCC

TGGGCTCTGGCGTGCACGGCGGCGTGGACTGGATGAGGAAGCTGGCCTTC

CGCTAeCGGCGGGTGAAGGAGATGTACAATACCTACAAGAACAACGTTGG

TGGGTTGATAGGCACTCCCAAAAGGGAGACCTGGCTACAGCTCCGAGCTG

AGCTGGAAGCTCTCACAGACCTCTGGCTGACCCACTCCCTGAAGGCACTA

AACCTCATCAACTCCCGGCCCAACTGTGTCAATGTGCTGGTCACCACCAC

TCAACTAATTCCTGCCCTGGCCAAAGTCCTGCTATATGGCCTGGGGTCTG

TGTTTCCTATTGAGAACATCTACAGTGCAACCAAGACAGGGAAGGAGAGC

TGCTTCGAGAGGATAATGCAGAGATTCGGCAGAAAAGCTGTCTACGTGGT

GATCGGTGATGGTGTGGAAGAGGAGCAAGGAGCGAAAAAGCACAACATGC

CTTTCTGGCGGATATCCTGCCACGCAGACCTGGAGGCACTGAGGCACGCC

CTGGAACTGGAGTATTTATAGCAGGATCAGCAGCATCTCCACCTGCCAT-3'

SEQ ID N° 6 :

5^'-ACTCTAGAAGGACCCACTCACTATAGGGCTCGAGCGGCCGCCCGATTGGT

TCTACTGTGGGTCTGGACTGATCTCCATGTCCTGTTGTGGGGCTTTTACA

GCCTTTGGATTGTGAAAACTGCTGAGAGAGACTTGCAATCCAGTCACATA AGTATAATAAAGAAATATTGGTCCTCATGGAAGAAGAGCAAGATTTACCA

GAGCAACCAGTGAAAAAAGCCAAGATGCAGGAATCAGGAGAGCAAACTAT

AAGTCAAGTAAGCAATCCAGATGTCAGTGATCAGAAGCCTGAAACATCAA

GCCTTGCTTCAAACCTTCCCATGTCAGAGGAAATTATGACATGCACCGAT

TACATCCCTCGCTCATCCAATGATTATACCTCACAAATGTATTCTGCAAA

ACCTTATGCACATATTCTCTCAGTTCCTGTTTCGGAAACTGCTTACCCTG

GACAGACTCAATACCAGACACTACAGCAGACTCAACCCTATGCTGTCTAC

CCTCAGGCAACCCAAACGTATGGACTACCTCCTTTTGGTGCATTGTGGCC

AGGTATGAAACCTGAAAGTGGTTTAATTCAGACTCCATCTCCAAGTCAAC

ACAGTGTTCTTACCTGCACTACAGGGTTAACCACAAGCCAGCCAAGCCCA

GCACATTATTCTTATCCCATTCAAGCTTCAAGCACAAATGCCAGCCTGAT

ATCTACTTCTTCTACAATTGCCAATATTCCAGCAGCAGCAGTAGCCAGCA

TCTCAAACCAGGATTATCCCACCTATACTATTCTTGGTCAGAATCAGTAC

CAGGCCTGCTACCCCAGCTCCAGCTTTGGAGTCACAGGTCAGACTAACAG

TGATGCAGAGAGCACCACATTAGCAGCAACCACATACCAGTCGGAGAAGC

CTAGTGTCATGGCGCCTGCACCTGCAGCACAGAGACTTTCCTCTGGAGAC

CCTTCTACAAGTCCATCTTTGTCCCAGACTACACCAAGTAAAGATACTGA

TGATCAGTCCAGGAAAAACATGACTAGCAAGAACCGGGGCAAGAGGAAAG

CTGATGCCACTTCTTCCCAAGACAGTGAATTAGAACGGGTATTTCTGTGG

GACTTGGATGAAACCATCATCATCTTCCACTCACTTCTTACTGGATCCTA

TGCCCAGAAATATGGAAAGGACCCAACAGTAGTGATTGGCTCAGGTTTAA

CAATGGAAGAAATGA I I I I I GAAGTGGCTGATACTCATCTATTTTTCAAT

GACTTAGAGGAGTGTGACCAGGTACATGTGGAAGATGTGGCTTCTGATGA

CAATGGCCAAGACTTGAGCAACTACAGTTTCTCAACAGATGGTTTCAGTG

GCTCAGGAGGTAGTGGCAGCCATGGTTCATCTGTGGGTGTTCAGGGAGGT

GTGGACTGGATGAGGAAACTAGCTTTCCGCTACCGGAAAGTGAGAGAAAT

CTATGATAAGCATAAAAGCAACGTGGGTGGTCTCCTCAGTCCCCAGAGGA

AGGAAGCACTGCAGAGATTAAGAGCAGAAATTGAAGTTTTAACAGATTCC

TGGTTAGGAACTGCATTAAAGTCCTTACTTCTCATCCAGTCCAGAAAGGA

TTGTGTGAATGTTCTGATCACTACCACCCAGCTGGTTCCAGCCCTGGCCA

AGGTTCTCCTATATGGACTAGGAGAAATATTTCCTATTGAGAACATCTAT

AGTGCTACCAAAATTGGTAAGGAGAGCTGCTTTGAGAGAATTGTGTCAAG

GTTTGGAAAGAAAGTCACATATGTAGTGATTGGAGATGGACGAGATGAAG

AAATTG€AGCCAA-3'

SEQ ID N° 7 :

MLLFPQVAVKTEPMSSSETASTTADGSLNNFSGSAIGSSSFSPRPTNQFS

PPQIYPSNRPYPHILPTPSSQTMAAYGQTQFTTGMQQATAYATYPQPGQP

YGISSYGALWAGIKTEGGLSQSQSPGQTGFLSYGTSFSTPQPGQAPYSYQ

MQGSSFTTSSGIYTGNNSLTNSSGFNSSQQDYPSYPSFGQGQYAQYYNSS

PYPAHYMTSSNTSPTTPSTNATYQLQEPPSGITSQAVTDPTAEYSTIHSP

STPIKDSDSDRLRRGSDGKSRGRGRRNNNPSPPPDSDLERVFIWDLDETI

IVFHSLLTGSYANRYGRDPPTSVSLGLRMEEMIFNLADTHLFFNDLEECD

QVHIDDVSSDDNGQDLSTYNFGTDGFPAAATSANLCLATGVRGGVDWMRK

LAFRYRRVKEIYNTYKNNVGGLLGPAKREAWLQLRAEIEALTDSWLTLAL

KALSLIHSRTNCVNILVTTTQLIPALAKVLLYGLGIVFPIENIYSATKIG

KESCFERIIQRFGRKWYWIGDGVEEEQGAKKHAMPFWRISSHSDLMAL HHALELEYL -ΛAZ

SEQ ID N° 8 :

MEMQDLTSPHSRLSGSSESPSGPKLGNSHINSNSMTPNGTEVKTEPMSSS

ETASTTADGSLNNFSGSAIGSSSFSPRPTHQFSPPQIYPSNRPYPHILPT

PSSQTMAAYGQTQFTTGMQQATAYATYPQPGQPYGISSYGALWAGIKTEG

GLSQSQSPGQTGFLSYGTSFSTPQPGQAPYSYQMQGSSFTTSSGIYTGNN

SLTNSSGFNSSQQDYPSYPSFGQGQYAQYYNSSPYPAHYMTSSNTSPTTP

STNATYQLQEPPSGITSQAVTDPTAEYSTIHSPSTPIKDSDSDRLRRGSD

GKSRGRGRRNNNPSPPPDSDLERVFIWDLDETIIVFHSLLTGSYANRYGR

DPPTSVSLGLRMEEMIFNLADTHLFFNDLEECDQVHIDDVSSDDNGQDLS

TYNFGTDGFPAAATSANLCLATGVRGGVDWMRKLAFRYRRVKEIYNTYKN

NVGGLLGPAKREAWLQLRAEIEALTDSWLTLALKALSLIHSRTNCVNILV

TTTQLIPALAKVLLYGLGIVFPIENIYSATKIGKESCFERIIQRFGRKW

YWIGDGVEEEQGAKKHAMPFWRISSHSDLMALHHALELEYL

SEQ ID N° 9 :

MVELVISPSLTVNSDCLDKLKFNRADAAVWTLSDRQGITKSAPLRVSQLF

SRSCPRVLPRQPSTAMAAYGQTQYSAGIQQATPYTAYPPPAQAYGIPSYS

IKTEDSLNHSPGQSGFLSYGSSFSTSPTGQSPYTYQMHGTTGFYQGGNGL

GNAAGFGSVHQDYPSYPGFPQSQYPQYYGSSYNPPYVPASSICPSPLSTS

TYVLQEASHNVPNQSSESLAGEYNTHNGPSTPAKEGDTDRPHRASDGKLR

GRSKRSSDPSPAGDNEIERVFVWDLDETIIIFHSLLTGTFASRYGKDTTT

SVRIGLMMEEMIFNLADTHLFFNDLEDCDQIHVDDVSSDDNGQDLSTYNF

SADGFHSSAPGANLCLGSGVHGGVDWMRKLAFRYRRVKEMYNTYKNNVGG

LIGTPKRETWLQLRAELEALTDLWLTHSLKALNLINSRPNCVNVLVTTTQ

LIPALAKVLLYGLGSVFPIENIYSATKTGKESCFERIMQRFGRKAVYWI

GDGVEEEQGAKKHNMPFWRISCHADLEALRHALELEYL

SEQ ID N° 10 :

MEEEQDLPEQPVKKAKMQESGEQTISQVSNPDVSDQKPETSSLASNLPMS

EEIMTCTDYIPRSSNDYTSQMYSAKPYAHILSVPVSETAYPGQTQYQTLQ

QTQPDAVYPQATQTYGLPPFGALWPGMKPESGLIQTPSPSQHSVLTCTTG

LTTSQPSPAHYSYPIQASSTNASLISTSSTIANIPAAAVASISNQDYPTY

TILGQNQYQACYPSSSFGVTGQTNSDAESTTLAATTYQSEKPSVMAPAPA

AQKLSSGDPSTSPSLSQTTPSKDTDDQSRKNMNSKNRGKKKADATSSQDS

ELERVFLWDLDETIIIFHSLLTGSYAQKYGKDPTWIGSGLTMEKMIFEV

ADTHLFSNDLKECDQVHVEDVAPNDKGQNLNNYSFSTNGFSGSGGSGSHG

SSVGVQGGVDWMRKLAFRYRKVREIYDKHKSNVGGLLSPQRKEALQKLKA

EIEVLTNSWLGTALKSLLLIQSKKNCVNVLITTTQLLPALAKVLLYGLGK

IFPIENIYSATKIGKESCFERIVTSLGKKLTYWIGDGRDEEIAAK

SEQ ID N° 11 :

5'-CTCAGCCATGTGCTCTGTATAATTAAGAGC-3'

SEQ ID N° 12 :

5'-AGGCACGGCACATGAATTTGTGCACGTGCT-3'

SEQ ID N° 13 :

5'-ATGAACAAGCACGAGCATTGC-3'

SEQ ID N° 14 :

5^'-GCAATGCTCGTGCTTGTTCAT-3' SEQ ID N° 15 :

5'-AGGCTAATCTTGGCACCATGG-3'

SEQ ID N° 16 :

5'-CACTGCTGTTTACGTAGCAGG-3'

SEQ ID N° 17 :

5'-TGAATAACAGCTTTCTCAGCC-3'

SEQ ID N° 18 :

5'-GACTATATAGTTCTTCTCCATT-3'

SEQ ID N° 19 :

5'-CTTTCAGCCTCTCCCAATGC-3'

SEQ ID N° 20 :

5'-ACCAACAAACTCCTGTCTCAC-3'

SEQ ID N° 21 :

5'-ACCTACTGATTGACATAGTTGA-3'

SEQ ID N° 22 :

5'-ACTATAAAAGGGAGATGGTCAC-3'

SEQ ID N° 23 :

5'-GTGACCATCTCCCTTTTATAGT-3'

SEQ ID N° 24 :

5'-TGCTGAGGTACTGGTGGTAA-3'

SEQ ID N° 25 :

5 -AAATCTGGAGGCTGGTATTC-3'

SEQ ID N° 26 :

5 -TGCTTTATGTTTCTCTTACGTC-3'

SEQ ID N° 27 :

5'-AGAGTACTGCACATATTCATCA-3'

SEQ ID N° 28 :

5 -TGCTGTGGCACATACAACCC-3'

SEQ ID N° 29 :

5'-TGCTGTGGCACATACAACCC-3'

SEQ ID N° 30 :

5' C ACTGAAGCAGAGTAACAACA3'

SEQ ID N° 31 :

S'CCAACAGAGGCTGTTACTATTS'

SEQ ID N° 32 :

5'GGGACTTTTGTGCAAGTGGT3'

SEQ ID N° 33 :

5'GACAACTGAAATCATAACCAC3'

SEQ ID N° 34 :

5TTATGATATATGTTCAGTTAGGG3' SEQ ID N° 35 :

5'CATACACAGGGACATTACATG3' SEQ ID N° 36 :

5OGCAGGTCACAAAGACCAAA3' SEQ ID N° 37 :

5ΑGATGGAACATGTGGGCACA3' Λ

SEQ ID N° 38 :

5OTGATGTGGTTGTTAATCGGT3' SEQ ID N° 39 :

5ΑCACAGAAGGTGACAACACTG3' SEQ ID N° 40 :

5'GAGATAAGATTGGGGAAGCAT3' SEQ ID N° 41 :

S'CCAATCCAGTTGCCATCATCS' SEQ ID N° 42 :

5'GCTATTTTCCTGTACCCACATT3' SEQ ID N° 43 :

5'GAAAGCTCTCACTTATAAACAG3' SEQ ID N° 44 :

5'GGCTCAGAAACCCAAACATAC3' SEQ ID N° 45 :

5'GTGCAACCACTGCATGAATAT3' SEQ ID N° 46 :

5ΑGCTGGCATTTCAATGATACT3' SEQ ID N° 47 : 5'GTGGCAGACACATAACGCTG3'

ANNEX 3

Untitled Sequence # 1 -> Full Restriction Map

DNA sequence 3731 b.p . TCTCCTTT TCT . . . GATGTACTGTAA linear

Positions of Restriction Endonucleases sites (unique sites underlined)

26 47 68

50

ScrF I Hph I Dde I EcoR II Eco57 I

BsmA I BstN I Alu I Alu I

SfaN I Eco57 I Mae III Mae III Dde I HinD ITT

AGG ICATCAAGACITGAAGGTGGATTGITCACACTICTI II I I I II I 560

TCCGTAGTTCTGACTTCCACCTAACAGTGTCΑGAGTCAGT^

4 I83 - 4I92 5I05 -I I 5I1I7-I . 5I3-9I - 5I5I3I -

511 521 542 554

513 521 556

518 521 ScrF I EcoR II Mnl I Nla IV Rsa I BstN I Ban I Alu I SfaN I

G ITACCCICTCAACICTGGACAOI-CACCATACAIGCTACCA^I 640

CATGGGGAGTTGGACCTGTCCGTGGTATC_TCGATGGT^

5 I61 I ^• 5I72 5I80 5I90 5I98• 566 580

572 572

Sec I ScrF I EcoR II BstN I Hae III

Xmn I Mnl I Mse I Bsr I Eas_I

GG IAAATAATTCACTCACAAATTCICTCrc.GATTITAA^ I II I 720

CCTTTATTAAGTGAGTGTTTAAGGAGACCrAAATTATCAA^

6 I42 ^♦ - 6I63 ' 6I72 • • 7I05 • 7I1I2I

712 712 713 715

800

746 774

Sau3A I Mbo I

Dpn I Alw I Hph I Mae III Alu I SfaN I Tthlll II BstY I Rsa I

CC^CCAATGCCACTTACCA IGCTTCAAGAACCGCCATCTO^I I I I M I 880

GGTGGTTACGGTGAATGGTCGAAGTTCTTGGCGGTAGACCGTAGTGGTCGGT^

|. . I I *| I • II • I ^■

819 840 851 862 877

843 857

863 863 863 863 cia i

Sau3A I

Hinf I Taq I Mnl I Mae II

Hinf I Mbo I Taq I Pml I

Mse I AlwN I Don I Hσa I BsaA I

AGCACAATCCACAGCCCATCAAC^CCCAT ITAAAGIATTCIAGIATTC^III I I I II 960

TCGTGTTAGGTGTCGGGTAGTTGTGGGTAATTTCTAAGTCTAAGACT

9 I0-9 I 9I18I 9I2I6I • 9I34I I- • 9I5I7•

914 926 937 957

920 928 939 958

926 927 Mbo II Sau3A I

Hae III Mbo I

Sau96 I Mnl I Hinf I BsmA I

Nla IV Hph I AlwN I Dpn I Fok I

TGGACG IGIGIGCCGIAAGAAACAATAATCCTTICACICTCCCCCIAGIATTCTGIATCTTGAGAG^ I I 1040

ACCTGCCCCGGCririTiυi ATTAGGAAGTGG

9 I6I6I ^• I • 9I8-9I 9l9-9l 1I007• • - 1I036I

967 992 1001 1040

968 1007

972 1007

Sau3A I

Mbo I

Dpn I

Sau96 I Al I

Ava II Nla IV

PpuM I BstY I

Nla IV BamH I

ECOO109 I Alw I Sty I

Bsr I Mnl I Eco57 I Sec I

AGACAATCATTGTΓΓTCCACTCCTTGCΠA ICIK-I^I^ I II I I 1120

TCTGTTAGTAACAAAAGGTGAGGAACGAATGACCCAGGATGCGGT^

1 I070II 1I096II 1I110 1I120

1073 1099 1120

1073 1099

1074 1099

1074 1100

1100 1100 1100

Mbo II Tthlll I Mbo II Mse I Dde I Mae III

CTTGGACK^GAATGG IAAGAAATGATTTTCAACTTGG I I I I I 1200

GAACCTGACGCTΓACCTTCTT ACTAAAAGTTGAACCGTCTGTGTGTAAATAAA

I . . . i . i i . i i .

1136 1176 1183 1194

1187 1196 • Dde I Sau96 I Ava II Eco57 I PpuM I Fnu4H I

Mbo II EcoO109 I Bbv I

AGTCCATATAGATGATGΥ-TCTTCAGATGATAAC 1280

TCAGGTATATCTACTAOUUGAAGTCTACTATTGCCT^

1 i2i20 ^• 1I2I39 I ^{• • •} , 1 i280

1221 1239 1280

1240 1240

1244 11 y

nu4H I

Bbv I Bsr I Fok I Hae III

;t I Bsr I Tthlll II Rsa I Bsr I Hae I

CTGCAGCAACCAGTGCTAACTTATGTTTGGCAACTGGTGTA^ 1360

GACGTCGTTGGTCACGATTGAATACAAACCGTTGACCACAT^

I I I - l - l l' - I I - II

1281 1290 1304 1319 1335 1350

1283 1313 1338 1351

1283

Alu I Sau96 I ScrF I

Mnl I Ava II Mnl I EcoR II

Mme I Tthlll II Dde I BstN I

I I I I I I I I

TACAGACGGGTAAAAGAGATTTACAACACCTACAAAAATAATGTTGGAG 1440

AT ?IX rGCCCATTTTCTCTAAATGTTGTGGATGTTTTTATTAC^

• 1I403I • 1I413 l- l 1l425I' 1I437• 1407 1418 1429 1437 1418 1437

1423 ScrF I EcoR II Pst I Hae III BstN I Sau96 I

Fnu4H I Sau96 I Pie I Bsr I

Bbv I Mnl I Hinf I HinC II Hae III

G ICITGCAGTTGIAGIGIGCCGAAATTGAAGCCCTGACCGIACTCICT^I^ I I 1520

CGACGTCAACT XCGGCTTTAACTTCGGGACπ -KX- GAGGACCAACTG^

1 I4I41 1I45I0I - 1I475I- 1I483 I- 1I492

1441 1452 1475 1489

1442 1453 1479 1492

1479 1479

Msp I

Hpa II ScrF I

Nci I Alu I

Ben I Ssp I Mae III Dde I Fok I

C ICICGGAC-AAACTGTGTGAIATATTTTAGITAAC-AACTACIT^I I 1600

GGGCCTGTTTGACACACTTATAAAATCATTGTTGATGAGTCGACT

1 I5I21 ^• 1 I53 ^•8 1 I547 ^• 1 I557 I 1 I564

1521 1560

1521

1522

Ssp I Alu I

GGAATTGTATTTCCAATAGAAAATATTTTACAGTGCAACTAA^ 1680

CCTTAACATAAAGGTΓATCTI'.ΎATAAATGTCACGTT^

• • j • • • I • _* •

1622 1655

Mbo II SfaN I

Mbo II Mbo II BstU I EcoN I

I I I I I I

GTTTGGAAGAAAAGTGGTGTATGTTGTTATAGGAGATGGTGTAGA 1760

CAAACCTTCTM^CCACATAC-AACAATATCCTCTA

I . . . . i i . . I l l *

1686 1724 1750 1758

1727 1753 Sau3A I

Mbo I

Dpn I HinP I

BstY I Nla III Sty I Hha I

Alw I Mnl I Sec I Rsa I Hae II

Mnl I Sau96 I Sau96 I Nla III Gsu I Eco47 TTT

Gsu I Gsu I Ava II Hae III pfiM r Bsr I Mae III

TCTGGAGGATCTCCAGCCACTCGGACCr ATGGCCCTGCACCATGCCTT^ 1840

AGACCTCCTAGAGGTCGGTGAGCCTGGAGTACCGGGACGTGGTACGGAA^

1762 1771 1783 1792 1801 1813 1825

1765 1783 1792 1802 1814 1830

1767 1786 1806 1819 1830

1767 1789 1806 1831

1768 1831

1768

Hae III Msp I

Fnu4H I Hpa II

Alu I Sec I Sau3A I Nae I

Pvu II ScrF I Mbo I BsmA I HinP I

NspB II EcoR II Dpn I Bsr I Hha I Gsu I

HinP I BstN I Alw I Sau96 I Hae II Mnl I

Hha I Bbv I Mae III BstY I Hae III SfaN I CfrlO T Dde I

I III I I II I I I I II II I II I

CTTTGACAGCGCACAGCTGCTCTGTGACCAGGGACAGATCCAGCAGGCC 1920

GAAACTGTCGCGTGTCGACGAGACACTGGTCCCTGTCTAG

1 I8-49 II 1I856^• 1I864I ^• 1I8I76^• 1I886I- I 1I897^• II 1I9I05I II 1I9-19

1849 1868 1877 1886 1902 1910

1854 1868 1877 1889 1903 1911

1854 1868 1877 1892 1903

1855 1868 1877 1905

1856 1906 1906 1908

^• 1I9I33I 1I94I0I I ^{• •} 1I965 1I97^•8 1I98I6I- 1I993

1934 1942 1988

1934 1943 1988

1934 1943 1989

1937 1946 HinC II Sea I Alu I Pie I Rsa I Pvu II Hinf I Bsr I

NspB II Mae I Hinf I Nla III

TTCATCAGAAC IAIGCTG ITTG IACTC ITA IGITACTGTG IAA I I 2080

AACTAGTCTTGTCGACAACTGACiATCATGACACTTAGGTO

^•II I I- I II • I I ^• - I

2011 2023 2033 2052

2011 2019 2037

2012 2019 2026 2016 2025

Sau96 I Ava II PpuM I EcoO109 I Mse I Mbo II BspM I Eco57 I

GTCTGCCACAT ITAACTACACGGTTCAAACCTGTGIAAGAAAIGIGAICCT^ I 2160

CAGACGGTGTAATTtaTGTGCα^AGTTTGGACA riCITI^CTGGAC

^•I ^• • I II I ^• I

2091 2114 2123 2134

2120 2120

2121 BsmA I Mnl I

Alu I AlwN I Bsr I

TATAAACA IGCTTCTCCAATACAGCAAACCTAATTGCACAACIAGIAGA^ I I 2240

ATATTTGT ICG.AAGAGGTTAT.GTCGTTTGGA.TTAACGTGTT.G|TCITCTGACT.TTAC^ . I . i 2168 2201 2228

2203 2234

Sec I ScrF I EcoR II BstN I

Mse I Mse I BsmA I

Dra I Alu I Mnl I Hph I Dra I Mnl I

I I I I I M I I

TTTTTAAAAAGATAAAAAAACAGCITTATTGAGGTATAATTCARC^ 2400

AAAAATTTTTCTATTTTΠ-ICTCGAAATAAC

I I - I - I - I ^• I I ^• - I I -

2323 2342 2351 2361 2377 2395

2324 2378 2398

Hae III Stu I Rsa I

Hae I Mnl I Rsa I

Mse I Mnl I Nla III Sea I

ACAATAGCATTAGAAATAAGTGAT ITAAAIGIGCICTCTGCICTCACAACIATGGCAAGI^ II 2480

TGTTATCGTAATCTTTATTCACTAATTTCCGGAGACGGAGT^^

• 2I424ll-2l431 I • 2I445 • I 2I4I57* 2428 2437 2453

2428 2458

2429 Mae II Mse I BsmA I Rsa I

Mse I Dra I Rsa I Dde I Nla III

CATAATAGTTT ITAACTATGTCTAATITITAA^I I I I I I 2560

GTATTATCAAAATT<-ATAC»GATTAAATTTGCATATT^^

• 2I491 - 2I5I05I -2I521 2l5-2l9 2I5-39I

2506 2531 2542

2510

ScrF I EcoR II BstN I

TGTAAATTTCΓAATAATGGATAATTCTTTTAΓΛTAT^ I 2640

AC^TTTAAACATTATTACCTATTAAGAAAATGTATAAOU^

• • • • • • I •

2630 2630 2630 HgiA I Bspl286 I Mae II Fnu4H I Bbv I &paτ, ^τ Mnl I Pml I Nla III BsaA I Nla III Dde I

I I I II I I I

AAGCATGAGGCAGCACGTGCACAAATTCATGTGCCGTGCCπ^ 2720

TTCGTACTCCGTCGTGCACGTGTTTAAGTACACGGCACGGAATAGACTCAAAAGCCATATTT

I I I II I ^• I ^• I

2644 2654 2668 2685

2647 2654 2650 2657 2650

2655 2657 2657

Nla III Bst T Fok I SfaN I I I I I

TrTπTAGATAATGTTGTCAAGACCAAAAGCATGGATGTC^ 2800

AAAAAATCTATTACAACAGTTCTGGTTTTCGTACCT^^

I ' l l * ^{• • •} I •

2744 2754 2799

2751 Hae III Alu I

EcoO109 I ≤ c_X

Mnl I HgiA I

Dde I Bspl286 I

Mbo II Sau96 I Mse I Baα_II Mnl I

I I M M I I I I

ATCAGΓΓCTTCTGAGGGCCITGATGAAATAACAC^^ 2880

TAGTCAAGAAGACTCCCGGAACTACTTTATTGTGTCGT^

I - I M i l ^• • • I ^• I I ^• I -

2807 2815 2844 2865 2879

2811 2865

2813 ^' 2865

2814 2865

2816 2866

Mae III Mbo II I I

TCACTTTTTCATTTCCΛCACTAATGTATTATATGT^ 2960

AGTGAAAAAGTAAAGGTGTGATTACATAATATACATTGATGAACCTΓTTΓTATTAATA^

2914 2948

Dde I

Hinf I HgiA I

Mse I Nla III Bspl286 I

Ase I BspH I BsmA I Nla III

AATATAGATGATAGTAGATATATTTTATTAATAAAATGGTTCATGAATCGGAGACT 3040

Msp I

Mse I Mbo II Hpa II Ase I Taq I Bbv TT SfaN I BspM II

TTA ¹T¹TAATTATCGTGTCTGCATTTTCT TICGATAAAGGIAAGACACACGIATGCTA^II 3120

AATAATTAATAGCAC-AGACCTAAAAGAAAGCTATTTCCTTC^

II • I- I • I • II ^•

3043 3069 3078 3088 3096

3044 3078 3097

3097 Nla III

Dde I Sty I

EcoN I Sec I

Bsu36 I Nco I

Mae III Dsa I

Mbo II Hph I Mnl I Hph I Mae II

ACTCCCTATGTGCGTATTTTCΓ ΓTT ICTTCCTGITICACCICI^I I II I 3200

TGAGGGATACACGCATAAAAGAGAAAGAAGGACAGTGGGACTCCTTCCAAGTAACGGTAACA^

3 I146• I3I154ll-3l161 • 3I185II- 3I1-99

3153 3188

3158 3188

3159 3188

3189 Nla III

Mnl I BspM I Rsa I SfaN I Hph I

GTTC ICTCTCCAICCTGCATTATGITACTACIATGACAGGI I 3280

CAAGGAGAGGTGGACGTAATACATGATGTACTGTCCGTAGTTAGACCCCTTTATTATTTTAATA

3 I204 -3I211 - 3l222 l- 3l236- • • 3I264 •

3228

Nla III Ssp I Tthlll II BspH I

TAAGAGTTTCTCCAAAAGTGGTCAGTTTGGCTGGGCA IATATTTTCTC^^ I II 3360

ATTCTCAAAGAGGTTTTCACCAGTCAAACCGACCC^

I . j . j i

3317 3336 3352

3353 Bbv II Fok I Mbo II Alu I

Dde I Pie I Mbo II

Bsu36 I Hinf I Mse I Alu I Ear I TACC ICITTAGIGATGIAGITICTTCIITAATCA^ I I I 3440

ATGGGAATCCTACTCAGAAGAAATTAGTTAGTATATAACCCGCCrTTTTTGTGGTCGAA^

3 I3I64 I- 3I37I3I • 3I382 • • 3I415 - 3I435I-

3365 3373 3435

3369 3376 3439

3375

AACTTAATCAAAAAAGAAGGAAAACCGAACTTCGTTTGTCAACTGGTAAAAATTGGA

3 I535 - 3I554 I -3I571 • - 3I595I

3560 3600

Hae III Hae I Rsa I Mbo II

TGCAGACTGAAAAGACAAAGTTTATCTT IGIGCCTTACTC^ I I 3680

ACGTCTGACTTTTCIXTITrCAAATAGAAC^

I I - • • ^• I ^• I -

3628 3667 3679

3629 Mse I Mse I

Ase I Mse I Rsa I

TTCA ITITAATTTTGTGTTITAAGTITAATAAAATTTATTTGTGATGITACTGTAA 3731

Restriction Endonucleases site usage

Aat II - BstN I 10 HinC II 3 Pie I 3

Ace I - BstU I 1 HinD III 1 Pml I 2

Afl II - BstX I 1 Hinf I 8 PpuM I 3

Afl III - BstY I 4 HinP I 4 Pst I 3

Aha II - Bsu36 I 2 Hpa I - Pvu I -

Alu I 18 CfrlO I 1 Hpa II 3 Pvu II 2

Alw I 5 Cla I 1 Hph I 11 Rsa I 17

AlwN I 4 Dde I 15 Kpn I - Rsr II -

Apa I - Dpn I 7 Mae I 2 Sac I 1

ApaL I 1 Dra I 5 Mae II 6 Sac II -

Ase I 3 Dra III - Mae III 10 Sal I -

Asp718 - Drd I - Mbo I 7 Sau3A I 7

Ava I - Dsa I 1 Mbo II 17 Sau96 I 12

Ava II 6 Eae I 1 Mlu I - Sea I 2

Avr II - Eag I - Mme I 2 ScrF I 11

BamH I 1 Ear I 1 Mnl I 27 Sec I 8

Ban I 1 Eco47 III 1 Mse I 1 SfaN I 9

Ban II 1 Eco57 I 7 Mse I 22 Sfi I -

Bbe I - EcoN I 2 Msp I 3 Sma I -

Bbv I 9 EcoO109 I 4 Nae I 1 SnaB I -

Bbv II 2 EcoR I - Nar I - Spe I -

Bel I 1 EcoR II 10 Nci I 1 Sph I -

Ben I 1 EcoR V - Nco I 1 Spl I 1

Bgl I - Esp I - Nde I 2 Ssp I 3

Bgl II - Fnu4H I 9 Nhe I - Stu I 1

BsaA I 3 Fok I 6 Nla III 14 Sty I 3

Bsm I 1 Fsp I 1 Nla IV 4 Taq I 3

BsmA I 8 Gdi II - Not I - Tthlll I 2

Bspl286 I 4 Gsu I 4 Nru I - Tthlll II 7

BspH I 2 Hae I 4 Nsi I - Xba I -

BspM I 2 Hae II 2 Nsp7524 I - Xca I -

BspM II 1 Hae III 11 NspE 1 II 2 Xho I -

Bsr I 12 Hga I 1 NspH [ I - Xcm I -

BssH II - HgiA I 4 PaeR7 I - Xma I -

BstB I - HgiE II - PflM I 1 Xmn I 1

BstE II - Hha I 4

Enryme Site Use Site position (Fragment length) Fragment order

ApaL I g/tgcac" 1 1( 2656) 2657 ( 1075)

BamH I g/gatcc 1 1( 1098) 1099 ( 2633)

Ban I g/gyrcc 1 1( 579) 580 ( 3152)

Ban II grgcy/c 1 1( 2864) 2865 ( 867)

Bel I t/gatca 1 1( 114) 115 ( 3617)

Ben I ccs/gg 1 1( 1520) 1521 ( 2211)

Bsm I gaatgc 1/-1 1 1( 398) 399 ( 3333)

BspM II t/ccgga 1 1< 3095) 3096 ( 636)

BstU I cg/cg 1 1( 1749) 1750 ( 1982)

BstX I ccannnnn/ntgg 1 1( 2743) 2744 ( 988)

CfrlO I r/ccggy 1 1( 1904) 1905 ( 1827)

Cla I at/cgat 1 1< 926) 927 ( 2805)

Dsa I c/crygg 1 1( 3187) 3188 ( 544)

Eae I y/ggccr 1 1( 711) 712 ( 3020)

Ear I ctcttc 1/4 1 1( 3434) 3435 ( 297)

Eco47 III agc/gct 1 1( 1829) 1830 ( 1902)

Fsp I tgc/gca 1 1( 1941) 1942 ( 1790)

Hga I gacgc 5/10 1 1( 933) 934 ( 2798)

HinD III a/agctt 1 1( 552) 553 ( 3179)

Mse I tgg/cca 1 1( 711) 712 ( 3020)

Nae I gcc/ggc 1 1( 1904) 1905 ( 1827)

Nci I cc/sgg 1 1( 1520) 1521 ( 2211)

Nco I c/catgg 1 1( 3187) 3188( 544)

PflM I ccannnn/ntgg 1 1( 1800) 1801 ( 1931)

Sac I gagct/c 1 1( 2864) 2865 ( 867)

Spl I c/gtacg 1 1( 446) 447 ( 3285)

Stu I agg/cct 1 1( 2427) 2428 ( 1304)

Xmn I gaann/nnttc 1 1( 641) 642 ( 3090)

Bbv II gaagac 2/6 1( 3077) 1 3078 ( 297) 3 3375 ( 357) 2 BspH I t/catga 2 1 ( 3000) 1 3001 ( 351) 3 3352 ( 380) 2

BspM I acctgc 4/8 2 1 ( 2122) 1 2123 ( 1088) 2 3211 ( 521) 3

Bsu36 I cc/tnagg 2 1 ( 3157) 1 3158 ( 206) 3 3364 ( 368) 2

EcoN I cctnn/nnnagg 2 1 ( 1757) 1 1758 ( 1400) 2 3158 ( 574) 3

Hae II rgcgc/y 2 1 ( 1829) 2 18301 72) 3 1902 ( 1830) 1

Mae I c/tag 2 1 ( 2022) 1 2023 ( 1452) 2 3475 ( 257) 3

Me I tccrac 20/18 2 1 ( 316) 3 317 ( 1086) 2 1403 ( 2329) 1

Nde I ca/tatg 2 1 ( 371) 2 372 ( 90) 3 462 ( 3270) 1

NspB II cmg/ckg 2 1 ( 1853) 1 1854 ( 157) 3 2011 ( 1721) 2

Pml I cac/gtg 2 1 ( 956) 3 957 ( 1697) 1 2654 ( 1078) 2

Pvu II cag/ctg 2 1 ( 1853) 1 1854 ( 157) 3 2011 ( 1721) 2

Sea I agt/act 2 1 ( 2024) 1 2025 ( 432) 3 2457 ( 1275) 2

Tthlll I gacn/nngtc 2 1 ( 1195) 2 1196 ( 793) 3 1989 ( 1743) 1

Ase I at/taat 3 1 ( 2986) 1 29871 56) 3 3043 ( 641) 2 36841 48) 4

BsaA I yac/gtr 3 1 ( 423) 4 424 ( 533) 3 957 ( 1697) 1 26541 1078) 2

HinC II gty/rac 3 1 ( 1482) 2 1483 ( 533) 3 2016 ( 1544) 1 35601 172) 4

Hpa II c/cgg 3 1 ( 1521) 1 1522 ( 384) 4 1906 ( 1191) 2 30971 635) 3

Msp I c/cgg 3 1 ( 1521) 1 1522 ( 384) 4 1906 ( 1191) 2 30971 635) 3

Pie I gagtc 4/5 3 1 1474) 1 1475 ( 544) 3 2019 ( 1354) 2 33731 359) 4

PpuM I rg/gwccy 3 1 ( 1072) 2 1073 ( 166) 4 1239 ( 881)-3 21201 1612) 1

Pst I ctgca/g 3 1 ( 1280) 2 1281 ( 161) 3 1442 ( 2158) 1 36001 132) 4

Ssp I aat/att 3 1 1537) 2 1538 ( 84) 4 1622 ( 1695) 1 33171 415) 3

Sty I c/cwwgg 3 1 1119) 2 1120 ( 686) 3 1806( 1382) 1 31881 544) 4

Taq I t/cga 3 1 927) 2 928 ( 9) 4 937 ( 2132) 1 30691 663) 3

AlwN I cagnnn/ctg 4 1 917) 3 918 ( 81) 5 999 ( 947) 2 19461 255) 4

2201 1531) 1

Bspl286 I gdgch/c 4 1 49) 5 50 ( 2607) 1 2657 ( 208) 3 28651 166) 4 3031 701) 2

BstY I r/gatcy 4 1 861) 2 862 ( 237) 4 1099 ( 668) 3 17671 109) 5 1876 1856) 1

EcoO109 I rg/gnccy 4 1 1072) 1 1073 ( 166) 5 12391 881) 3 21201 694) 4 2814 918) 2

Gsu I ctggag 16/14 4 1 1761) 2 1762 ( 9) 5 1771 ( 43) 4 18141 97) 3 1911 1821) 1

Hae I wgg/ccw 4 1 711) 3 712 ( 638) 4 1350 { 1078) 2 24281 1200) 1 3628 104) 5

HgiA I gwgcw/c 4 1 49) 5 50 ( 2607) 1 2657 ( 208) 3 28651 166) 4 30311 701) 2

Hha I gcg/c 4 1 1830) 1 1831 ( 18) 5 1849 ( 54) 3 19031 40) 4 1943 ( 1789) 2

HinP I g/cgc 4 1 1830) 1 1831 ( 18) 5 1849 ( 54) 3 19031 40) 4 1943 ( 1789) 2

Nla IV ggn/ncc 4 1 579) 2 580 ( 386) 3 966 ( 107) 4 10731 26) 5 1099 ( 2633) 1

Alw I ggatc ... 4/5 5 1 862) 2 863 ( 236) 4 1099( 1) 6 11001 667) 3 1767 ( 110) 5 1877 ( 1855) 1

Dra I ttt/aaa 5 1( 228) 3 229 ( 2094) 1 23231 54) 6 23771 128) 5 2505 ( 1003) 2 3508 ( 224) 4

Ava II g/gwcc 6 1 1073) 2 1074 ( 166) 6 12401 178) 5 14181 365) 3

1783 < 205) 4 1988 ( 133) 7 21211 1611) 1

Fok I ggatg 9/13 6 1( 797) 2 798 ( 238) 6 10361 302) 5 13381 226) 7

1564 ( 1190) 1 2754 ( 615) 3 33691 363) 4

Mae II a/cgt 6 1( 424) 6 425 ( 533) 3 9581 1035) 1 19931 517) 5

2510 ( 145) 7 2655 ( 544) 2 31991 533) 4

Dpn I ga/tc 7 1( 115) 4 116 ( 747) 2 8631 63) 8 9261 81) 7

1007 ( 93) 6 1100 ( 668) 3 17681 109) 5 18771 1855) 1

Eco57 I ctgaag 16/14 7 1( 75) 7 76 ( 416) 4 4921 64) 8 5561 554) 3

1110 ( 111) 6 1221 ( 913) 2 21341 1322) 1 34561 276) 5

Mbo I /gate 7 1( 115) 4 116 ( 747) 2 8631 63) 8 9261 81) 7

1007 ( 93) 6 1100 ( 668) 3 17681 109) 5 18771 1855) 1

Sau3A I /gate 7 1( 115) 4 116 ( 747) 2 8631 63) 8 9261 81) 7

1007 ( 93) 6 1100 ( 668) 3 17681 109) 5 18771 1855) 1

Tthlll II caarca 11/9 7 1( 126) 7 127 ( 253) 4 3801 471) 2 8511 453) 3

1304 ( 109) 8 1413 ( 1923) 1 33361 218) 5 35541 178) 6

BsmA I gtctc 1/5 8 1( 510) 4 511 ( 529) 3 10401 852) 1 18921 311) 6

2203 ( 67) 9 2270 ( 128) 8 23981 133) 7 25311 480) 5

3011 ( 721) 2

Hinf I g/antc 8 1( 913) 2 914 ( 6) 9 9201 81) 7 10011 474) 4

1475 ( 544) 3 2019 ( 14) 8 20331 972) 1 30051 368) 5

3373 ( 359) 6

Sec I c/cnngg 8 1( 714) 2 715 ( 405) 5 11201 686) 3 18061 62) 8 7 31881 544) 4

Bbv I gcagc 8/12 9 1 191) 7 192 176) 8 368 406) 5 774 506) 3 1280 3)10 1283 158) 9 1441 415) 4 1856 794) 2 2650 821) 1 3471 261) 6 Fnu4H I gc/ngc 9 1 191) 7 192 176) 8 368 406) 5 774 506) 3 1280 3)10 1283 158) 9 1441 415) 4 1856 794) 2 2650 821) 1 3471 261) 6 SfaN I gcatc 5/9 9 1 482) 4 483 115) 9 598 242) 6 840 913) 1 1753 144) 8 1897 43)10 1940 859) 2 2799 289) 5 3088 148) 7 3236 496) 3

BstN I cc/wgg 10 1 436) 437 84) 8 521 51)10 572 143) 7

715 722) 1437 42)11 1479 389) 4 1868 66) 9

1934 345) 2279 351) 5 2630 1102) 1 EcoR II /ccwgg 10 1 436) 437 84) 8 521 51)10 572 143) 7

715 722) 1437 42)11 1479 389) 4 1868 66) 9

1934 345) 2279 351) 5 2630 1102) 1 Mae III /gtnac 10 1 85) 86 419) 3 505 12)11 517 340) 5

857 337) 1194 353) 4 1547 278) 7 1825 39)10

1864 1050) 2914 239) 8 3153 579)_2

Hae III gg/cc 11 1 712) 2 713 255) 968 383) 5 1351 102) 9

1453 39)11 1492 300) 1792 94)10 1886 22)12

1908 521) 3 2429 387) 2816 813) 1 3629 103) 8 Hph I ggtga 8/7 11 1 285) 5 286 232) 518 228 ) 7 746 97)

843 146) 8 989 948) 1937 41) 11 1978 383)

2361 793) 2 3154 31)12 3185 79 ) 10 3264 468) ScrF I cc/ngg 11 1 436) 3 437 84) 8 521 51) 10 572 143)

715 722) 2 1437 42)11 1479 42 ) 12 1521 347)

1868 66) 9 1934 345) 6 2279 351) 4 2630 1102)

Bsr I actgg 1/-1 12 1 139)10 140 149) 8 289 416) 2 705 365) 1070 220) 5 1290 23)12 1313 22)13 1335 154) 1489 324) 4 1813 76)11 1889 148) 9 2037 191) 2228 1504) 1

Sau96 I g/gncc 12 1 966) 1 967 107) 8 1074 166) 1240 178) 5 1418 34)12 1452 40)11 1492 291) 1783 9)13 1792 94)10 1886 102) 9 1988 133) 2121 694) 3 2815 917) 2

Nla III catg/ 14 1 46)11 47 1742) 1 1789 13)15 1802 250) 5 2052 393) 2 2445 94) 9 2539 105) 8 2644 24)14 2668 83)10 2751 251) 4 3002 26)13 3028 161) 6 3189 39)12 3228 125) 7 3353 379) 3

Dde I c/tnag 15 1 40)15 41 200) 8 241 272) 5 513 26)16

539 644) 1 1183 61)14 1244 181) 9 1425 132)11

1557 362) 4 1919 610) 2 2529 156)10 2685 126)12

2811 223) 6 3034 125)13 3159 206) 7 3365 367) 3

Mbo II gaaga 8/7 17 1 94)11 95 877) 1 972 164) 6 1136 51)15 1187 33)17 1220 466) 3 1686 38)16 1724 3)18 1727 387) 4 2114 693) 2 2807 141) 8 2948 130) 9 3078 68)12 3146 230) 5 3376 59)13 3435 100)10 3535 144) 7 3679 53)14

Rsa I gt/ac 17 1 426) 4 427 21)15 448 113)11 561 163) 8 724 153)10 877 442) 3 1319 500) 2 1819 207) 7 2026 265) 6 2291 162) 9 2453 5)18 2458 63)13 2521 21)16 2542 680) 1 3222 373) 5 3595 72)12 3667 56)14 3723 9)17

Alu I ag/ct 18 1 67)16 68 137)11 205 204) 5 409 133)13

542 12)19 554 36)17 590 153)10 743 76)15

819 604) 1 1423 137)12 1560 95)14 1655 200) 6

1855 157) 8 2012 156) 9 2168 174) 7 2342 524) 3

2866 549) 2 3415 24)18 3439 293) 4

Mse I t/taa 22 1 19)20 20 43)18 63 111)11 174 56)14 230 442) 2 672 237) 6 909 267) 5 1176 915) 1 2091 233) 7 2324 54)16 2378 46)17 2424 67)12 2491 15)21 2506 338) 3 2844 144) 8 2988 56)15 3044 338) 4 3382 127) 9 3509 62)13 3571 114)10 3685 12)22 3697 5)23 3702 30)19

Mnl I cctc 7/7 27 1 25)23 26 135)10 161 193) 8 354 105)14 459 107)13 566 97)16 663 276) 5 939 53)19 992 104)15 1096 311) 3 1407 22)24 1429 21)25 14501 315) 2 17651 21)26 17861 124)11 19101 55)18

19651 21)27 19861 248) 6 22341 117)12 23511 44)20

23951 36)22 24311 6)28 24371 210) 7 26471 166) 9

28131 66)17 28791 282) 4 31611 43)21 32041 528) 1

460 sites found

No Sites found for the following Restriction Endonucleases

Aat II gacgt/c Drd I gacnnnn/nngtc NspH I rcatg/y

Ace I gt/mkac Eag I c/ggccg PaeR7 I c/tcgag

Afl II c/ttaag EcoR I g/aattc Pvu I cgat/cg

Afl III a/crygt ECOR V ga /ate Rsr II cg/gwccg

Aha II gr/cgyc Esp I gc/tnagc Sac II ccgc/gg

Apa I gggcc/c Gdi II yggccg -5/-1 Sal I g/tcgac

Asp718 g/gtacc HgiE II accnnnnnnggt Sfi I ggccnnnn/nggcc

Ava I c/ycgrg Hpa I gtt/aac Sma I ccc/ggg

Avr II c/ctagg Kpn I ggtac/c SnaB I tac/gta _

Bbe I ggcgc/c Mlu I a/cgcgt Spe I a/ctagt

Bgl I gccnnnn/nggc Nar I gg/cgcc Sph I gcatg/c

Bgl II a/gatct Nhe I g/ctagc Xba I t/ctaga

BssH II g/cgcgc Not I gc/ggccgc Xca I gta/tac

BstB I .tt/cgaa Nru I tcg/cga Xho I c/tcgag

BstE II g/gtnacc Nsi I atgca/t Xcm I ccannnnn/nnnntgg

Dra III cacnnn/gtg Nsp7524 I r/catgy Xma I c/ccggg

ANNEX 4

DNA sequence 4321 b.p. AAACCAATAAGG ... GATGTACTGTAA linear

Positions of Restriction Endonucleases sites (unique sites underlined)

Alu I

I

AAACCAATAAGG'TTAGGACAAGAGAATAGCTGTGG'I^TGCG'rTGCAAAAACCAAAAAAAAAAAAAAAAAAAAAAAGAAAG 80

T'TTGG'rTATTCCAATCCTGTTCTCTTATCGACACCAAACGCAACtf

• • I • • • • • •

28 Sty I Tthlll II

Sec I HinP I

Nla IV Hha I —

Mnl I Nla III Fsp I Fok I

Sec I Nco I Fnu4H I Mnl I

Ava I Dsa I Bbv I Taq I Hinf I Mae I

II I I II I II I I I I I I

CCCCGAGGCTCCATGGGCAGACCTAO^AGGCTGCGCAAACAAATCGAGTiGATGAGATTCTGCTGTTTCTTTGTCTAGGGT 160 GGGGCTCCGAGGTACCCGTCTGGATGTTCCGACGCGTTTG'ITTAGCTCCCTACTCTAAGACGACAAAGAAACAGATCCCA

II I I -II • I II I ^• I I I- I - I

82 91 110 124 135 154

83 91 110 126

85 92 112 129

87 113

91 113

91 116

Tthlll II Fnu4H I HinP I

Bbv I Hha I HinP I

SfaN I NspB II Hae II Hha I Mae III

Dde I Fnu4H I Eco47 III Alu I Tthlll II Alu I

I I II I I II I I I I I

TCTCAGATGCTATCTGCCGCrGCTG'TTrGGTGGGGAAGGAGC 240

AGAGTCTACGATAGACGGCGACGACAAACCACCCCTTCCTCGCGACCCGCG'ITTCGACAATGG'ITTGTCTTGCCACCCTC

I I - II I- I • II I • I I • I • I-

162 176 200 214 223 239

166 177 200 208 218

179 201 208

179 201 184

Bsr I ..

Nla IV Mnl I Gsu I Mme I Mse I

I I I I I I

CTGATGGCTCCGAGTTTGGGGCGAGGTAGAAACTCTC^GTGCCAC'ITCCGACTTTAAGCCTTCCTGTTGCCGTCCACTG 320 GACTACCGAGGCTCAAACCCCGCTCCATC'TTTGAGAGGTCACGGTGAAGGCTGAAATTCGGAAGGACAACGGCAGGTGAC

I ^• - I - I I - I • I •

246 263 275 288 295

277 HinP I

Nla IV

Nar I.

Hae II

Bbe I

ScrF I

Hae III

Gdi II

Eae I Hha I

Sec I Fnu4H I ScrF I NspB II Ban I EcoR II Ava I Fnu4H I Aha II

BstN I Mae II Fnu4H I Bbv I EcoR II Mbo II Afl III Dde I Bbv I Fnu4H I BstN I

TC _ΪCGGGTTTCTTCCTGGGGAACACGTTTTCGCTCAGTCGCTCGGCAGCCCGAGCCTGCGGCAGCGGCCAGGCGCCTGCC 400 ACCGCCCAAAGAAGGACCCCTTGTGCAAAAGCGAGTCAGCGAGCCGTCGGGCTCGGACGCCGTCGCCGGTCCGCGGACGG

330 342 353 365 378 388

334 344 365 381 388

334 369 381 391

334 382 391

334 384 385 392 385 386 388 391 391 391 391 392

Fnu4H I BstU I Sec I Sac II NspB II Dsa I

HinP I Fnu4H I Dde I HinP I

Alu I Hha I HinP I HinP I BstU I

HinP I Mn] . I Hha I Fnu4H I Hha I Bspl286 I

Hha I Pst I Hae II Hae II Bbv I Hae II Ban II Hha I

CCCTGCGCCGAGCTTTCCCCTGCAGAGGCGCTCCACTCCCAGAAGCGCCGCGGCTGCACCAGAGCGCCTGAGAGCCCCCG 480 GGGACGCGGCTCGAAAGGGGACGTCTCCGCGAGGTGAGGGTCTTCGCGGCGCCGACGTGGTCTCGCGGACTCTCGGGGGC

405 420 427 444 453 463 472 480

405 425 445 453 464 472

411 428 445 464 479 428 447 468 480

448

449

45C 1

ScrF I EcoR II

Rsa I BstN I Taq I Bsr I

BstU I Fok I Mnl I £s B_I Mse I

CGCGTACCCATCCAGGAGCAAAACTATGTCAGGAATGGAGGTTTGCTAACCCAGAAAATTCGAAGGAACACATTAAACTG 560 GCGCATGGGTAGGTCCTCGTTTTGATACAGTCCTTACCTCCAAACGATTGGGTCT'ITTAAGCTTCCTTGTGTAATTTGAC

481 489 518 539 553

484 492 540 557

492

Fnu4H I HinP I

Bbv I Hha I

SfaN I Hae II Tthlll II

Fok I Eco47 III Bsr I Mnl I BspM I

GTGGATGCAGCAGATGTAAGCGCTGTGCAAACATCTCAAGCCAGTTCAGATGTTGCTGTTTCCTCAAGTTGCAGGTCTAT 640 CACCTACGTCGTCTACATTCGCGACACG'TTTGTAGAGTTCGGTCAAGTCTACAACGACAAAGGAGTTCAACGTCCAGATA

563 579 601 622 631

564 579 588

567 580

567 580 Sau3A I

Mbo I Sau96 I

Dpn I Nla IV

BstY I Bsr I

Alw I Dde I Hinf I Hae III Mae III

_GGAAATG_CA_G I_GIAT_CTAACCAGCCCGCATAGCCGTCITGAGTGGTAGTAGTGIAATCCCCICAGTGIGCCCCAAACTCGGITAACT 720

_CCTTTAC_GT_CCTAGATTGGTCGGGCGTATCGGCAGACTCACCATCATCACTTAGGGGGTCACCGGGGTTTGAGCCATTGA

|| • I l l - l I

650 675 690 702 715

650 697

651 702

651 702 651

Pie I

Hinf I Nla IV Fnu4H I

Nla III Ban I Mse I Bbv I Alu I

CTCATATAAATAGTAATTCC IATGIACTCCCAATGIGCACCGAAGTITAAAACAGAGCCAATGAGICAGCAGTGAAACAIGCTTCA 800

_GAGTATATTTATCATTAAGGTACTGAGGGTTACCGTGGCTTCAATTTTGTCTCGGTTACTCGTCGTCACTTTGTCGAAGT | | . I . I . .| . I

740 753 763 781 794

743 753 781

743 Mse I Bsr I

Dra I Dde I Hph I

ACGACAGCCGACGGGTCT ITITAAACAATTTCITCAGGTTCAGCAATTGGGAGCAGTAGTTTCAGCCCACGACCAACTICACICA 880 TGCTGTCGGCTGCCCAGAAATTTGTTAAAGAGTCCAAGTCGTTAACCCTCGTCATCAAAGTCGGGTGCTGGTTGAGTGGT

II- I ^{• • •} - I I -

818 830 875

819 878

Fnu4H I Mme I Mnl I Bbv I

GTTCTCTCCACCACAGATTTACCCTT ICCAACAGACCATACCCACATATTCTCCCTACCCCTTCICTCACAAACTATGGICTG 960 CAAGAGAGGTGGTGTCTAAATGGGAAGGTTGTCTGGTATGGGTGTATAAGAGGGATGGGGAAGGAGTGTTTGATACCGAC _• • I • • _{• •} j _• I _•

906 943 957 957

Rsa I ScrF I

Mae II EcoR II Rsa I

Nde I Tthlll II Bsm I Alu I isaA I BstN I ?Pl I

C IATATGGGCIAAACACAGTTTACCACAGGIAATGCAACAAIGCTACAGCCTATGCCIAICGITACCCACAGCICAGGACAGCCIGITAC 1040 GTATACCCGTTTGTGTCAAATGGTGTCCTTACGTTGTTCGATGTCGGATACGGTGCATGGGTGTCGGTCCTGTCGGCATG

9 I61 9I6-9 • 9I88• 9I98• ^■ 1I0I13I • 1I026• 1I0I36•

1014 1026 1037 1016 1026

ScrF I Hph I Dde I EcoR II Nde I BsmA I BstN I

Mnl I -~ SfaN I Eco57 I Mae III Mae III

GGCATTTC ICTCIATATGGTGCATTGTGGGCAGGICATCAAGACITGAAGGTGGATTGITCACAGITCITCAGITICACICTGGACAGAC 1120 CCGTAAACWAGTATACCACGTAACACCCGTCCGTAGTTCTGACTTCCACCTAACAGTGTCAGAGTCAGTGGACCTGTCTG

1 I04-8I ^• - 1I072 -1I081 • 1I094 I I 1I1I06I

1051 1100 1110

1102 1110 1107 1110 ScrF I Eco57 I EcoR II Alu I Alu I Rsa I BstN I Nla IV Dde I HinD III Mnl I Ban I Alu I SfaN I

I I II I I I I I I I

AGGATTTCTCAGCTATGGCACAAGCTTCAGTACCCCTCAACCTGGACAGGCACCATACAGCTACCAGATGCAAGGTAGCA 1200 TCCTAAAGAGTCGATACCGTGTTCGAAGTCATGGGGAGTTGGACCTGTCCGTGGTATGTCGATGGTCTACGTTCCATCGT

1 I12-8I • 1I1I42I I 1I155 -I 1I1-69 1I1-79 1I187■

1131 1143 1150 1161 1169 1145 1161 1161 Xmn I Mnl I Mse I

GTT-RAACAACATCATCAGGAATATATACAGG IAAATAATTCACTCACAAATTCICTCTGGATTITAATAGTTCACAGCAGGAC 1280 CAAAATGTTGTAGTAGTCCTTATATATGTCCTTΓATTAAGTGAGTGTTTAAGGAGACCTAAATTATCAAGTGTCGTCCTG

• • • I • • I _• I _• •

1231 1252 1261 Sec I ScrF I EcoR II BstN I Hae III Mse I

Hae I Hph I

Bsr I Eae I Rsa I Alu I

TATCCGTCTTATCC ICAGTTTT IGIGC ICAGGGTCAG ITACGCACAGTATTATAACA IGCTICACCGTATCCAGCACATTATATGAC 1360 ATAGGCAGAATAGGGTCAAAACCGGTCCCAGTCATGCGTGTCATAATATTGTCGAGTGGCATAGGTCGTGTAATATACTG . j .|| I . I . . i i

1294 1301 1313 1332

1301 1335

1301 1302 1304 1304 1304 1304 Fnu4H I Hph I

Bbv I Fok I Alu I SfaN I Tthlll II

CAG ICAGCAACACCAGCCCAACGACACCIATCCACCAATGCCACTTACCAIGCTTCAAGAACCGCCATCTGGICATICACCAGCCI 1440 GTCGTCGTTGTGGTCGGGTTGCTGTGGTAGGTGGTTACGGTGAATGGTCGAAGTTCTTGGCGGTAGACCGTAGTGGTCGG

1 I363 1I387• • 1I40•8 • 1I4-29I -1I440

1363 1432

Sau3A I bo i ia i

Dpn I Sau3A I

Alw I Hinf I Taq I

BstY I Hinf I Mbo I

Mae III Rsa I Mse I AlwN I Dpn I

I I I I I I I I I I I

AAGCAGTTACAGATCCCACAGCAGAGTACAGCACAATCCACAGCCCATCAACACCCATTAAAGATTCAGATTCTGATCGA 1520 TTCGTCAATGTCTAGGGTGTCGTCTCATGTCGTGTTAGGTGTCGGGTAGTTGTGGGTAATTTCTAAGTCTAAGACTAGCT

1446 1466 1498 1507 1515

1451 1503 1515

1452 1509 1517

1452 1515

1452 1516

1452

Mbo II Sau3A I

Mnl I Mae II Hae III Mbo I

Taq I Pml I Sau96 I Mnl I Hinf I

Hβft.I. BsaA I Nla IV Hph I AlwN I Dpn I

TTGCGTCGAGGTTCAGATGGGAAATCACGTGGACGGGGCCGAAGAAACAATAATCCTTCACCTCCCCCAGATTCTGATCT 1600 AACGCAGCTCCAAGTCTACCCTTTAGTGCACCTGCCCCGGCTTCTTTGTTATTAGGAAGTGGAGGGGGTCTAAGACTAGA

1 I523I I - • 1I5I46• 1I5I5I5 -I • I .57-8I 1I58I8 1I596 •

1526 "^* 1546 1556 1581 1590 1528 1547 1557 1596

1561 1596

Sau96 I

Ava II

PpuM I

Nla IV

BsmA I EcoO109 I Fok I Bsr I

TGAGAGAGTGTTCATCTGGGACTTG IGATGIAGACAATCATTGTTTTCCACTCCTTGCTTAICTGIGIGTCCTACGCCAACAGAT 1680 ACTCTCTCACAAGTAGACCCTGAACCTACTCTGTTAGTAACAAAAGGTGAGGAACGAATGACCCAGGATGCGGTTGTCTA

I I- • • I- II 1625 1659

1629 1662

1662 1662 1663 1663 Sau3A Mbo I Dpn I Alw I Nla IV BstY I

BamH I

Alw I Sty I

Mnl I Eco57 I Sec I Mbo II

AT_GGGAG_GI_GIAT_CCA_CCCACTT_CAGTTTCCCTTGGACTGCGAATGGAAGAAATGATTTTCAACTTGGCAGACACACATTTA 1760 TA_CC_CTCCCTA_GGT_GGGT_GAAGTCAAAGGGAACCTGACGCTTACCTTCTTTACTAAAAGTTGAACCGTCTGTGTGTAAAT

1685 1699 1709 1725

1688 1709

1688 1688 1688

1689

Dde I

Sau96 I

Ava II

Mbo II Tthlll I Eco57 I PpuM I Mse I Dde I Mae III Mbo II EcoO109 I

TTTTTTAATGACTTAGAAGAATGTGACCAAGTCCATATAGATGATG'ITTCTTCAGATGATAACGGACAGGACCTAAGCAC 1840 AAAAAATTACTGAATCTTC'ΓTACACTGGTTCAGGTATATCTACTACAAAGAAGTCTACTATTGCCTGTCCTGGATTCGTG

1765 1772 1783 1809 1828

1776 1785 1810 1828 1829 1829

1833

Fnu4H I Bbv I Pst I

Fnu4H I Bsr I

Bbv I Bsr I Tthlll II Rsa I

ATATAACTTTGGAACAGATGGCTTTCCTGCTGCAGCAACCAGTGCTAACTTATGTTTGGCAACTGGTGTACGGGGCGGTG 1920 TATATTGAAACCTTGTCTACCGAAAGGACGACGTCGTTGGTCACGATTGAATACAAACCGTTGACCACATGCCCCGCCAC

II I I- - I - I I -

1869 1879 1893 1908

1869 1902

1870 1872 1872 Fok I Hae III Mnl I

Bsr I Hae I Mme I

TGGA ICTGIGATGAGAAAGTTIGIC^CTTCCGCTACAGACGGGTAAAAGAGATTTACAACACCTACAAAAATAATGITTGGIAGGT 000 ACCTGACCTACTCTTTCAACCGGAAGGCGATGTCTGCCCATTTTCTCTAAATGTTGTGGATGTTTTTATTACAACCTCCA

1 I924I - 1I9I39 • • • • • - 1I992I -

1927 1940 1996

Pst I Fnu4H I ScrF I

Alu I Bbv I EcoR II

Sau96 I ScrF I Hae III BstN I

Ava II Mnl I EcoR II Sau96 I Pie I Bsr I

Tthlll II Dde I BstN I Mnl I Hinf I HinC II

CT IGCTTGIGTCCAIGCITAAGIAGGGAAGCICTGGICITGCAGTTGIAGIGIGCCGAAATTGAAGCCCTGACCGIACTCICTGGITTGACAICT 2080 GACGAACCAGGTCGATTCTCCCTTCGGACCGACGTCAACTCCCGGCTTTAACTTCGGGACTGGCTGAGGACCAACTGTGA

2 I002 I ^• I 2I014I • 2I026II 2l0-3l9l • - 2l064l - 2l072 I •

2007 2018 2026 2041 2064 2078

2007 2026 2042 2068

2012 2030 2068

2030 2068 2031 Hpa II ScrF I _Sau96 I Nci I Alii I

_Hae in Ben I Ssp I Mae III Dde I Fok I

I I I I I I I I

_GGCCCT_GAAA_GCACTCT_CGCTCATTCACTCCCGGACAAACTGTGTGAATATTTTAGTAACAACTACTCAGCTCATCCCAG 2160 _CC_GGGA_CTTT_CGT_GA_GAG_CGA_GTAAGTGAGGGCCTGTTTGACACACTTATAAAATCATTGTTGATGAGTCGAGTAGGGTC

I • • II • I • I • I I- I ^• ₂₀81 2110 2127 2136 2146 2153

₂081 2110 2149

2110 2111 2111

Ssp I

_CATTG_GCGAAAGTCCTGCTGTATGGGTTAGGAATTGTATTTCCAATAGAAA IATATTTACAGTGCAACTAAAATAGGAAAA 2240 _GTAACCGCTTTCAGGACGACATACCCAATCCTTAACATAAAGGTTATCTTTTATAAATGTCACGTTGATTTTATCCTTTT • • • • • 1 • • •

2211

Mbo II Alu I Mbo II Mbo II

GAAA IGCTGTTTTGAGAGAATAATTCAAAGGTTTGG IAAGAAAAGTGGTGTATGTTGTTAAGGAGATGGTGTAG IAAGIAAGA 2320 CTTTCGACAAAACTCTCTTATTAAGTTTCCAAACCTTCTTTTCACCACATACAACAATATCCTCTACCACATCTTCTTCT

I • I • • - I I -

2244 2275 2313

231£_ Sau3A I Mbo I Dpn I

BstY I Nla III Sty I

Mnl I Mnl I Sec I

SfaN I Gsu I Gsu I Sau96 I Sau96 I Nla III

BstU I EcoN I Alw I Ava II Hae III PflM I

I I I I I I I I I I I I I I I

ACAAGGAGCAAAAAAGCACGCGATGCCCTTCTGGAGGATCTCCAGCCACTCGGACCTCATG&CCCTGCACCATGCCTTGG 2400 TGTTCCTCGTTTTTTCGTGCGCTACGGGAAGACCTCCTAGAGGTCGGTGAGCCTGGAGTACCGGGACGTGGTACGGAACC

I - I I - I I I I I ^• I I I - I I I I

2339 2347 2356 2372 2381 2390

2342 2351 2360 2372 2381 2391

54 2375 2395

2356 2378 2395

2357

Fnu4H I

HinP I Alu I Sec I Sau3A I Bsr I Hha I Pvu II ScrF I Mbo I Sau96 I Rsa I Hae II NspB II EcoR II Dpn I Nla IV Gsu I Eco47 III HinP I BstN I Alw I Hae III

Bsr I Mae III Hha I Bbv I Mae III BstY I EcoO109 I

I I I I I I I I I I I I I I I I I

AACTGGAGTACCTGTAACAGCGCTCGGCACTTTGACAGCGCACAGCTGCTCTGTGACCAGGGACAGATCCAGCAGGCCCC 2480 TTGACCTCATGGACATTGTCGCGAGCCGTGAAACTGTCGCGTGTCGACGAGACACTGGTCCCTGTCTAGGTCGTCCGGGG

II I • I II • I • III • I I • II • II I-

2402 2414 2438^' 2445 2453 2465 2474 2403 2419 2438 2457 2466 2475

2408 2419 2443 2457 2466 2475 2420 2443 2457 2466 2475 2420 2444 2457 2466 2479

2445

Hae III Msp I Hpa II Nae I SfaN I HinP I Hph I AlwN I

Hha I Gsu I ScrF I HinP I SfaN I CfrlO I EcoR II Hha I BsmA I Hae II Mnl I Dde I BstN I Fsp I Mnl I

I I II II I II I

AGTCTCGCATCAGCGCCGGCCTCCAGAACTTAGCAATTTCCGCCTGGTGATGCGCAGTTGCTGTCAGTCTTGACCTCTGC 2560 TCAGAGCGTAGTCGCGGCCGGAGGTCTTGAATCGTTAAAGGCGGACCACTACGCGTCAACGACAGTCAGAACTGGAGACG

I I • II II I II I- - I I l-ll I • • I •

2482 2492 2500 2509 2523 2531 2554

2487 2495 2523 2532

2493 2501 2523 2532

2493 2526 2535

2495 ₂529

2496 2496 2498 Tthlll I HinC II Sea I

Sau96 I Alu I Pie I Rsa I

Ava II Pvu II Hinf I Bsr I

Hph I Mnl I Mae II NspB II Mae I Hinf I

_CTTT_GTG I_GT_GAAT_GG IAG IGIACCA ICGTCTATTTCATCAGAAC IAIGCTG ITTG IACTC ITA IGITACTGTG IAATCICAGTGAAAATAAGC 2640 _GAAA_CA_CCA_CTTA_CCT_CCT_GGTGCAGATAAAGTAGTCTTGTCGACAACTGAGATCATGACACTTAGGTCACTTTTATTCG

I • I I I ^• I ^• I I I h i l l - I I ^■

2567 2575 2582 2600 2612 2622

₂577 2600 2608 2626

₂577 2601 2608 2615

2578 2605 2614

Sau96 I Ava II PpuM I EcoO109 I Nla III Mse I Mbo II BspM I

I I I I I I

_CAT_GA_GAATGTTTTAGCACAGCGTTATGTGTCTGCCACATTAACTACACGGTTCAAACCTGTGAAGAAAGGACCTGCAAA 2720 _GTA_CTCTTACAAAATCGTGTCGCAATACACAGACGGTGTAATTGATGTGCCAAGTTTGGACACTTCTTTCCTGGACGTTT

I • ^{• •} I ^{• •} I I I I

₂641 2680 2703 2712

2709 2709 .2710 2710 _ BsmA I Eco57 I Alu I AlwN I

I I I I

CGCTTCAGTTGTTAGCATTTTCAATGTGATATAAACAGCTTCTCCAATACAGCAAACCTAATTGCACAACAGAGACTGAA 2800 GCGAAGTCAACAATCGTAAAAGTTACACTATATTTGTCGAAGAGGTTATGTCGTTTGGATTAACGTGTTGTCTCTGACTT

2 I723 ' ' 2I757- • • 2I79I0 -

2792 Sec I ScrF I EcoR II Mnl I BstN I

Bsr I BsmA I Sec I Rsa I

I I I I I I

ATGTGTTTCCTGAATACCAGTGGAGGAATTTTCTTGTAAAGAAGGTTTACTTTTTGGTGTCTCATACCCAGGGTAATCTG 2880 TACACAAAGGACTTATGGTCACCTCCTTAAAAGAACATTTCTTCCAAATGAAAAACCACAGAGTATGGGTCCCATTAGAC

I ^• I ^{• •} • I- II • I

2817 2859 2867 2880

2823 2868

2868 2868 2868 Mse I Dra I Alu I Mnl I Hph I I I I I I

TACATCTCTACTTATTTATGAACAGAC'TTTTTTTAAAAAGATAAAAAAACAGC'I TATTGAGGTATAATTCACCCACCAG 2960 ATGTAGAGATGAATAAATACTTGTCTGAAAAAAATTTTTCTATr ri GTCGAAATAACTCCATATTAAGTGGGTGGTC

^{• •} II • -I I I

2912 2931 2940 2950

2913

Hae III BsmA I Stu. I

Mse I Mbo II Hae I Mnl I

Dra I Ear I Mse I Mnl I Nla III

ACTTT TTAAACATCAAATAATT AALGACAAT GC I 11 I I I

TGAAAAAA'ITTGTAGTTTATTAACTTCTCTGTTATCGTAATCTTTATTCACTAATTTCCGGAGACGGAGTGTTGTACCGT

I I ^• - I I - ^• - I N I I • I

2966 2984 3013 3020 3034

2967 2984 3017 3026

2987 3017

3018

Rsa I Mae II

Sea I Mse I BsmA I

Rsa I Mse I Dra I Rsa I Dde I

I I I I I I I I I I

AGTACAGTACTTTGAATTTTAGCACATTGCATAATAGTTTTAAGTATGTCTAATTTAAACGTATAATATGTACATCACTG 120 TCATGTCATGAAACTTAAAATCGTGTAACGTATTATCAAAATTCATACAGATTAAATTTGCATATTATACATGTAGTGAC

I II ^{• • •} I - II I- I I I

3042 3080 3094 3110 3118

3046 3095 3120

3047 3099 Rsa I

Nla III A_GA_CAAT_C IAT_GITA_CAGAAAGAATTTTTGGTGTAAATTTGTAATAATGGATAATTCTTTTACATATTGTTTAGGGAAATGA 3200

T_CT_GTTA_GTA_CAT_GTCTTTCTTAAAAACCACATTTAAACATTATTACCTATTAAGAAAATGTATAACAAATCCCTTTACT

| _*| • • _* • • • •

3128

3131

HgiA I

Bspl286 I

Mae II

Fnu4H I

BstN I Nla III BsaA I Nla III Dde I

I I I I I I I I I

TATTGAAA_GGTAG_CAATGCCTGGATAGTGAAGCATGAGGCAGCACGTGCACAAATTCATGTGCCGTGCCTTATCTGAGTT 3280 ATAA_CTTT_CCATCGTTACGGACCTATCACTTCGTACTCCGTCGTGCACGTGTTTAAGTACACGGCACGGAATAGACTCAA

I - ^• I I I - I I I ^• I ^• - I

3219 3233 3243 3257 3274

3219 3236 3243

3219 3239 3246

3239

3244

3246

Nla III

BstX I Fok I

TTCGGTATAAATATGTAGATAATGGATTTTTTTTTAGATAATGTTGTCAAGACCAAAAGCATGGATGTCAAGTGTCAGTA 3360 AAGCCATATTTATACATCTATTACCTAAAAAAAAATCTATTACAACAGTτCTGGTTTTCGTACCTACAGTTCACAGTCAT

• • • • * I I 1 * *

3333 3343 3340 Hae III EcoO109 I Mnl I Dde I SfaN I Mbo II Sau96 I Mse I

I I I M M I

AGGATTTTGTTTTCTAAAATTTTTTCCTGCATCAGTTCTTCTGAGGGCCTTGATGAAATAACACAGCAGTTTCTTAAACA 3440 TCCTAAAACAAAAGATTTTAAAAAAGGACGTAGTCAAGAAGACTCCCGGAACTACTTA'iτGTGTCGTCAAAGAATTTGT

I- I -I MM • • • I •

3389 3397 3405 3434

3401 3403 3404 3406 Alu I Sac., I HgiA I Bspl286 I Ban II Mnl I Mae III

I I I I

ATTTGAAACAAAATGAXSCTCTCCTACCACCTCACTTTTTCATTTCCACACTAATGTATTATATGTAACTACTTGGAAAAA 3520 TAAACTTTGTTTTACTCGAGAGGATGGTGGAGTGAAAAAGTAAAGGTGTGATTACATAATATACATTGATGAACCTTTTT

I I - I ^• • • • I ^•

3455 3469 3504

3455 3455 3455 3456

Hinf I Mse I Nla III

Mbo II Ase I BspH I

I II II I

ATAATTATTCAAATGCTTCTTCCCACAAAGAATATAGATGATAGTAGATATATTTTATTAATAAAATGGTTCATGAATCG 3600 TATTAATAAGTTTACGAAGAAGGGTGTTTCTTATATCTACTATCATCTATATAAAATAATTATTTTACCAAGTACTTAGC

I ^• • • II • -II I -

3538 577 3591

3578 3592

3595 Dde I HgiA I

Bspl286 I Mse I Mbo II

BsmA I Nla III Ase I Taq I Bbv II SfaN I

_G IA_GA_CTAA_CAAAGTTTT_CIATGITGCITCAGAATTAITITAATTATCGTGTCTGCATTTTCTTTICGATAAAGGIAAGACACACGIAT 3680 _CT_CTGATT_GTTT_CAAAA_GTACACGAGTCTTAATAATTAATAGCACAGACGTAAAAGAAAGCTATTTCCTTCTGTGTGCTA

I • I - I I ^• I I ^{• •} I - I ^• I ^•

3601 3618 3633 3 3665599 3 3666688 3678

3621 3634 3668 3621 3624

Dde I EcoN I

Msp I Bsu36 I

Hpa II Mae III

BSPM II Mbo II Hph I Mnl I

_GCTAAT M_CC_GGAAATCAGCAAACTTTGCATTACTCCCTATGTGCGTATTTTCTCTTTICTTCCTGITICACCICITGIAGGAAGGTT 3760

_CGATTA_GGCCTTTAGTCGTTTGAAACGTAATGAGGGATACACGCATAAAAGAGAAAGAAGGACAGTGGGACTCCTTCCAA

II • • • •

3686 3I736' I3I744ll-3l751

3687 3743

3687 3748 3748 3749

Nla III

Sty I

Sec I

Nco I

Dsa I Mnl I Nla III

Hph I Mae II BspM I Rsa I SfaN I

I I I I I I I I I

CATTGCCATTGTCATCACCATGGAAACAACGTTCCTCTCCACCTGCATTATGTACTACATGACAGGCATCAATCTGGGGA 3840 GTAACGGTAACAGTAGTGGTACCTTTGTTGCAAGGAGAGGTGGACGTAATACATGATGTACTGTCCGTAGTTAGACCCCT

3 I775II- 3I7-89 I - 3I801 - 3l812 l - 3l826-

3778 3794 3818

3778

3779

Hph I Ssp I

AATAATAAAATTAT ICACCTTTGTCAGACCATAAGAGTTTCTCCAAAAGTGGTCAGTTTGGCTGGGCAIATATTTTCTCTCA 3920 TTATTATTTTAATAGTGGAAACAGTCTGGTATTCTCAAAGAGGTTTTCACCAGTCAAACCGACCCGTTATAAAAGAGAGT • I • • • • _• I ■ •

3854 3907

Bbv II Fok I Mbo II Nla III Dde I Pie I Tthlll II BspH I Bsu36 I Hinf I Mse I

I I I I I I I I I I

TCTAACAAACACAATC ATTGTCATGAAATTACCCTTAGGATGAGTCTTCTTTAATCAATCATATATTGGGCGGAAAAAA 4000 AGATTGTTTGTGTTAOTTAACAGTACnraAATGGGAATCCTACTCAGAAGAAATTAG'raAGTATATAACCCGCCTTTTTT

3 I926• - 3I9I42 • II I- - I II • I

3954 3963 3972

3943 3955 3963

3959 3966 3965

Alu I Mae I

Mbo II Fnu4H I

Alu I Ear I Eoo57 I Bbv I

CACCA IGCTrTGACCCGAAGTAGTTG IAAGAIGCTACTTCATTCTTTTC ITGAAGTTGTGTGTTG ICTGCITAGAAATAGTCATTT 4080 GTGGTCGAAACTGGGCTTCATCAACTTCTCGATGAAGTAAGAAAAGACTTCAACACACAACGACGATCTTTATCAGTAAA

I • - I I- • I • -I I -

4005 4025 4046 4061

4025 4061

4029 4065

Mse I HinC II

Dra I Mbo II Tthlll II

I I I I I

GTGAATTATCCAAATTGTTTAAATTCACAATTGAATTAGT'ITI TCTTCCTTTTGGCTTGAAGCAAACAGTTGACCATTT 4160 CACTTAATAGGTTTAACAAATTTAAGTGTTAACTTAATCAAAAAAGAAGGAAAACCGAACTTCGTTTGTCAACTGGTAAA

4099 4150 Pst I Hae III

Mse I Rsa I Hae I

TTAACCTTTTCATTTTATGTTTTTGTACTCTGCAGACTGAAAAGACAAAGTTTATCTTGGCCTTACTGTATAAAGGTATG 4240 AATTGGAAAAGTAAAATACAAAAACATGAGACGTCTGACTTTTCTGTTTCAAATAGAACCGGAATGACATATTTCCATAC

4161 4185 4218

4190 4219

Mse I Ase I Mse I

Rsa I Mbo II Mse I Rsa I

CTGTGTCCACCGTTGTGTACAGAATTTTTCTTCATTAATTTTGTGTTTAAGTTAATAAAATTTATTTGTGATGTACTGTA 4320 GACACAGGTGGCAACACATGTCTTAAAAAGAAGTAATTAAAACACAAATTCAATTATTTTAAATAAACACTACATGACAT

4257 4269 4287 4313

4274 4292 4275

A 4321 T

Restriction Endonucleases site usage

Aat II - BstN I 13 HinC II 3 Pie I 4

Ace I - BstU I 4 HinD III 1 Pral I 2

Afl II - BstX I 1 Hinf I 11 PpuM I 3

Afl III 1 BstY I 5 HinP I 14 Pst I 4

Aha II 1 Bsu36 I 2 Hpa I - Pvu I -

Alu I 21 CfrlO I 1 Hpa II 3 Pvu II 2

Alw I 6 Cla I 1 Hph I 11 Rsa I 18

AlwN I 4 Dde I 18 Kpn I - Rsr II -

Apa I - Dpn I 7 Mae I 3 Sac I 1

ApaL I 1 Dra I 5 Mae II 7 Sac II 1

Ase I 3 Dra III - Mae III 11 Sal I -

Asp 18 - Drd I - Mbo I 7 Sau3A I 7

Ava I 2 Dsa I 3 Mbo II 18 Sau96 I 13

Ava II 6 Eae I 2 Mlu I - Sea I 2

Avr II - Eag I - Mme I 3 ScrF I 14

BamH I 1 Ear I 2 Mnl I 30 Sec I 11

Ban I 3 Eco47 III 3 Mse I 1 SfaN I 11

Ban II 2 Eco57 I 6 Mse I 22 Sfi I -

Bbe I 1 EcoN I 2 Msp I 3 Sma I -

Bbv I IS EcoC-109 I 5 Nae I 1 SnaB I -

Bbv II 2 EcoR I - Nar I 1 Spe I -

Bel I - EcoR II 13 Nci I 1 Sph I -

Ben I 1 EcoR V - Nco I 2 Spl I 1

Bgl I - Esp I - Nde I 2 Ssp I 3

Bgl II - Fnu4H I 20 Nhe I - Stu I 1

BsaA I 3 Fok I 9 Nla III 15 Sty I 4

Bsm I 1 Fsp I 2 Nla rv 10 Taq I 5

BsmA I 8 ... Gdi II 1 Not I - Tthlll I 2

Bspl286 I 4 Gsu I 5 Nru I - Tthlll II 10

BspH I 2 Hae I 4 Nsi I - Xba I -

BspM I 3 Hae II 8 Nsp7524 I - Xca I -

BspM II 1 Hae III 13 NspBi II 5 Xho I -

Bsr I 15 Hga I 1 NspH [ I - Xc I -

BssH II - HgiA I 3 PaeR7 I - Xma I -

BstB I 1 HgiE II - PflM [ I 1 Xmn I 1

BstE II - Hha I 14

Enzyme Site Use Site position (Fragment length) Fragment order

Afl III a/crygt 1 11 341) 2 342( 3980) 1

Aha II gr/cgyc 1 11 390) 2 391{ 3931) 1

ApaL I g/tgcac 1 K 3245) 1 3246( 1076) 2

BamH I g/gatce 1 K 1687) 2 1688( 2634) 1

Bbe I ggcgc/e 1 11 390) 2 391( 3931) 1

Ben I ccs/gg 1 11 2109) 2 21101 2212) 1

Bsm I gaatgc 1/-1 1 11 987) 2 988( 3334) 1

BspM II t/ccgga 1 11 3685) 1 3686( 636) 2

BstB I tt/cgaa 1 K 538) 2 539( 3783) 1

BstX I ccannnnn /ntgg 1 11 3332) 1 3333( 989) 2

CfrlO I r/ccggy 1 11 2494) 1 24951 1827) 2

Cla I at/cgat 1 11 1515) 2 15161 2806) 1

Gdi II yggccg -5/-1 1 11 384) 2 385{ 3937) 1 Hga I gacgc 5/10 1 1S2:.)

HinD III a/agctt 1 1141) 2 11421 3180)

Mse I tgg/cca 1 1300) 2 13011 3021)

Nae I gcc/ggc 1 2494) 1 24951 1827)

Nar I gg/cgcc 1 390) 2 3911 3931)

Nci I cc/sgg 1 2109) 2 21101 2212)

PflM I ccannnn/ntgg 1 2389) 1 23901 1932)

Sac I gagct/c 1 3454) 1 34551 867)

Sac II ccgc/gg 1 447) 2 4481 3874)

Spl I c/gtacg 1 1035) 2 10361 3286)

Stu I agg/cct 1 3016) 1 30171 1305)

Xmn I gaann/nnttc 1 1230) 2 12311 3091)

Ava I c/ycgrg 2 81) 3 821 287) 3691 3953)

Ban II grgcy/c 2 471) 3 4721 2983) 34551 867) Bbv II gaagac 2/6 2 3667) 1 36681 297) 39651 357) BspH I t/catga 2 3590) 1 35911 351) 39421 380) Bsu36 I cc/tnagg 2 3747) 1 37481 206) 39541 368) Eae I y/ggccr 2 1 384) 3 3851 916) 13011 3021) Ear I ctcttc 1/4 2 1 2983) 1 29841 1041) 4025( 297) EcoN I cctnn/nnnagg 2 1 2346) 1 2347( 1401) 37481 574) Fsp I tgc/gca 2 1 111) 3 1121 2419) 25311 1791)

Nco I c/catgg ₂ 1 90) 3 911 3687) 3778 ( 544) Nde I ca/tatg 2 1 960) 2 9611 90) 10511 3271) Pml I cac/gtg 2 1 1545) 2 15461 1697) 3243 ( 1079) Pvu II cag/ctg 2 1 2442) 1 24431 157) 26001 1722) Sea I agt/act 2 1 2613) 1 26141 432) 30461 1276) Tthlll I gacn/nngtc 2 1 1784) 1 17851 793) 25781 1744 )^—2

Ase I at/taat 1 3576) 1 35771 56) 36331 641) 42741 48) Ban I g/gyrcc 1 390) 3 3911 362) 753 ( 416) 11691 3153) BsaA I yac/gtr 1 1012) 3 10131 533) 15461 1697) 32431 1079) BspM I acctgc 4/8 1 630) 3 6311 2081) 2712( 1089) 38011 521) Dsa I c/crygg 1 90) 4 911 357) 448( 3330) 37781 544) Eco47 III age/get 1 199) 4 2001 379) 579( 1840) 24191 1903) HgiA I gwgcw/c 1 3245) 1 32461 209) 34551 166) 36211 701) HinC II gty/rac 1 2071) 1 20721 533) 26051 1545) 41501 17₂) Hpa II c/cgg 1 2110) 1 21111 385) 24961 1191) 36871 635) Mae I c/tag 1 153) 4 1541 2458) 26121 1453) 40651 257) Mme I tccrac 20/18 1 287) 4 288( 618) 906( 1086) 19921 2330) Msp I c/cgg 1 2110) 1 21111 385) 24961 1191) 36871 635) PpuM I rg/gwccy 1 1661) 1 16621 166) 18281 881) 27091 1613) Ssp I aat/att 1 2126) 1 21271 84) 22111 1696) 3907( 415)

AlwN I cagnnn/ctg 4 1 1506) 2 15071 81) 5 15881 947) 3 25351 255) 4 2790 1532) 1 Bspl286 I gdgch/c 4 1 471) 3 472( 2774) 1 32461 209) 4 34551 166) 5 3621 701) 2

BstU I cg/cg 4 1 448) 3 4491 30) 4 4791 2) 5 4811 1858) 2 2339 1983) 1

Hae I wgg/ccw 4 1 1300) 1 13011 638) 4 19391 1078) 3 30i7( 1201) 2 4218 104) 5 Pie I gagtc 4/5 4 1 742) 3 7431 1321) 2 20641 544) 4 26081 1355) 1 3963 359) 5 Pst I ctgca/g~ 4 1 419) 3 4201 1450) 2 1870( 161) 4 20311 2159) 1 4190 132) 5 Sty I c/cwwgg 4 1 90) 5 911 1618) 1 1709( 686ι 3 23951 1383) 2 3778 544) 4

BstY I r/gatcy 5 1 649) 4 650( 801) 2 14511 237) 5 16881 668) 3 2356 109) 6 24651 1857) 1

Dra I ttt/aaa 5 1 817) 3 8181 2094) 1 29121 54) 6 29661 128) 5 3094 1004) 2 40981 224) 4

EcoO109 I rg/gnccy 5 1 1661) 1 16621 166) 6 18₂8( 646) 4 24741 235) 5 2709 695) 3 34041 918) 2

Gsu I ctggag 16/14 5 1 274) 3 2751 2076) 1 23511 9) 6 23601 43) 5 2403 98) 4 25011 1821) 2

NspB II cmg/ckg 5 1 176) 4 177( 205) 3 382{ 66) 6 4481 1995) 1 2443 157) 5 26001 1722) 2

Taq I t/cga 5 1 123) 5 1241 416) 4 540( 977) 2 1517( 9) 6 1526 2133) 1 36591 663) 3

Alw I ggatc 4/5 6 1 649) 650( 802) 2 14521 236) 5 1688( 1) 7 1689 667) 23561 110) 6 24661 1856) 1

Ava II g/gwcc 6 1 1662) 1663 ( 166) 6 18291 178) 5 2007( 365) 3 2372 205) 25771 133) 7 27101 1612) 2

Eco57 I ctgaag 16/14 6 1 1080) 1081 ( 64) 7 11451 554) 4 1699( 111) 6 1810 913) 27231 1323) 1 40461 276) 5

Dpn I ga/tc 1 650) 4 6511 801) 2 14521 63) 8 1515( 81) 7 1596 93) 6 16891 668) 3 2357( 109) 5 24661 1856) 1

Mae II a/cgt 1 343) 7 3441 670) 2 10141 533) 4 15471 1035) 1 2582 517) 6 30991 145) 8 32441 545) 3 37891 533) 5 Mbo I /gate 7 11 650) 4 6511 801) 2 14521 63) 8 15151 81) 7

15961 93) 6 16891 668) 3 23571 109) 5 24661 1856) 1

Sau3A I /gate 7 K 650) 4 6511 801) 2 1452 ( 63) 8 15151 81) 7

15961 93) 6 16891 668) 3 2357( 109) 5 24661 1856) 1

BsmA I gtctc 1/5 8 11 1099) 1 11001 529) 4 16291 853) 2 24821 310) 6

27921 67) 9 28591 128) 8 29871 133) 7 31201 4811 5

36011 721) 3

Hae II rgcgc/y 8 11 199) 3 2001 191) 4 3911 36) 7 4271 17) 9

4441 19) 8 4631 116) 5 5791 1840) 1 24191 73) 6

24921 1830) 2

Fok I ggatg 9/13 9 K 128) 9 1291 360) 5 4891 74)10 563 ( 824) 2

13871 238) 7 16251 302) 6 19271 226) 8 21531 1190) 1

3343 ( 616) 3 39591 363) 4

Nla IV ggn/ncc 10 11 86) 9 87( 159) 6 2461 145) 7 3911 311) 5

7021 51)10 7531 416) 3 11691 386) 4 15551 107) 8

16621 26)11 16881 787) 2 24751 1847) 1

Tthlll II caarca 11/9 10 K 115) 8 116( 68)10 1841 39)11 2231 365) 5

588( 381) 4 969( 471) 2 14401 453) 3 18931 109) 9

20021 1924) 1 39261 218) 6 41441 178) 7

Hinf I g/antc 11 K 134) 8 1351 555) 3 6901 53)10 7431 760) 2

15031 6)12 15091 81) 9 15901 474) 5 20641 544) 4

26081 14)11 26221 973) 1 35951 368) 6 39631 359) 7

Hph I ggtga 8/7 11 K 874) 2 875( 232) 6 11071 228) 7 13351 97) 9

14321 146) 8 15781 948) 1 25261 41)11 25671 383) 5

29501 794) 3 37441 31)12 37751 79)10 38541 468) 4

Mae III /gtnac 11 11 217)10 2181 497) 3 7151 379) 4 10941 12)12

11061 340) 6 14461 337) 7 17831 353) 5 21361 278) 8

24141 39)11 24531 1051) 1 35041 239) 9 3743 ( 579) 2

Sec I c/cnngg 11 11 82) 9 831 8)11 911 243) 7 3341 114) 8

448{ 856) 2 13041 405) 6 17091 686) 3 23951 62)10

24571 410) 5 28671 1)12 28681 910) 1 37781 544) 4

SfaN I gcatc 5/9 11 K 165) 8 1661 398) 5 5641 508) 3 1072 ( 115)11

11871 242) 7 14291 913) 1 23421 145)10 2487{ 42)12

25291 860) 2 33891 289) 6 36781 148) 9 38261 496) 4

BstN I cc/wgg 13 11 333) 7 3341 54)12 3881 104) 9 4921 534) 3

10261 84)10 11101 51)13 11611 143) 8 13041 7₂₂) 2

20261 42)14 20681 389) 4 24571 66)11 2523 ( 345) 6

28681 351) 5 32191 1103) 1

EcoR II /ccwgg 13 11 333) 7 3341 54)12 3881 104) 9 492( 534) 3

10261 84)10 11101 51)13 11611 143) 8 13041 7₂₂) 2

20261 42)14 20681 389) 4 24571 66)11 25231 345) 6

28681 351) 5 32191 1103) 1

Hae III gg/cc 13 K 385) 5 3861 316) 7 7021 600) 2 13021 255) 9

15571 383) 6 19401 102)11 20421 39)13 20811 300) 8

23811 94)12 24751 23)14 24981 520) 3 3018{ 388) 4

34061 813) 1 42191 103)10

Sau96 I g/gncc 13 11 701) 3 7021 854) 2 15561 107) 9 16631 166) 7

18291 178) 6 20071 34)13 20411 40)12 20811 291) 5

23721 9)14 23811 94)11 24751 102)10 2577( 133) 8

27101 695) 4 34051 917) 1

Hha I gcg/c 14 11 112) 4 1131 88) 6 2011 7)15 208( 184) 3

3921 13)14 4051 23) 9 428( 17)12 4451 19)10

4641 16)13 4801 100) 5 580( 1840) 1 2420 ( 18)11

24381 55) 7 24931 39) 8 25321 1790) 2

HinP I g/cgc 14 11 112) 4 1131 88) 6 2011 7)15 208 ( 184) 3

3921 13)14 4051 23) 9 428( 17)12 4451 19)10

4641 16)13 4801 100) 5 580( 1840) 1 24201 18)11

24381 55) 7 24931 39) 8 25321 1790) 2

ScrF I cc/ngg 14 11 333) 7 3341 54)12 388( 104) 9 4921 534) 3

10261 84)10 11101 51)13 11611 143) 8 13041 722) 2

20261 42)14 20681 42)15 21101 347) 5 24571 66)11

25231 345) 6 28681 351) 4 3219( 1103) 1

Bbv I gcagc 8/12 15 11 109)12 1101 69)14 179( 186) 8 365( 16)15

3811 72)13 4531 114)11 567 ( 214) 7 7811 176) 9

9571 406) 5 13631 506) 3 18691 3)16 1872( 158)10

20301 415) 4 24451 794) 2 32391 822) 1 40611 261) 6

Bsr I actgg 1/-1 15 11 276) 6 277( 280) 5 557( 44)14 601 ( 96)12

6971 181) 9 8781 416) 2 12941 365) 3 16591 220) 7

18791 23)15 19021 22)16 19241 154)10 20781 324) 4

24021 77)13 24791 147)11 26261 191) 8 2817( : 1505) 1

Nla III catg/ 15 11 91)11 921 648) 2 740( 1638) 1 23781 13)16

23911 250) 6 26411 393) 3 30341 94)10 31281 105) 9

32331 24)15 32571 83)12 3340( 252) 5 35921 26)14

36181 161) 7 37791 39)13 38181 125) 8 3943 ( 379) 4

Dde I c/tnag 18 11 161)11 1621 191) 9 3531 115)17 4681 207) 7 6751 155)13 830( 272) 5 11021 26)19 11281 644) 1

17721 61)18 18331 181)10 20141 132)14 21461 363) 4

25091 609) 2 31181 156)12 32741 127)15 34011 223) 6

36241 125)16 37491 206) 8 39551 367) 3

Mbo II gaaga 8/7 18 11 329) 5 330( 1231) 1 15611 164) 8 17251 51)16

17761 33)18 18091 466) 2 22751 38)17 23131 3)19

23161 387) 4 2703 ( 281) 6 29841 413) 3 33971 141)10

35381 130)11 36681 68)13 37361 230) 7 39661 59)14

40251 100)12 41251 144) 9 42691 53)15

Rsa I gt/ac 18 11 483) 4 4841 532) 2 10161 21)16 10371 113)12

11501 163) 9 13131 153)11 14661 442) 5 19081 500) 3

24081 207) 8 26151 265) 7 28801 162)10 30421 5)19

30471 63)14 31101 21)17 31311 681) 1 38121 373) 6

41851 72)13 42571 56)15 43131 9)18

Fnu4H I gc/ngc 20 K 109)12 110( 66)13 176( 3)16 1791 186) 8

3651 13)15 3781 3)17 3811 3)18 3841 63)14

4471 3)19 4S0( 3)20 453( 114)11 567( 214) 7

7811 176) 9 957( 406) 5 13631 506) 3 18691 3)21

18721 158)10 20301 415) 4 24451 794) 2 32391 822) 1

40611 261) 6

Alu I ag/ct 21 11 27)19 28( 186) 8 2141 25)20 2391 172)10

4111 383) 4 7941 204) 6 998( 133)15 11311 12)22

11431 36)18 11791 153)13 13321 76)17 14081 604) 1

20121 137)14 21491 95)16 22441 200) 7 24441 157)11

26011 156)12 2757( 174) 9 29311 525) 3 34561 549) 2

40051 24)21 40291 293) 5 —

Mse I t/taa 22 11 294) 5 2951 258) 7 5531 210)10 7631 56)16

819( 442) 2 12611 237) 8 14981 267) 6 17651 915) 1

26801 233) 9 29131 54)18 29671 46)19 3013 ( 67)14

30801 15)21 30951 339) 3 34341 144)11 35781 56)17

36341 338) 4 39721 127)12 40991 62)15 41611 114)13

42751 12)22 42871 5)23 42921 30)20

Mnl I 7/7 30 K 84)20 85( 41)26 1261 137)11 2631 162)10

425( 93)19 5181 104)16 6221 321) 2 9431 105)15

10481 107)14 11551 97)18 12521 276) 6 15281 53)24

15811 104)17 16851 311) 4 19961 22)27 20181 21)28

20391 315) 3 23541 21)29 23751 125)12 25001 54)23

25541 21)30 25751 248) 7 2823 ( 117)13 29401 80)21

30201 6)31 30261 210) 8 32361 167) 9 3403 ( 66)22

34691 282) 5 37511 43)25 37941 528) 1

587 sites found

No Sites found for the following Restriction Endonucleases

Aat II gacgt/c EcoR I g/aattc Pvu I cgat/cg

Ace I gt/mkac EcoR V gat/atc Rsr II cg/gwccg

Afl II c/tcaag- Esp I gc/tnagc Sal I g/tcgac

Apa I gggcc/c HgiE II accnnnnnnggt Sfi I ggccnnnn/nggcc

Asp718 g/gtacc Hpa I gtt/aac Sma I ccc/ggg

Avr II c/ctagg Kpn I ggtac/c SnaB I tac/gta

Bel I t/gatca Mlu I a/cgcgt Spe I a/ctagt

Bgl I gccnnπn/nggc Nhe I g/ctagc Sph I gcatg/c

Bgl II a/gatct Not I gc/ggccgc Xba I t/ctaga

BssH II g/cgcgc Nru I tcg/cga Xca I gta/tac

BstE II g/gtnacc Nsi I atgca/t Xho I c/tcgag

Dra III cacnnn/gtg Nsp7524 I r/catgy Xcm I ccannnnn/nnnntgg

Drd I gacnnnn/nngtc NspH I rcatg/y Xma I c/ccggg

Eag I c/ggccg PaeR7 I c/tcgag ANNEX 5

DNA sequence 3859 b.p. TCTCCTTTTTCT ... GATGTACTGTAA linear

Positions of Restriction Endonucleases sites (unique sites underlined)

I 80

Sau3A I

Mbo I

Dpn I

Mae III Mbo II Bleeil TT IIII Bsr I

GCAGAG ITAACAACATICTTCTAArrT'lTTTACCCCTIGIATCACAGGTGCIAAACATCTCAAGCICAGTTCAGATGTTGCTGTTT 160 CGTCrrCATTGTTGTAGAAGATTAAAAAAATGGGGACTAGTGTCCACGTTTGTAGAGTTCGGTCAAGTCTACAACGACAAA

8 I6 • 9I5 1I1I5 1I27' 1I40

116 116 116 Sau3A I Mbo I Dpn I BstY I Bsr I

Mnl I BspM I Alw I Dde I Hinf I

C ICTCAAGTTGICACKn'CTATGGAAATGCAGIGIATCTAACCAGCCCGCATAeCCGTCITGAGTGGTAGTAGTGIAATCCCCICAGT 2 0 GGAGTTCAACGTCCAGATACCTTTACGTCCTAGATTGGTCGGGCGTATCGGCAGACTCACCATCATCACTTAGGGGGTCA

1 I61 1I70 • 1I8I9 • • 2I14 • 2I2-9 I •

189 236 190 190 190

Sau96 X Pie I

Nla IV Hinf I Nla IV Fnu4H I

Hae III Mae III Nla III Ban I Mse I Bbv I

G IGCCCCAAACTCGGITAACTCTCATATAAATAGTAATTCCtATGiACTCCCAATGlGCACCGAAGTlTAAAACAGAGCCAATGAGI 320 CCOGGGTTTGAGCCATTGAGAGTATATTTATCATTAAGGTACTGAGGGTC^ 2 I41 ' 2I54 —• • 2I7-9I - 2I92 - 3I02 • 3I20

241 282 292 320

241 282

Mse I Alu I Dra I Dde I

CAGCAGTGAAACA IGCTTCAACGACAGCCGACGGGTCTIrITAAACAATTTCITCA∞ 400

480

417

Nde I Rsa I

Fnu4H I Mae II

Mnl I Bbv I Tthlll II Bsm T Alu I BsaA I

TC ICTCACAAACTATGGIC GCIATATGGGCIAAACACAGTTTACCACAGGIAATGCAACAAIGCTACAGCCTATGCCIAICGITACCC 560 AGGAGTGrraGATACCGACGTATACCCGTTTGTGTCAAATGGTGTCCTTACGTTGTTCGATGTCGGATACGGTGCATGGG

4 '82 ^• 4I96 I 5 I08 ^{• •} 5 I27 ^• 5 I37 ^{• •} 5 I5I2 I

496 553

500 555 ScrF I

EcoR II Rsa I Nde I BsmA I

BstN I Sol I Mnl I SfaN I Eco57 I Mae III

A_CA_GC I_CA_GGACA_GCC I_GITAC_GGCATTT_C ICTCIATATGGTGCATTGTGGGCAGG ICATCAAGACITGAAGGTGGATTG ITCACAGIT 640 T_GT_CGGT_CCTGT_CG_GCAT_GCCGTAAAG_GAGTATACCACGTAACACCCGTCCGTAGTTCTGACTTCCACCTAACAGTGTCA

I . M • I I ^• -I I - I I-

565 575 587 611 620 633

565 576 590 639

565

ScrF I

EcoR II ScrF I

Hph I Eco57 I EcoR II

Mae III Alu I Alu I Rsa I BstN I Nla IV

Dde I BstN I Dde I HinD III Mnl I Ban I Alu I

_C IT_CA_G ITI_CA_C ICT_GGACA_GACA_GGATTTC ITCAIGCTATGGCACA IAIGC ITTCAG ITACCC ICTCAAC ICTGGACAG IGCACCATACA IGC 720 GAGTCAGT_GGACCTGTCTGTCCTAAAGAGTCGATACCGTGTTCGAAGTCATGGGGAGTTGGACCTGTCCGTGGTATGTCG

I I I I - ^• I I - I I I I - I I I ^• I ^•

641 649 666677 668811 669944 770088 718

645 670 682 689 700 708 646 684 700 649 700 649 SfaN I Xmn I Mnl I Mse I

I I I I

TACCAGATGCAAGGTAGCAGTTTTACAACATCATCAGGAATATATACAGGAAATAATTCACTCACAAATTCCTCTGGATT 800 ATGGTCTACGTTCCATCGTCAAAATGTTGTAGTAGTCCTTATATATGTCCTTTATTAAGTGAGTGTTTAAGGAGACCTAA

7 I26 • • • • 7I70 • -7I91 8I00

Sec I ScrF I EcoR II BstN I Hae III Msg I

Hae I Hph I

Bsr I Ea£_I Rsa I Alu I

I I I I I I I

TAATAGTTCACAGCAGGACTATCCGTCTTATCCCAGTTTTGGCCAGGGTCAGTACGCACAGTATTATAACAGCTCACCGT 880 ATTATCAAGTGTCGTCCTGATAGGCAGAATAGGGTCAAAACCGGTCCCAGTCATGCGTGTCATAATATTGTCGAGTGGCA

^• I II I - I • -I I -

833 840 852 871

840 874

840 841 843 843 843 843 Fnu4H I Bbv I Fok I Alu I

I I I

ATCCAGCACATTATATGACCAGCAGCAACACCAGCCCAACGACACCATCCACCAATGCCACTTACCAGCTTCAAGAACCG 960 TAGGTCGTGTAATATACTGGTCGTCGTTGTGGTCGGGTTGCTGTGGTAGGTGGTTACGGTGAATGGTCGAAGTTCTTGGC

• " | • • _ | • • | » •

902 926 947

902

Sau3A I

Mbo I

Dpn I

Alw I

Hph I Mae III

SfaN I Tthlll II BstY I Rsa I Mse I

I I I I II I I

CCATCTGGCATCACCAGCCAAGCAGTTACAGATCCCACAGCAGAGTACAGCACAATCCACAGCCCATCAACACCCATTAA 1040 GGTAGACCGTAGTGGTCGGTTCGTCAATGTCTAGGGTGTCGTCTCATGTCGTGTTAGGTGTCGGGTAGTTGTGGGTAATT

I - I I - I I I ^• I • • • I •

68 979 990 1005 1037

971 985

991 991 991 991 Cla I

Sau3A I Mbo II Hinf I Taq I Mnl I Mae II Hae III AlwN I Mbo I Taq I Pml I Sau96 I Mnl I Hinf I Dpn I H a I BsaA I Nla IV Hph I

A_G IATT_CMAGATTCTGIAITICGATT_GICGTIC_GIA_GGTTCAGATGGGAAATCIAICGTGGACGIGIGIGCCGIAAGAAACAATAATCCTTiCACl 1120 T_CTAA_GT_CTAA_GACTA_GCTAA_CG_CA_GCT_CCAAGTCTACCCTTTAGTGCACCTGCCCCGGCTTCTTTGTTATTAGGAAGTG i i i . i l l • I I I • • I I • I I I I - I I

1042 1054 1062 1085 1094 1117

1046 1054 1065 1085 1095 1120

1048 1056 1067 1086 1096 1054 1100 1055

Sau3A I

Mbo I

Hinf I BsmA I

AlwN I Dpn I Fok I Bsr I

_CT_CCC_CC .A_G.ATT_CT_G.AT_CTT_GAGAGAGTGTTCATCTGGGACTTG IGATGIAGACAATCATTGTTTTCCACTCCTTGCTTA ICT 1200 _GA_GGGGGT_CTAA_GA_CTAGAACTCTCTCACAAGTAGACCCTGAACCTACTCTGTTAGTAACAAAAGGTGAGGAACGAATGA 1l12l7- 1l135 - • - 1I164I - ' ' 1I19*8

1129 1168

1135 1135

Sau3A I

Mbo I —

Dpn I

Alw I

Sau96 I Nla IV

Ava II BstY I

PpuM I BamH I

Nla IV Alw I Sty I

EcoO109 I Mnl I Eeo57 I See I Mbo II

I I I I I I I I

GGGTCCTACGCCAACAGATATGGGAGGGATCCACCCACTTCAGTTTCCCTTGGACTGCGAATGGAAGAAATGATTTTCAA 1280 CCCAGGATGCGGTTGTCTATACCCTCCCTAGGTGGGTGAAGTCAAAGGGAACCTGACGCTTACCTTCTTTACTAAAAGTT

II ^• - I II - I ^• I ^• • I ^•

1201 1224 1238 1248 1264

1201 1227 1248

1201 1227

1202 1227

1228 1228 1228 1228

Mbo II Tthlll I Eco57 I

Mse I Dde I Mae III Mbo II I I I I I II

CTTGGCAGACACACATTTATTTTTTAATGACTTAGAAGAATGTGACCAAGTCCATATAGATGATGTTTCTTCΛGATGATA 13 60 GAACCGTCTGTGTGTAAATAAAAAATTACTGAATC'ITCraACACTGGTTCAGGTATATCTACTACAAAGAAG'I CTACTAT

, ^• I - I I ^• I I ^• I I -

1304 1311 1322 1348 1315 . 1324 1349

Dde I Fnu4H I

Sau96 I Bbv I

Ava II Pst I

PpuM I Fnu4H I

EcoO109 I Bbv I Bsr I Tthlll II

I I I I I I I I

ACGGACAGGACCTAAGCACATATAACTTTGGAACAGATGGCTTTCCTGCTGCAGCAACCAGTGCTAACTTATGTTTGf'A 1440 TGCCTGTCCTGGATTCGTGTATATTGAAACCTTGTCTACCGAAAGGACGACGTCGTTGGTCACGATTGAATACAAACCGT ll - l ^{• • •} ll-l I • - I

1367 1408 1418 1432

1367 1408

1368 1409

1368 1411

1372 1411

Rsa I Fok I Hae III

I Bsr I Hae I

ACTGGTGTACGGGGCGGTGTGGACTGGATGAGAAAGTTGGCCTTCCGCTACAGACGGGTAAAAGAGATTTACAACACCTA 1520 TGACCACATGCCCCGCCACACCTGACCTACTCTTTCAACCGGAAGGCGATGTCTGCCCATTTTCTCTAAATGTTGTGGAT

1 I441 I - - 1I463I - 1I4I7-8

1447 1466 1479 Pst I

Fnu4H I

Alu I Bbv I

Sau96 I ScrF I Hae III

Mnl I Ava II Mnl I EcoR II Ξau96 I

Mme I Tthlll II Dde I BstN I Mnl I

_CAAAAATAAT_GTTG_GA_GGTCTGCTT_GGTCCA_GCTAA_GAG_GGAAG_CCTGGCTGCAGTTGAGGGCCGAAATTGAAGCCCTGA 1600 _GTTTTTATTA_CAA_CCT_CCA_GAC_GAA_CCA_GGTCGATTCTCCCTTCGGACCGACGTCAACTCCCGGCTTTAACTTCGGGACT

1531 1541 1553 1565 1578

1535 1546 1557 1565 1580 1546 1565 1581

1551 1569 1569 1570

ScrF I Msp I

EcoR II Hpa II

BstN I Sau96 I ScrF I

Pie I Bsr I Nei I

Hinf I HinC II Hae III Be, I Ssp I Mae III

_CCG IA_CT_CI_CT_GGITTGACAICTGIGCCCTGAAAGCACTCTCGCTCATTCACTCICICGGACAAACTGTGTGAIATATTTTAGITAACA 1680 _GGCT_GAG_GACCAACTGTGACCGGGACTTTCGTGAGAGCGAGTAAGTGAGGGCCTGTTTGACACACTTATAAAATCATTGT

1 I603I -1I611 I 1I620 • • 1I6I49 1I666^• 1I675

1603 1617 1649

1607 1620 1649 —

1607 1650

Alu I Dde I Fok I Ssp I

ACTAC ITCAIGCTCIATCCCAGCATTGGCGAAAGTCCTGCTGTATGGGTTAGGAATTGTATTTCCAATAGAAAIATATTTACAG 1760 TGATGAGTCGAGTAGGGTCGTAACCGCTTTCAGGACGACATACCCAATCCTTAACATAAAGGTTATCTTTTATAAATGTC

1l685l - 1l692 - • • • ' 1I750

1688

Alu I Mbo II

TGCAACTAAAATAGGAAAAGAAA IGCTGTTTTGAGAGAATAATTCAAAGGTTTGGIAAGAAAAGTGGTGTATGTTGTTATAG 1840 ACGTTGATTTTATCCTTTTCΓΓTCGACAAAATCTCTTATTAAGTTTCCAAACCTTCTTTTCACCACATACAACAATATC

1783 1814

Sau3A I Mbo I Dpn I BstY I Nla III

Mnl I Mnl I

Mbo II SfaN I Gsu I Gsu I Sau96 I Sau96 I

Mbo II BstU I EcoN I Alw I Ava II Hae III

GAGATGGTGTAG IAAGIAAGAACAAGGAGCAAAAAAGCACIGCGIATGCCICTTCITGGIAGIGIATCITCCAGCCACTCGIGACICTCIATGI 1920 CTCTACCACATCTTCTTCTTGTTCCTCGTTTTTTCGTGCGCTACGGGAAGACCTCCTAGAGGTCGGTGAGCCTGGAGTAC ^• 1I852I- * ^• 1I87-8I- 1I886I I 1I8I95I- -I I I I

1855 1881 90 1899 1911 192

1893 1914

1895 1917

1896

Fnu4H I

HinP I Alu I Sec I

Sty I Hha I Pvu II ScrF I

Sec I Rsa I Hae II NspB II EeoR II

Nla III Gsu I Eco47 ITT HinP I BstN I

PflM I Bsr I Mae III Hha I Bbv I Mae III

GCCCTGCACCATGCCTTGGAACTGGAGTACCTGTAACAGCGCTCGGCACTTTGACAGCGCACAGCTGCTCTGTGACCAGG 2000 CGGGACGTGGTACGGAACCTTGACCTCATGGACATTGTCGCGAGCCGTGAAACTGTCGCGTGTCGACGAGACACTGGTCC

1I9I29 I -1I9I41 I • 1I953 II- • I • III . i i .

1977 1984 1992 1930 1942 1958 1977 1996

1934 1947 1958 1982 1996 1934 1959 1982 1996

1959 1983 1996

1984 Hae III

Msp I

Hpa II

Sau3A I Nae I Hph I AlwN I

Mbo I BsmA I HinP I ScrF I HinP I

Dpn I Bsr I Hha I Gsu I EcoR II Hha I

Alw I Sau96 I Hae II Mnl I BstN I Fsp I

BstY I Hae III SfaN i CfrlO i Dde I Sec I SfaN I

_GA_CA_GAT_CCA_GCAG_GC_CCA_GT_CTC_GCATCAGCGCCGGCCTCCAGAACTTAGCAATTTCCGCCCTGGTGATGCGCAGTTGC 2080 CTGTCTAG_GT_CGT_CC_GGGTCA_GAGCGTAGTCGCGGCCGGAGGTCTTGAATCGTTAAAGGCGGGACCACTACGCGTCAACG

M - I I I I II II I II- I • -II I I II I

2004 2014 2025 2033 2047 2061 2068

2005 2014 2030 2038 2062 2070

2005 2017 2031 2039 2062 2071

2005 2020 2031 2062 2071

2005 2033 2065 2074 2034 2034 2036

Tthlll I HinC II Sea I Sau96 I Alu I Pie I Rsa I Ava II Pvu II Hinf I

Mnl I Hph I Mnl I Mae II NspB II Mae I

I I I II I II I I I II

TGTCAGTCTTGACCTCTGCCTTTGTGGTGAATGGAGGACCACGTCTATTTCATCAGAACAGCTGTTGACTCTAGTACTGT 2160 ACAGTCAGAACTGGAGACGGAAACACCACTTACCTCCTGGTGCAGATAAAGTAGTCTTGTCGACAACTGAGATCATGACA

• I • I ^• I II -I • II I I -I II

2093 2106 2114 2121 2139 2151

2116 2139 2147 2116 2140 2147 2154

2117 2144 2153

TACACGGTTCAAACCTG 2240 CTTAGGTCACTTTTATTCGGTACTCTTACAAAATCGTGTCGCAATACACAGACGGTGTAATTGATGTGCCAAGTTTGGAC

I I - I • • I-

2161 2180 2219

2165

Sau96 I Ava II PpuM I EcoO109 I Mbo II BspM I Eco57 I Alu I

I II I I I

TGAAGAAAGGACCTGCAAACGCTTCAGTTGTTAGCATTTTCAATGTGATATAAACAGCTTCTCCAATACAGCAAACCTAA 2320 ACTTCITTCCTGGACGTTTGCGAAGTCAACAATCGTAAAAGTTACACTATATTTGTCGAAGAGGTTATGTCGTTTGGATT

I ll-l • I • . i .

2242 2251 2262 2296

2248 2248 2249

AACGTGTTGTCTCTGACTTTACACAAAGGACTTATGGTCACCTCCTTAAAAGAACATTTCTTCCAAATGAAAAACCACAG l - l ^{• •} I ^• I • • • I •

2329 2356 2398

2331 2362

Sec I ScrF I EeoR II

BstN I Mse I

Sec I Rsa I Dra I Alu I Mnl I

II I II I I

TCATACCCAGGGTAATCTGTACATCTCTACTTATTTATGAACAGACTTTTTTTAAAAAGATAAAAAAACAGCTTTATTGA 2480 AGTATGGGTCCCATTAGACATGTAGAGATGAATAAATACTTGTCΓGAAAAAAATTTTTCTATTTTTTTGTCGAAATAACT ll - l- ^{• •} -II ^• I I-

2406 2419 2451 2470 2479

2407 2452

2407 2407 2407

≤£U I Mse I BsmA I Hae I

Hph I Dra I Mnl I Mse I Mnl I

GGTATAATT ICACCCACCAGACTTTT ITITAAACATCAAATAATTG IAGGIAGACAATAGCATTAGAAATAAGTGAT ITAAA IGIGC IC 2560 CCATATTAAGTGGGTGGTCTGAAAAAATTTGTAGTTTATTAACTCCTCTGTTATCGTAATCTTTATTCACTAATTTCCGG

| . I I • - I I - ^{• •} I I I I -

₂489 2505 2523 2552 2559

2506 2526 2556

2556 2557 Rsa I Mae II

Rsa I Mse I

Mnl I Nla III Sea I Mse I Dra I

I I I I I I I I I

TCTGCCTCACAACATGGCAAGTACAGTACTTTGAATTTTAGCACATTGCATAATAGTTTTAAGTATGTCTAATTTAAACG 2640 AGACGGAGTGTTGTACCGTTCATGTCATGAAAC'I AAAATCGTGTAACGTATTATCAAAATTCATACAGATTAAATTTGC

I • I -I II • • • I- ^• II I ^•

2565 2573 2585 2619 2633

2581 2634

2586 2638

BsmA I Rsa I Rsa I Dde I Nla III

TATAATATG ITACATCAC ITGIAGACAATC IATGITACAGAAAGAATTTTTGGTGTAAATTTGTAATAATGGATAATTCTTT AC 2720 ATATTATACATGTAGTGACTCTGTTAGTACATGTCTTTCTTAAAAACCACATTTAAACATTATTACCTATTAAGAAAATG

I- I I- I I - - - - -

2649 2657 2667

2659 2670

HgiA I Bspl286 I Mae II Fnu4H I ScrF I Bbv I ApaL I

EcoR II Mnl I Pml I

BstN I Nla III BsaA I Nla III

I I I I I I I I

ATATTGTTTAGGGAAATGATATTGAAAGGTAGCAATGCCTGGATAGTGAAGCATGAGGCAGCACGTGCACAAATTCATGT 2800 TATAACAAATCCCTTTACTATAACTTTCCATCGTTACGGACCTATCACTTCGTACTCCGTCGTGCACGTGTTTAAGTACA

2758 2772 2782 2796

2758 2775 2782

2758 2778 2785

2778

2783 2785 2785

Nla III

Dde I BstX I

I

GCCGTGCriTATCTGAGTTTTCGGTATAAATATGTAGATAATGGATTTTTTTTTAGATAATGTTGTCAAGACCAAAAGCA 2880 CGGCACGGAATAGACTCAAAAGCCATATTTATACATCTATTAC AAAAAAAAATCTATTACAACAGTTCTGGTTTTCGT

• • • • • • • j | ■

2813- 2872

2879 Hae III EcoO109 I Mnl I Dde I Fok I SfaN I Mbo II Sau96 I

TG IGATGTO^GTGTCAGTAAGGATTTTG'I TCTAAAATTTTTTCCTGICATCAGTTICTTCITGMAGMGGCCTTGATGAAATAAC 2960 ACCTACAGTTCACAGTCATTCCTAAAACAAAGATTTTAAAAAAGGACGTAGTCAAGAAGACTCCCGGAACTACTTTATTG I . . I . I |.||||

2882 2927 2935 2943

2939 2941 2942 2944 Alu I Sac I HgiA I Bspl286 I Mse I Baη II Mnl I

A_CA_GCA_GTTT_CT ITAAA_CAATTT_GAAA_CAAAATGIAIGCTCTCCTACCACICTCACTTTTTCATTTCCACACTAATGTATTATA 3040 T_GT_CGT_CAAAGAATTTGTTAAACTTTGTTTTACTCGAGAGGATGGTGGAGTGAAAAAGTAAAGGTGTGATTACATAATAT

• I • ^• II ^• I ^•

₂972 2993 3007

2993 2993 2993 2994

Mse I Mae III Mbo II Ase I

TG ITAACTACTTGGAAAAAATAATTATTCAAATGCTTICTTCCCACAAAGAATATAGATGATAGTAGATATATTTTAITITAAT 3120 ACATTGATGAACCTTTTTTATTAATAAGTTTACGAAGAAGGGTGTTTCTTATATCTACTATCATCTATATAAAATAATTA

I • • • I • • ' * I I ^• 3042 3076 3115

3116 Dde I Hinf I HgiA I

Nla III Bspl286 I Mse I

BspH I BsmA I Nla III Ase I Taq I

II I I I I I II I

AAAATGGTTCATGAATCGGAGACTAACAAAGTTTTCATGTGCTCAGAATTATTAATTATCGTGTCTGCATTTTCTTTC«A 3200 TTTTACCAAGTACTTAGCCTCTGATTGTTTCAAAAGTACACGAGTCTTAATAATTAATAGCACAGACGTAAAAGAAAGCT

II I I- - l l- l -II • • I •

3129 3139 3156 3171 3197

3130 3159 3172

3133 3159 3162

Msp I

Mbo II Hpa II

Bbv II SfaN I BSPM II Mbo II

I I II I

TAAAGGAAGACACACGATGCTAATCCGGAAATCAGCAAACTTTGCATTACTCCCTATGTGCGTATTTTCTCTTTCTTCCT 3280 ATTTCCTTCTGTGTGCTACGATTAGGCCTTTAGTCGTTTGAAACGTAATGAGGGATACACGCATAAAAGAGAAAGAAGGA

3206 3216 3224 3274

3206 3225 3225

Nla III

Dde I Sty I

EcoN I Sec I

Bsu36 I Ncς> I

Hph I Mnl I Ds I Mnl I Nla III lae III Hph I Mae II BspM I Rsa I

GTCACCCTGAGGAAGGTTCATTGCCATTGTCATCACCATGGAAACAACGTTCCTCTCCACCTGCATTATGTACTACATGA 3360 CAGTGGGACTCCTTCCAAGTAACGGTAACAGTAGTGGTACCTTTGTTGCAAGGAGAGGTGGACGTAATACATGATGTACT

II II I- • - I II - I • I I- | l -

3281 ^"~ 33331133 33332277 3339 3350

3282 3289 33331166 33333322 3356

3286 3316 3286 3316 3287 3316

3317

SfaN I Hph I

I I

CAGGCATCAATCTGGGGAAATAATAAAATTATCACCTTTGTCAGACCATAAGAGTTTCTCCAAAAGTGGTCAGTTTGGCT 3440 GTCCGTAGTTAGACCCCTTTATTATTTTAATAGTGGAAACAGT ΓGGTATTCTCAAAGAGGTTTTCACCAGTCAAACCGA

I • • • j • _{• • * •}

3364 3392

Bbv II Fok I Mbo II Nla III Dde I Pie I Ssp I Tthlll II BspH I Bsu36 I Hinf I Mse I

I I II II I I II I

GGGCAATATTTTCTCTCATCTAACAAACACAATCCATTGTCATGAAATTACCCTTAGGATGAGTCTTCTTTAATCAATCA 3520 CCCGTTATAAAAGAGAGTAGATTGTTTGTGTTAGGTAACAGTACTTTAATGGGAATCCTACTCAGAAGAAATTAGTTAGT

I ^{• •} I ^• II • II I -I II I

3445 3464 3480 3492 3501 3510

3481 3493 3501

3497 3504 3503 Alu I Mbo II Fnu4H I

Alu I Ear ,ι Eco57 I Bbv I

TATATTGGGCGGAAAAAACACCAGCTTTGACCCGAAGTAGTTGAAGAGCTACTTCATTCTTTTCTGAAGTTGTGTGTTGC 3600 ATATAACCCGCCTTTTTTGTGGTCGAAACTGGGCTTCATCAACTTCTCGATGAAGTAAGAAAAGACTTCAACACACAACG

3543 3563 3584 3599 3563 3599

3567

Mse I

Mae I Dra I Mbo II i ' ' '

TGCTAGAAATAGTCATTTGTGAATTATCCAAATTGTTTAAATTCACAATTGAATTAGTTTTTTCTTCCTTTTGGCTTGAA 3680

ACGATCTTTATCAGTAAACACTTAATAGGTTTAACAAATTTAAGTGTTAACTTAATCAAAAAAGAAGGAAAACCGAACTT

3603 3636 3663 3637

HinC II Pst I Hae III Tthlll II Mse I Rsa I Hae I

GCAAACAGTTGACCATT'ITTAACCTTTTCATTTTATGTTTTTGTACTCTGCAGACTGAAAAGACAAAGTTTATCTTGGCC 3760 CGTTTGTCyUiCTGGTAAAAATTGGAAAAGTAAAATACAAAAACATGAGACGTCTGACTTTTCTGTTTCAAATAGAACCGG

3682 3699 3723 3756

3688 3728 3757

Mse I Ase I Mse I —

Rsa I Mbo II Mse I

TTACTGTATAAAGGTATGCTGTGTCCACCGTTGTGTACAGAATTTTTCTTCATTAATTTTGTGTTTAAGTTAATAAAATT 3840 AATGACATATTTCCATACGACACAGGTGGCAACACATGTCTTAAAAAGAAGTAATTAAAACACAAATTCAATTATTTTAA

3795 3807 3825

3812 3830 3813

Rsa I

I TATTTGTGATGTACTGTAA 3859 ATAAACACTACATGACATT

^• I

3851

Restriction Endonucleases site usage

Aat II - BstN I 10 HinC II 3 Pie I 4

Ace I - BstU I 1 HinD III 1 Pml I 2

Afl II - BstX I 1 Hinf I 10 PpuM I 3

Afl III - BstY I 5 HinP I 4 Pst I 3

Aha II - Bsu36 I 2 Hpa I - Pvu I -

Alu I 18 CfrlO I 1 Hpa II 3 Pvu II 2

Alw I 6 Cla I 1 Hph r 11 Rsa I 17

AlwN I 4 ^""* Dde I 16 Kpn I - Rsr II -

Apa I - Dpn I 8 Mae I- 2 Sac I 1

ApaL I 1 Dra I 5 Mae II 6 Sac II -

Ase I 3 Dra III - Mae III 11 Sal I -

Asp718 - Drd I - Mbo I 8 Sau3A I 8

Ava I - Dsa I 1 Mbo II 17 Sau96 I 13

Ava II 6 Eae I 1 Mlu I - Sea I 2

Avr II - Eag I - Mme I 2 ScrF I 11

BamH I 1 Ear I 1 Mnl I 27 Sec I 8

Ban I 2 Eco47 III 1 Mse I 1 SfaN I 9

Ban II 1 Eco57 I 7 Mse I 22 Sfi I -

Bbe I - EcoN I 2 Msp I 3 Sma I -

Bbv I 9 EcoO109 I 4 Nae I 1 SnaB I -

Bbv II 2 EcoR I - Nar I - Spe I -

Bel I 1 EcoR II 10 Nci I 1 Sph I -

Ben I 1 EcoR V - Nco I 1 Spl I 1

Bgl I - Esp I - Nde I 2 Ssp I 3

Bgl II - Fnu4H I 9 Nhe I - Stu I 1

BsaA I 3 Fok I 6 Nla III 15 Sty I 3

Bsm I 1 Fsp I 1 Nla IV 6 Taq I 3

BsmA I 8 Gdi II - Not I - Tthlll I 2

Bspl286 I 4 Gsu I 4 Nru I - Tthlll II 7

BspH I 2 Hae I 4 Nsi I - Xba I -

BspM I 3 Hae II 2 Nsp7524 I - Xea I -

BspM II 1 Hae III 12 NspEi II 2 Xho I -

Bsr I 13 Hga I 1 NspH [ I - Xcm I -

BssH II - HgiA I 4 PaeR7 I - Xma I -

BstB I - HgiE II - PflM [ I 1 Xmn I 1 Enzyme Site Use Site position (Fragment length) Fragment order

ApaL I g/tgcac 1 1( 2784) 1 2785( 1075) 2

BamH I g/gatcc 1 1( 1226) 2 1227 ( 2633) 1

Ban II grgcy/c 1 1( 2992) 1 2993( 867) 2

Bel I t/gatca 1 K 114) 2 115 ( 3745) 1

Ben I ccs/gg 1 1( 1648) 2 1649 ( 2211) 1

Bsm I gaatgc 1/-1 1 1( 526) 2 527{ 3333) 1

BspM II t/ccgga 1 1( 3223) 1 3224( 636) 2

BstU I cg/cg 1 1( 1877) 2 1878( 1982) 1

BstX I ccannnnn /ntgg 1 1( 2871) 1 2872( 988) 2

CfrlO I r/ccggy 1 1( 2032) 1 2033 ( 1827) 2

Cla I at/cgat 1 1( 1054) 2 1055( 2805) 1

Dsa I c/crygg 1 1( 3315) 1 3316( 544) 2

Eae I y/ggccr 1 1( 839) 2 840( 3020) 1

Ear I ctcttc 1/4 1 1( 3562) 1 3563{ 297) 2

Eco47 III agc/gct 1 1( 1957) 1 1958 ( 1902) 2

Fsp I tgc/gca 1 1( 2069) 1 2070( 1790) 2

Hga I gacgc 5/10 1 1( 1061) 2 1062 ( 2798) 1

HinD III a/agctt 1 1( 680) 2 681( 3179) 1

Mse I tgg/cca 1 1( 839) 2 840( 3020) 1

Nae I gcc/ggc 1 1( 2032) 1 2033 { 1827) 2

Nci I cc/sgg 1 1( 1648) 2 1649( 2211) 1

Nco I c/catgg 1 1( 3315) 1 3316( 544) 2

PflM I ceannnn/ntgg 1 1( 1928) 2 1929( 1931) 1

Sac I gagct/c 1 1( 2992) 1 2993 ( 867) 2

Spl I c/gtacg 1 K 574) 2 575( 3285) 1

Stu I agg/cct 1 1( 2555) 1 2556( 1304) 2

Xmn I gaann/nnttc 1 1( 769) 2 770( 3090) 1

Ban I g/gyrcc 2 1( 291) 3 292 ( 416) 2 708( 3152) 1

Bbv II gaagac 2/6 2 1( 3205) 1 3206( 297) 3 3503 ( 357) 2

BspH I t/catga 2 1( 3128) 1 3129( 351) 3 3480( 380) 2

Bsu36 I cc/tnagg 2 1( 3285) 1 3286 ( 206) 3 3492 ( 368) 2

EcoN I cctnn/nnnagg 2 1( 1885) 1 1886( 1400) 2 3286( 574) 3

Hae II rgcgc/y 2 1( 1957) 1 1958( 72) 3 2030( 1830) 2

Mae I c/tag 2 1( 2150) 1 2151 ( 1452) 2 3603 ( 257) 3

Mme I tccrac 20/18 2 1( 444) 3 445( 1086) 2 1531( 2329) 1

Nde I ca/tatg 2 1{ 499) 2 500( 90) 3 590( 3270) 1

NspB II cmg/ckg 2 1( 1981) 1 1982 ( 157) 3 2139( 1721) 2

Pml I cac/gtg 2 1( 1084) 2 1085( 1697) 1 2782 ( 1078) 3

Pvu II cag/ctg 2 1( 1981) 1 1982 ( 157) 3 2139( 1721) 2

Sea I agt/act 2 1( 2152) 1 2153 ( 432) 3 2585( 1275) 2

Tthlll I gacn/nngt :c 2 1( 1323) 2 1324 ( 793) 3 2117 ( 1743) 1

Ase I at/taat 3 1( 3114) 1 3115( 56) 3 3171( 641) 2 3812 ( 48) 4

BsaA I yac/gtr 3 K 551) 3 552( 533) 4 1085 ( 1697) 1 2782 ( 1078) 2

BspM I acctgc 4/8 3 K 169) 4 170( 2081) 1 2251( 1088) 2 3339( 521) 3

HinC II gty/rac_^ 3 1( 1610) 1 1611 ( 533) 3 2144 ( 1544) 2 3688( 172) 4

Hpa II c/cgg 3 1( 1649) 1 1650( 384) 4 2034{ 1191) 2 3225 ( 635) 3

Msp I c/cgg 3 1( 1649> 1 1650( 384) 4 2034( 1191) 2 3225( 635) 3

PpuM I rg/gwecy 3 1( 1200) 2 1201( 166) 4 1367( 881) 3 2248( 1612) 1

Pst I ctgca/g 3 1( 1408) 2 1409( 161) 3 1570( 2158) 1 3728 ( 132) 4

Ssp I aat/att 3 1( 1665) 2 1666 ( 84) 4 1750( 1695) 1 3445 ( 415) 3

Sty I c/cwwgg 3 1( 1247) 2 1248 ( 686) 3 1934 ( 1382) 1 3316( 544) 4

Taq I t/cga 3 1( 1055) 2 1056( 9) 4 1065( 2132) 1 3197( 663) 3

AlwN I cagnnn/ctg 4 1( 1045) 2 1046( 81) 5 1127 ( 947) 3 2074 ( 255) 4

2329( 1531) 1

Bspl286 I gdgch/c 4 K 49) 5 50( 2735) 1 2785 ( 208) 3 2993 ( 166) 4 3159( 701) 2

EcoC-109 I rg/gnecy 4 1( 1200) 1 1201( 166) 5 1367 ( 881) 3 2248( 694) 4 2942 ( 918) 2

Gsu I ctggag 16/14 4 1( 1889) 1 1890( 9) 5 1899( 43) 4 1942 ( 97) 3 2039( 1821) 2

Hae I wgg/ccw 4 1( 839) 3 840( 638) 4 1478( 1078) 2 2556( 1200) 1 3756( 104) 5

HgiA I gwgew/c 4 K 49) 5 50( 2735) 1 2785( 208) 3 2993 ( 166) 4 3159( 701) 2

Hha I gcg/c 4 1( 1958) 1 1959( 18) 5 1977 ( 54) 3 2031( 40) 4 2071 ( 1789) 2

HinP I g/cgc 4 1( 1958) 1 1959( 18) 5 1977 ( 54) 3 2031 ( 40) 4 2071( 1789) 2

Pie I ^• gagtc 4/5 4 1( 281) 5 282( 1321) 2 1603 ( 544) 3 2147( 1354) 1 3501( 359) 4

BstY I r/gatcy 5 1( 188) 5 189( 801) 2 990( 237) 4 1227 ( 668) 3 1895( 109) 6 2004( 1856) 1 Dra I ttt/aaa 5 1( 356) 3 357( 2094) 1 24511 54) 6 25051 128) 5

2633 ( 1003) 2 36361 224) 4

Alw I ggatc 4/5 6 K 188) 5 1891 802) 2 9911 236) 4 12271 1) 7

1228 ( 667) 3 18951 110) 6 20051 1855) 1

Ava II g/gwcc 6 1( 1201) 2 12021 166) 6 13681 178) 5 15461 365) 3

1911 ( 205) 4 21161 133) 7 22491 1611) 1

Fok I ggatg 9/13 6 1( 925) 2 9261 238) 6 11641 302) 5 14661 226) 7

1692( 1190) 1 28821 615) 3 34971 363) 4

Mae II a/cgt 6 1( 552) 2 553( 533) 4 10861 1035) 1 21211 517) 6

2638 ( 145) 7 2783 ( 544) 3 33271 533) 5

Nla IV ggn/ncc 6 1( 240) 4 2411 51) 6 2921 416) 2 7081 386) 3

1094 ( 107) 5 12011 26) 7 12271 2633) 1

Eco57 I ctgaag 16/14 7 K 75) 7 761 544) 4 6201 64) 8 6841 554) 3

1238 ( 111) 6 13491 913) 2 22621 1322) 1 35841 276) 5

Tthlll II caarca 11/9 7 K 126) 7 1271 381) 4 5081 471) 2 9791 453) 3

1432 ( 109) 8 15411 1923) 1 34641 218) 5 36821 178) 6

BsmA I gtctc 1/5 8 1( 638) 3 639{ 529) 4 11681 852) 1 20201 311) 6

2331 ( 67) 9 23981 128) 8 25261 133) 7 26591 480) 5

3139 ( 721) 2

Dpn I ga/tc 8 1( 115) 4 1161 74) 8 1901 801) 2 991( 63) 9

1054 ( 81) 7 11351 93) 6 12281 668) 3 1896( 109) 5

2005 ( 1855) 1

Mbo I /gate 8 K 115) 4 1161 74) 8 190( 801) 2 9911 63) 9

1054 ( 81) 7 11351 93) 6 12281 668) 3 1896( 109) 5

2005 ( 1855) 1

Sau3A I /gate 8 1( 115) 4 1161 74) 8 1901 801) 2 9911 63) 9

1054 ( 81) 7 11351 93) 6 12281 668) 3 18961 109) 5

2005 ( 1855) 1

Sec I c/cnngg 8 K 842) 2 8431 405) 5 12481 686) 3 19341 62) ^• 8

1996 ( 65) 7 20611 345) 6 24061 1) 9 24071 909) 1

3316 ( 544) 4

Bbv I gcagc 8/12 9 1( 319) 6 3201 176) 8 496( 406) 5 902( 506) 3

1408 ( 3)10 14111 158) 9 15691 415) 4 19841 794) 2

2778 ( 821) 1 35991 261) 7

Fnu4H I gc/ngc 9 1( 319) 6 3201 176) 8 4961 406) 5 9021 506) 3

1408 ( 3)10 14111 158) 9 15691 415) 4 19841 794) 2

2778 ( 821) 1 35991 261) 7

SfaN I gcatc 5/9 9 K 610) 3 6111 115) 9 726( 242) 9681 913) 1

1881 ( 144) 8 20251 43)10 20681 859) 29271 289) 5

3216 ( 148) 7 33641 496) 4

BstN I cc/wgg 10 K 564) 3 5651 84) 8 6491 51)10 700( 143) 7

843 ( 722) 2 15651 42)11 16071 389) 4 1996( 66) 9

2062 ( 345) 6 24071 351) 5 27581 1102) 1

EcoR II /ccwgg 10 1( 564) 3 5651 84) 8 649( 51)10 700( 143) 7

843 ( 722) 2 15651 42)11 16071 389) 4 19961 66) 9

2062 ( 345) 6 24071 351) 5 27581 1102) 1

Hinf I g/antc 10 K 228) 7 2291 53) 9 282( 760) 2 10421 6)11

1048 ( 81) 8 11291 474) 4 16031 544) 3 2147( 14)10

2161 ( 972) 1 31331 368) 5 35011 359) 6

Hph I ggtga 8/7 11 K 413) 4 4141 232) 6 6461 228) 7 8741 97) 9

971 ( 146) 8 11171 948) 1 20651 41)11 21061 383) 5

2489 ( 793) 2 32821 31)12 33131 79)10 33921 468) 3

Mae III /gtnac 11 1( 85)10 861 168) 9 2541 379) 3 633( 12)12

645 ( 340) 5 9851 337) 6 13221 353) 4 16751 278) 7

1953 ( 39)11 19921 1050) 1 30421 239) 8 32811 579) 2

ScrF I cc/ngg 11 1( 564) 3 5651 84) 8 6491 51)10 7001 143) 7

843 ( 722) 2 15651 42)11 16071 42)12 16491 347) 5

1996 ( 66) 9 20621 345) 6 24071 351) 4 27581 1102) 1

Hae III gg/cc 12 1( 240) 8 2411 600) 2 8411 255) 7 10961 383) 5

1479 ( 102)10 15811 39)12 16201 300) 6 19201 94)11

2014 ( 22)13 20361 521) 3 25571 387) 4 29441 813) 1

3757 ( 103) 9

Bsr I actgg 1/-1 13 1( 139)10 140( 96)11 2361 181) 7 417( 416) 2

833 ( 365) 3 11981 220) 5 14181 23)13 14411 22)14

1463( 154) 8 16171 324) 4 19411 76)12 20171 148) 9

2165( 191) 6 23561 : 1504) 1

Sau96 I g/gncc 13 11 240) 5 2411 854) 2 10951 107) 9 12021 166) 7

13681 178) 6 15461 34)13 15801 40)12 16201 291) 4

1911 ( 9)14 19201 94)11 20141 102)10 21161 133) 8

22491 694) 3 29431 917) 1

Nla III catg/ 15 11 46)12 471 232) 6 2791 1638) 1 19171 13)16

1930( 250) 5 21801 393) 2 25731 94)10 26671 105) 9

2772 ( 24)15 27961 83)11 28791 251) 4 31301 26)14

31561 161) 7 33171 39)13 33561 125) 8 34811 379) 3 Dde I c/tnag 16 11 40)16 411 173) 9 214( 155)11 3691 272) 5

6411 26)17 667( 644) 1 13111 61)15 13721 181) 8

15531 132)12 16851 362) 4 20471 610) 2 26571 156)10

28131 126)13 29391 223) 6 31621 125)14 32871 206) 7

34931 367) 3

Mbo II gaaga 8/7 17 11 94)11 95( 1005) 1 11001 164) 6 12641 51)15

13151 33)17 13481 466) 3 18141 38)16 18521 3)18

18551 387) 4 22421 693) 2 29351 141) 8 30761 130) 9

32061 68)12 32741 230) 5 35041 59)13 35631 100)10

36631 144) 7 38071 53)14

Rsa I gt/ac 17 11 554) 2 5551 21)15 576( 113)11 6891 163) 8

8521 153)10 10051 442) 4 14471 500) 3 19471 207) 7

21541 265) 6 24191 162) 9 25811 5)18 25861 63)13

26491 21)16 26701 680) 1 33501 373) 5 37231 72)12

37951 56)14 38511 9)17

Alu I ag/ct 18 11 67)16 68( 265) 5 3331 204) 6 537( 133)13

6701 12)19 6821 36)17 7181 153)11 8711 76)15

9471 604) 1 15511 137)12 16881 95)14 1783 ( 200) 7

19831 157) 9 21401 156)10 22961 174) 8 24701 524) 3

29941 549) 2 35431 24)18 35671 293) 4

Mse I t/taa 22 11 19)20 201 43)18 631 239) 6 3021 56)14

358{ 442) 2 8001 237) 7 10371 267) 5 13041 915) 1

22191 233) 8 24521 54)16 25061 46)17 25521 67)12

26191 15)21 26341 338) 3 29721 144)-9 31161 56)15

31721 338) 4 35101 127)10 36371 62)13 36991 114)11

38131 12)22 38251 5)23 38301 30)19

Mnl I cctc 7/7 27 11 25)23 26( 135)10 1611 321) 2 4821 105)14

5871 107)13 6941 97)16 7911 276) 6 10671 53)19

11201 104)15 12241 311) 4 15351 22)24 15571 21)25

15781 315) 3 18931 21)26 19141 124)11 20381 55)18

2093 ( 21)27 21141 248) 7 23621 117)12 24791 44)20

25231 36)22 25591 6)28 25651 210) 8 27751 166) 9

29411 66)17 30071 282) 5 32891 43)21 33321 528) 1

478 sites found

No Sites found for the following Restriction Endonucleases

Aat II gacgt/c Drd I gacnnnn/nng c NspH I rcatg/y Ace I gt/mkac Eag I c/ggccg PaeR7 I c/tcgag Afl II c/ttaag EcoR I g/aattc Pvu I cgat/cg

Afl III a/crygt EcoR V gat/atc Rsr II cg/gwccg

Aha II gr/cgyc Esp I gc/tnagc Sac II ccgc/gg

Apa I gggcc/c Gdi II yggccg -5/-1 Sal I g/tcgac

Asp718 g/gtacc HgiE II accnnnnnnggt Sfi I ggccnnnn/nggcc

Ava I c/ycgrg Hpa I gtt/aac Sma I ccc/ggg

Avr II c/ctagg"^* Kpn ggtac/c SnaB I tac/gta

Bbe I ggcgc/c Mlu a/cgcgt Spe I a/ctagt

Bgl I gccnnnn/nggc Nar gg/cgcc Sph gcatg/c

Bgl II a/gatct Nhe g/ctagc Xba t/ctaga

BssH II g/cgcgc Not gc/ggccgc Xca gta/tac

BstE 1 I tt/cgaa Nru tcg/cga Xho c/tcgag

BstE II g/gtnacc Nsi atgca/t Xcm ccannnnn/nnnntgg

Dra III cacnnn/gtg Nsp7524 I r/catgy Xma c/ccggg ANNEX 6

DNA sequence 1649 b.p. GGAAATGGTAGA ... TCCACCTGCCAT linear

Positions of Restriction Endonucleases sites (unique sites underlined)

Sau3A I Mbo I

Dpn I Mae I Mse I

See I Hph I Mnl I Eco57 I Hga I

II I I I I I I

GGAAATGGTAC-AACTAGT^TCTCΑCCC-AGCCT 80

CCI ΓACCATCTΓGATCACTAGAGT^^

• II I- I -I • • - I I- I

13 23 31 62 80 14 69

19 19 19

Sau3A I

Mbo I

Dde Dpn I

Dde I Sau96 I Mbo II BstY I

Fnu4H I Pie I Hph I Nla IV Ear I EsL.Il Bbv I Hinf I Mae III SfaN I Hae III Alu I Gsu I

ACG 1CTGCTGTGTGG 1ACICT1X_AG1TGACA^ 1 1 1 1 I II I II 160

TGCGACGACACACCTGAGACTCACTGTCIX^^

I - l l - l - I I - I ^• I ^• I II I II-

83 94 102 114 127 147 154

83 94 117 127 149 158

98 127 150 158

134 159

159 159 Sau3A I Fnu4H I Mbo I

Nla III Dpn I

Stv I Alw I

Mae II Sec I Hga I Nla IV

Aϋ_HI Nco I Hae III BstY I

Pml I Mnl I Dsa I Gdi II BamiLI.

BsaA I BstX I Bbv I Eae I Rsa I Alw I

II I I II I II I I II

TCTTGCCCAOSTGTCCrrcCCCCK 240

AGAACGGGTO-ACAGGAGGGGGCGGTCGGAAGG^

II- I - I II- -I -II I ^• I ^• II

168 192 203 211 223 234

168 175 1 19988 2 21111 234

169 1 19988 2 21i2: 234

169 1 19988 217 234

198 235

199 235

203 235 235

Alu I

Gsu I Xmn_ι Mbo II Alu I M Mnnll II H Hiinnff II SfaN I Bbv II

I II I I I I

GCAOCTACCCCCATTACAGCTTACC^ 20

CGTCCGATGGGGGTAATGTCGAATt-OSTGGAGGTC^

I- II ^• I • - l - l l

259 269 288 303 314

270 288 314

320 Hae III Mse I Hae I ScrF I EcoR II BstN I Dde I Alu I Sec I Mnl I Nla TV Eco57 I Hph Bspl286 I BstX I Eae I Hinf I Alu I Gsu I Mnl I Bsr I Ban II

GCTTGAACCATTCCCCΌGCCAGAGTCGAT^ 400

CGAACTTGGTAAGGGGACCGGTCTCACCTAAGGAGTCGA^

328 337 348 356 364 378 386 394

334 352 362 370 380 394

335 353 368

335

337

338

Msp I

Hpa II

CfrlO I HgiA I

Fnu4H I Bspl286 I

SfaN I Mnl I Bsr I Bbv I ApaL I

TACACCTACCAGATGCACGGCAC-AA(AGGGTTC^^ 480

ATGTGGATGGTCTACGTGCCGTCTTGTCCCAAGATA^

412 441 451 461 479

461 479

464 479

465

Msp I

Hpa II

ScrF I

ScrF I Nci I

EcoR II Ben I Rsa I

BstN I Sec I Bsr I Bsr Fok I Mnl I

GCACCAGGACΓATCCTTCCTACCCCGGCTTCCCCCAGAGCCAGTACCCCCAGTATO 560 CGTGGTCCΓGATAGGAAGGATGGGGCCGAAGGGGGTCTCGGTC^

484 502 520 529 543 554

484 503 523

484 503

503

" "* 504

504

Hae III

Gdi II

Eae I ScrF I

Msp I EcoR II

Hpa II BstN I

ScrF I SfaN I Gsu I

Nci I AlwN I Mnl I

Ben I Fnu4H I ScoN I SfaN I

Mae II Bbv I Mnl I Mae II Mae II Mnl I Mae II

ACGTCCCGGCCAGCΛGCATCTGCCCTTCGCCCCTC CCACG^ 640

TGC-AGGGCCGGTCGTCGTAGACGGGAAGCGGGGAGAGGTGCAGGT^

561 573 592 599 609 620 633

565 573 613 623

565 574 613

565 576 614

566 616

567 616

567

568 Mae III Pie I Sau96 I BsmA I

Hinf I Hph I Ava II Mnl I

AACCAGAGT-TCCG IAGITCACTTGCTOI STGAATACAAC I I I 720

TTGGTCTCAAGGCIK^GTGAACGACCACITO

• l l - l - ^• I • • I - I

653 665 686 707

653 686 711

655

Mnl I

ScrF I

Nci I Msp I Msp I

Msp I Hpa II Hpa II

Hpa II CfrlO I ScrF I

Ben I Mnl I Nci I

Fnu4H I Hae III Sec I Dde I Ben I

[ae III Sau9β I Alu I Hae III Mnl Mae III Sec I

CAGGCCG<^CCGG<_CCTCCGATGGGAAGCTCCGAGGCCGGTCTAAGAGGAG 800

GTCCG XX?rcGCCCGGAGGCTACCCrrcCGAGGCTC

I I I I I I • I -I I I I I • I I • I - I I I

723 732 747 755 766 774 783

724 733 751 762 784

730 753 784

730 756 784

730 757 785

730

735

BsmA I Fok I

Fok I SfaN I 1 1 I I AGATTGAGCGTGTGTTCGTGTGGGACTTGGATGA^^ 880

TCTAACTCGCACA(^ΛG aCΑCCCI\3AA^ j . I . . . I I

829 875

833 876

Mbo II

Mae II HinP I Sau3A I Aha II Hha I Hae III Mbo II Mbo I Aat II Fsp I Hae I Ear I Dpn I

AGATACGGGAAGGACACCAOSIAICGTCCGTIGICGCATTI^I I I I 960

TCTATG^CCTTCCΓGTX-GTGCΓGCAGG^^

^• 9I0I1 9 M09 9M16 - 9 I2-9 9 I37 I-

901 910 917 929 937

902 910 937

939

Sau3A I

Mnl I Mbo I

Gsu I Dpn I BsmA I Hae III

ScrF I Alw I Mae II Mse I

1040

AGACAAGAAσiτACIGGACCTCCTAACACTGGTCTAGσiX_CAA

9 ¹66 ^* 9 9 H7766 I 9 9 l8877- l l 9 9 I99'99 1 1 I00I0077 I ^• 1 I0I2;7 1 I037 ^•

976 993 1007 1027

976 994 1008 1027

977 994 1010 1028

980 994 994 Nla IV

ScrF I

EcoR II Bspl286 I

BstN I Ban II

Sec I Sec I Fnu4H I

Sau96 I ScrF I HgiA I

Nla IV EcoR II Bspl286 I

US2E-II Hae III Xcjn_I BstN I ApaL I

G ^(^TACAACITCTC ICGCr3ACGGCITCCACAGTrrC I II I I I I I I 1120

CGTGTATGTTGAAG-GGCGACTGCCGAAGG G C-AAGCCGGGGTC

1056 1078 1089 1099 1112

1078 1099 1112

1081 1099 1119

1082 1102

1082

1085

Hph I

Hae III Msp I

Fok I Alu I Hpa II

Bsr I Mnl I Hae I CfrlO I Rsa I Mai

Q3CσiX-<aCTGGATGAG<-AAGClX-GCC K:CGrrACCGGCG 1200

CαSCACCTGACCTACTCCTTCGACOXSAAG^

I -I I I II - ll - l - I • - I

1128 1135 1143 1155 11 1194

1131 1140 1156

1144 1156

1162

ScrF I Alu I

EcoR II Dde I

Hae III

Msp I

Hpa II

* "* ScrF I Hph I Nci I Mae III

Eco57 I Mnl I Ben I 2≤££_H

I I II I III

CCCACTCCCTGAAGGCACTAAAO_TC^TC^ 1360

GGGTGAGGGACTTCOπ'GATTTGGAGTAGTTGAGGGCα^^

I- ^• I ^• II I ^{• •} III

1289 1303 1314 1339

1314 1340

1314 1341

1315 1315 1317 1317 Hae III Mse I Hae I Sec I Eae I ScrF I ScrF I EcoR II EcoR II BstN I BstN I Hae III Sec I Hae I

CCTGCCCTGGCCAAAGTCCTGCTATATXSGCCTGGGGTCTGTGT^ 1440

GGACGGGACCGGTTTCAGGACGATATACCGGACCCV^GACACAAAGGATAACTCTTG^

1365 1387 1366 1388 1366 1390 1366 1390 1368 1390 1368 1390 1368 1369

Sau3A I

Mbo I

Fnu4H Mae II Mnl Bbv I Mnl Ace I Hph I Hph I Mbo II Alu I Taσ .I Hinf I Alu I BsaA I Dpn I Ear I

GAAGGAGAGCrarrTCGAGAGGATAATGCAGAGAT^ 1520

CΓTCCTCTCGAOSAAGCΓCΓCCTATTACGTCT^

1448 1455 1473 1486 1493 1501 1517 1449 1459 1490 1498 1505 1517

1449 1494 1520

1501

Mnl I

Bαl I Gsu I ScrF I Nla III ScrF I EcoR II SEH_I EcoR II Mnl I BstN I Nsp7524 I ECOR V V BstN I Dde I Sec I

AGGAGCAAGGAGCGAAAAAGCACAACATGCCTTTCTGGCG^ 1600 TCCTCGTTCCTO-CTTTTTCGTGTTGT^^

1545 1561 1579 1589 1599 1545 1579 1591 1600 1546 1579 1600 1549 1580 1600

1583

Fnu4H I Bbv I Sau3A I Mbo I

Gsu I Dpn I Bsr I Alw I SfaN I BSPM I

CTGGAACTG<AGTATTTATAGC-AGGATCAGCAGCA^ 1649

GACCΓTGACCTCATAAATATCGTCCTAGTCGTCGTAGAGGTGGACGGTA

1606 1624 1633 1641 1607 1625 1625 1625

1630 1630

Restriction Endonucleases site usage

Aat II 2 BstN I 11 HinC II - Pie I

Ace I 1 BstU I - HinD III - Pml I

Afl II - BstX I 2 Hinf I 5 PpuM I

Afl III 1 BstY I 3 HinP I 1 Pst I

Aha II 2 Bsu36 I - Hpa I - Pvu I

Alu I 13 CfrlO I . 3 Hpa II 8 Pvu II

Alw I 4 Cla I - Hph I 8 Rsa I

AlwN I 1 Dde I 6 Kpn I - Rsr II

Apa I - Dpn I 7 Mae I 1 Sac I ApaL I Dra I Mae II 10 Sac II Ase I Dra III Mae III 5 Sal I Asp718 Drd I Mbo I 7 Sau3A I 7 Ava I Dsa I Mbo II 6 Sau96 I 5 Ava II 1 Eae I Mlu I Sea I Avr II Eag I Mme I ScrF I 16 BamH I 1 Ear I Mnl I 22 Sec I 10 Ban I Eco47 III Msc I 3 SfaN I 7 Ban II 2 Eco57 I Mse I 2 Sfi I Bbe I EcoN I Msp I 8 Sma I Bbv I 6 EcoO109 Nae I SnaB I Bbv II 1 EcoR I Nar I Spe I 1 Bel I EcoR II 11 Nci I 5 Sph I Ben I 5 EcoR V 1 Nco I 1 Spl I Bgl I 1 Esp I 1 Nde I Ssp I Bgl II 1 Fnu4H I 8 Nhe I Stu I BsaA I 2 Fok I 4 Nla III 2 Sty I 1 Bsm I Fsp I 1 Nla IV 5 Taq I 1 BsmA I 4 Gdi II 2 Not I Tthlll I Bspl286 4 Gsu I 7 Nru I Tthlll II BspH I Hae I 6 Nsi I Xba I BspM I 1 Hae II Nsp7524 I 1 Xca I BspM II Hae III 14 NspB II 1 Xho I Bsr I 6 Hga I 2 NspH I 1 Xcm I 1 BssH II HgiA I 2 PaeR7 I Xma I BstB I HgiE II PflM I Xmn I 1 BstE II Hha I

Enzyme S te Use Site position (Fragment length) Fragment order

Ace I gt/mkac 1 1 ( 1489) 1 1490 ( 160) 2

Afl III a/crygt 1 1 ( 168) 2 169 ( 1481) 1

AlwN I cagnnn/ctg 1 1 ( 573) 2 574 ( 1076) 1

Ava II g/gwcc 1 1 ( 685) 2 686 { 964) 1

BamH I g/gatcc 1 1 ( 233) 2 234 ( 1416) 1

Bbv II gaagac 2/6 1 1 ( 313) 2 314 ( 1336) 1

Bgl I gccnnnn/nggc 1 1 1548) 1 1549 ( 101) 2

Bgl II a/gatct 1 1 ( 157) 2 158 ( 1492) 1

BspM I acctgc 4/8 1 1 ( 1640) 1 1641 { 9) 2

BstE II g/gtnacc 1 1 ( 1338) 1 1339 ( 311) 2

Dsa I c/crygg 1 1 [ 197) 2 198 ( 1452) 1

EcoN I cctnn/nnnagg 1 1 612) 2 613 ( 1037) 1

EcoR V gat/ te 1 1 1560) 1 1561 ( 89) 2

Esp I gc/tnagc 1 1 1246) 1 1247 ( 403) 2

Fsp I tgc/gca 1 1 908) 1 909 ( 741) 2

Hha I gcg/c 1 1 909) 1 910 ( 740) 2

HinP I g/cgc 1 1 909) 1 910 ( 740) 2

Mae I c/tag 1 1 13) 2 14 ( 1636) 1

Nco I c/catgg 1 1 197) 2 198 ( 1452) 1

Nsp7524 I r/catgy 1 1 1544) 1 1545 ( 105) 2

NspB II cmg/ckg 1 1 1055) 1 1056 ( 594) 2

NspH I rcatg/y 1 1 1544) 1 1545 ( 105) 2

Pral I cac/gtg 1 1 167) 2 168 ( 1482) 1

Spe I a/ctagt 1 1 12) 2 13 ( 1637) 1

Sty I c/cwwgg 1 1 197) 2 198 ( 1452) 1

Tag I t/cga 1 1 1454) 1 1455 ( 195) 2

Xcm I ccannnnn/nnnntgg 1 1 1088) 1 1089 ( 561) 2

Xmn I gaann/nnttc 1 1 287) 2 288 ( 1362) 1

Aat II gacgt/c 2 1 900) 1 901 ( 106) 3 1007 ( 643) 2

Aha II gr/cgyc 2 1 900) 1 901 ( 106) 3 1007 ( 643) 2

ApaL I g/tgcac 2 1 478) 3 479 ( 633) 1 1112 ( 538) 2

Ban II grgcy/c 2 1 393) 3 394 ( 708) 1 1102 ( 548) 2

BsaA I yac/gtr 2 1 167) 2 168 ( 1325) 1 1493 ( 157) 3

BstX I ccannnnn/ntgg 2 1 191) 2 192 ( 136) 3 328 ( 1322) 1

Gdi II yggccg -5/-1 2 1 210) 3 211 ( 356) 2 567 ( 1083) 1

Hga I gacgc 5/10 2 1 79) 3 80( 137) 2 217 ( 1433) 1

HgiA I gwgcw/c 2 1 478) 3 479 ( 633) 1 1112 ( 538) 2

Mse I t/taa 2 1 68) 3 69 ( 968) 1 1037 ( 613) 2

Nla III catg/ 2 1 198) 2 199 ( 1347) 1 1546 ( 104) 3

Pie I gagtc 4/5 2 . 1 93) 3 94 ( 559) 2 653 ( 997) 1

BstY I r/gatcy 3 1 157) 3 158 ( 76) 4 234 ( 759) 1 993 ( 657) 2

CfrlO I r/ccggy 3 1( 463) 2 464 ( 292) 4 756 ( 399) 3 1155 ( 495) 1 Ear I ctcttc 1/4 3 1 148) 3 149 780) 1 929 ( 588) 2 1517 { 133) 4 Eco57 I ctgaag 16/14 3 1 61) 4 62 308) 3 370 ( 919) 1 1289( 361) 2 Mse I tgg/cca 3 1 336) 3 337 690) 1 1027 ( 341) 2 1368 ( 282) 4 Rsa I gt/ac 3 1 222) 4 223 300) 3 523 ( 651) 1 1174{ 476) 2

Alw I ggatc 4/5 4 1 233) 234 1) 5 235( 759) 1 994 ( 630) 2 1624 26)

BsmA I gtctc 1/5 4 1 710) 711 122) 5 833 ( 177) 4 1010 ( 216) 3 1226 424) Bspl286 I gdgch/c 4 1 393) 394 85) 4 479 ( 623) 1 1102 ( 10) 5 1112 538)

Fok I ggatg 9/13 4 1 542) 543 286) 3 829 ( 47) 5 876( 255) 4 1131 519)

Ben I ccs/gg 5 1 502) 503 62) 565 ( 165) 4 730 ( 54) 6

784 530) 1314 336)

Eae I y/ggccr 5 1 210) 211 126) 337( 230) 4 567 ( 460) 1

1027 341) 1368 282)

Hinf I g/antc 5 1 93) 94 194) 288 ( 60) 6 348 ( 305) 2

653 820) 1473 177)

Mae III /gtnac 5 1 101) 102 553) 655 ( 119)_5 774 ( 213) 4

987 353) 1340 310)

Nci I cc/sgg 5 1 502) 503 62) 565 ( 165) 4 730 ( 54) 6

784 530) 1314 336)

Nla IV ggn/ncc 5 1 126) 127 107) 234 ( 128) 3 362 ( 716) 1

1078 7) 1085 565)

Sau96 I g/gncc 5 1 126) 127 559) 686 ( 46) 6 732 ( 346) 2

1078 239) 1317 333)

Bbv I gcagc 8/12 1 82) 83 120) 203 ( 258) 461( 112) 5 573 876) 1449 181) 1630 ( 20)

Bsr I actgg 1/-1 1 385) 386 65) 451 ( 69) 520( 9) 7 529 599) 1128 478) 1606 ( 44)

Dde I c/tnag 1 97) 98 36) 134 ( 219) 353 ( 409) 2 762 486) 1248 341) 1589 ( 61)

Hae I wgg/ccw 1 336) 337 579) 916 ( 111) 1027 ( 116) 5 1143 225) 1368 19) 1387 ( 263)

Mbo II gaaga 8/7 1 149) 150 164) 314 ( 615) 929 ( 10) 7 939 27) 966 551) 1517 { 133)

Dpn I ga/tc 7 1 18) 8 19 140) 159 ( 76) 5 235 ( 702) 937 57) 6 994 507) 1501 ( 124) 4 1625 ( 25)

Gsu I ctggag 16/14 7 1 153 ) 4 154 116) 270 ( 94) 6 364 ( 250) 614 363 ) 2 977 603) 1580 ( 27 ) 8 1607 ( 43)

Mbo I /gate 7 1 18 ) 8 19 140) 159 ( 76) 5 235 ( 702) 937 57 ) 6 994 507) 1501 ( 124) 4 1625 ( 25)

Sau3A I /gate 7 1 18 ) 8 19 140) 159 ( 76) 5 235 ( 702) 937 57) 6 994 507) 1501 ( 124) 4 1625 ( 25)

SfaN I gcatc -~ 5/9 7 1 113 ) 5 114 189) 303 ( 109) 6 412 ( 164) 576 47-) 7 623 252) 875 ( 758) 1 1633 ( 17)

Fnu4H I gc/ngc 1 82) 8 83 120) 203 ( 258) 461 ( 112) 7

573 151) 724 395) 1119 ( 330) 1449 ( 181) 4

1630 20)

Hpa II c/cgg 1 464) 465 39) 7 504 ( 62) 566 ( 164)

730 27) 757 28) 8 785 ( 371) 1156 ( 159)

1315 335)

Hph I ggtga 8/7 8 1 22) 23 94) 117 ( 263) 380( 285)

665 497) 1162 179) 1341 ( 157) 1498 ( 7)

1505 145)

Msp I c/cgg 1 464) 465 39) 7 504 ( 62) 566( 164)

730 27) 757 28) 8 785 ( 371) 1156 ( 159)

1315 335)

Mae II a/cgt 10 1 168) 169 392) 561( 38 ) 8 599 ( 10)10

609 24) 633 269) 902 ( 97 ) 7 999 ( 9)11

1008 186) 1194 300) 1494 ( 156) 6

Sec I c/cnngg 10 1 197) 198 136) 334( 168) 6 502 ( 249)

751 32) 783 298) 1081 ( 18 ) 11 1099 ( 266)

1365 25)10 1390 209) 1599 ( 51) 8

BstN I cc/wgg 11 1 334) 2 335 149) 4 484 ( 132 ) 6 616 ( 360)

976 106) 8 1082 17)12 1099 ( 131 ) 7 1230 ( 136)

1366 24)10 1390 189) 3 1579 ( 21 ) 11 1600 ( 50)

EcoR II /ccwgg 11 1 334) 2 335 149) 4 484 { 132 ) 6 616 ( 360)

976 106) 8 1082 17)12 1099 ( 131 ) 7 1230 ( 136)

1366 24)10 1390 189) 3 1579 ( 21 ) 11 1600 ( 50) Alu I ag/ct 13 1( 146) 5 147 ( 112) 6 259 ( 61) 8 320 ( 36)10

356( 12)11 368 ( 379) 2 747 ( 393) 1 1140 ( 99) 7

1239 ( 7)12 1246 ( 5)14 1251 ( 7)13 1258 ( 190) 3

1448 ( 38) 9 1486 ( 164) 4

Hae III gg/cc 14 1( 126) 6 127 ( 85) 9 212 ( 126) 7 338 ( 230) 2

568 ( 155) 5 723 ( 10)15 733 ( 22)13 755 ( 162) 4

917 ( 111) 8 1028 ( 50)12 1078 ( 66)10 1144 ( 173) 3

1317 ( 52)11 1369 ( 19)14 1388 ( 262) 1

ScrF I cc/ngg 16 K 334) 1 335( 149) 4 484( 19)16 503 ( 62) 9

565 ( 51)12 616 ( 114) 6 730( 54)10 784 ( 192) 2

976 ( 106) 7 1082 ( 17)17 1099 ( 131) 5 1230 ( 84) 8

1314 ( 52)11 1366 ( 24)14 1390 ( 189) 3 1579 ( 21)15

1600 ( 50)13

Mnl I cctc 7/7 22 K 30)16 31 ( 144) 4 175 ( 94) 7 269 ( 83) 9

352 ( 26)18 378 ( 63)10 441 ( 113) 6 554 ( 38)14

592 ( 21)19 613 ( 7)23 620 ( 87) 8 707 ( 28)17

735 ( 18)20 753 ( 13)21 766 ( 214) 1 980 ( 155) 3

1135 ( 135) 5 1270 ( 33)15 1303 ( 156) 2 1459 ( 61)12

1520 ( 63)11 1583 ( 8)22 1591 ( 59)13

325 sites found

No Sites found for the following Restriction Endonucleases

Afl II c/ttaag Eco47 III age/get Pst I ctgca/g

Apa I gggcc/c ECOO109 I rg/gnccy Pvu I cgat/cg

Ase I at/taat EcoR I g/aattc Pvu II cag/ctg

Asp718 g/gtacc Hae II rgcgc/y Rsr II cg/gwccg

Ava I c/ycgrg HgiE II accnnnnnnggt Sac I gagct/c

Avr II c/ctagg HinC II gty/rac Sac II ccgc/gg

Ban I g/gyrcc HinD III a/agctt Sal I g/tcgac

Bbe I ggcgc/c Hpa I gtt/aac Sea I agt/act

Bel I t/gatca Kpn I ggtac/c Sfi I ggccnnnn/nggcc

Bsm I gaatgc 1/-1 Mlu I a/cgcgt Sma I ccc/ggg

BspH I t/catga Mme I tccrac 20/18 SnaB I tac/gta

BspM II t/ccgga Nae I gcc/ggc Sph I gcatg/c

BssH II g/cgcgc Nar I gg/cgcc Spl I c/gtacg

BstB I tt/cgaa Nde I ca/tatg Ssp I aat/att

BstU I cg/cg Nhe I g/ctagc Stu I agg/cct

Bsu36 I cc/tnagg Not I gc/ggccgc Tthlll I gacn/nngtc

Cla I at/cgat Nru I tcg/cga Tthlll II caarca 11/9

Dra I ttt/aaa_^ Nsi I atgca/t Xba I t/ctaga

Dra III cacnnn/gtg PaeR7 I c/tcgag Xca I gta/ ae

Drd I gacnnnn/nngtc PflM I ccaπnnn/ntgg Xho I c/tcgag

Eag I c/ggccg PpuM I rg/gwccy Xma I c/ccggg

ANNEX 7

DNA sequence 1813 b.p. ACTCTAGAAGGA ... AATTGCAGCCAA linear

Positions of Restriction Endonucleases sites (unique sites underlined)

2di_H

Eaσ I

Eae I

BsmA I Mae III

Dde I Bsr I

CCTGTTgKX.GKTriTACArc I I I I 160

GGACAACACCCCGAAAATG"rCGGAAACCTAACA^

. I I . | |

122 140

128 143

Nla III

Dpn I Bel I Nla III

AGCAAACTATAAGTCAAGTAAGCAATCCAιGATX-T^^ II I 320

TCG ITGATATTCAGTTC^TTCGTTAGGTCTACAGT^

• • • 11 * * * *

278 320

279

Nla III

NspH I Mnl I

Mnl I N Nsspp77552244 II F Fookk II Fok I Mnl I

ATGTCAGAGGAAATTATGACATGC-ACCGAT^^ I I 400

TA ΛiGTCTCCTTTAATACTGTACGTGGCTAATGTAGGGAG^

I . | | . I I . I | .

327 3 33399 3 35533 364 379

339 357

340 ScrF I EcoR II AlwN I BstN I Pie I Pie I

Dde I Sec I Hinf I Hinf I

ACCTTATX-CACATATTCTC MTCAGTTCCTGTTT^^ II I I 480

TGGAATACGTGTATAAGAGAGTCAAGGACAAAGCCTTTGACGAAT^

|.| • II • I . |.

419 446 455 479 421 447 455 479

447

ScrF I EcoR II

560

547 549 549

640

GGACI IX-ΑCCAAATTAAGTCTGAGGTAGAGGTTC-AGTTGTC

^• I -I I - I II ^•

573 581 595 612 626

581 626 627

Tthlll II Alu I

HinD III EcoR V Mb

GCCAAGCCCAG<^CΑTTATTCTTATCCCATTCAAG^ 720

CGGTTCGG<?rcGTX-TAATAAGAATAGGGTAAGTTCGAAGTTC

. || |« • I ♦ I •

673 698 708

... 674

679-

Fnu4H I ScrF I

Bbv I ScrF I EcoR II

735 758 800

735 800

Hae III Alu I Mae III stu I Gsu I Pie I HσiA I Fnu4H I

Hae I Alu I Hinf I SfaN I Bspl286 I Bbv I

CAGGCCTCCTACCCCAGCTCCAGCTTTG<3ACTCA^ 880

GTCCGGACGATGGGGTCGAGGTCGJvJ-ACCTCAG^

II • I I • I l-l • - I I - I

802 816 829 852 860 873

802 818 829 860 873

803 822 831 HinP_l

HbS

Nla IV

Nar I

Has_ιι

Bbe I Fnu4H I

Ban I Bbv I BsmA I

Mme I Aha II Pst I Gsu I Bsr I Mae I L III BspM I BsmA I Mnl I

CACATAC ICACTI CGGAGAAGCCT I AGTGTCATGGCGCCT -^ 960

GTGTATGGTCAGCCTCTTCGC-ATCACAGTACC^^

I I -I l -ll I I I • I I I I *

887 901 908 920 932 940

890 911 922 943

911 924 946

911 924

911

912

Sec I ScrF I

Sau3A I Nci I

Mbo I ScrF I Msp I

Dpn I EcoR II Mae I Hpa II

Bel I BstN I Nla III Bcn_I

GTCCATCTTTσrCCCAGACTACACCL^GTAAAGATACTGATGATCAGTC 1040

CAGGTAGAAACAGGGTCTGATGTGGTTCATTTCTATGACT

• 1I0I01 1I0-09 1I0-19 I ^• 1I034

1002 1009 1024 1034

1002 1009 1034

1002 1034

1034 1034 Alu I M l I SfaN I Mbo II Fok I

AAG IAGGAAAIGCRGIATGCCACTTICTICCCAAGACAGTX^ I 1120

TTCTCCI ΓCGACTACGGTGAAGAAGGGTTCTGT^

1 I043 I- 1 1I005533 ^• 1 1I006622 ^{• • •} 1I106

1049

Sau3A

Mbo 1

Dpn I

Alw I

Nla IV Sau96 I

GTAGAAGGTGAGTGAAGAATGACCTAGGATACGGGTCTTTATACCTTTCCTGG^

1 I123 1 I140 I I ^• 1 I154 1 I1I6-8 ^• 1 I190 1I197-

1143 1168

1143 1169

1144

1144 lb J

Nla III

NspH I

Nsp7524 I

Rsa I

ScrF I

1280

Hae I Mnl I Mbo II Eae I Dde I

G IAAC»TG ∞CTKrrGATGACAAT IGI∞ I I 1360

Sty I Sec I Nco I Dsa I Fnu4H I Fok I Alu I Msp I

Bbv I Mnl I Bsr I Mnl I Mae I Hpa II

I II I I I I I I I

TAGTGGCAGCCΑTGGTTCATΛ-TK_TGGGTGT^ 1440

ATCACCffTCGGTACC-AAGTAGACACCCACAAGTCCCTCCACACCTGACCTAC^

I II ^{• •} I ^• I I ^• I l-l - I

1366 1396 1405 1412 1419 1433

1366 1408 1421 1433

1370 1370 1370 1370 1371

Dde I

ScrF I EcoR II

1600

1549 Sty I

Sec I

Hae III

Mse I

Hae I

Eae I

ScrF I

Sau3A Alu I EcoR II Mbo I Pvu II EcoN T Dpn I NsoB IT BstN I Xmn T Bel I BstX I Nla IV Sec I Mae I Ssp I

TTGTGTGAATGTTCTX_ATCACTACCACCC^^ 1680 AAa^CACTTACaAGACTAGTGATGGTGGGTCGACCAA

1615 1624 1634 1642 1668 1676 1616 1629 1643 1674 1616 1629 1643 1616 1630 1643 1643 1645 1645 1645 1646 1648 1648 Fnu4H I Bbv I

HσiE II Alu I

TTCCTATTGAGAACaTCTATAGTGCTACCAAAATTGGTAAGG 1760 AAGGATA CTCTKn' G TATC^GAa ΛT^^^

• • i • * 1 1 * * * *

1707 1725

Restriction Endonucleases site usage

Aat II - BstN I 8 HinC II 2 Pie I 4

Ace I 1 BstU I - HinD III 1 Pml I -

Afl II - BstX I 3 Hinf I 7 PpuM I 2

Afl III 1 BstY I 1 HinP I 1 Pst I 2

Aha II 1 Bsu36 I 1 Hpa I 1 PVU I -

Alu I 7 CfrlO I - Hpa II 2 Pvu II 1

Alw I 2 Cla I - Hph I - Rsa I 2

AlwN I 1 Dde I 7 Kpn I - Rsr II -

Apa I - Dpn I 5 Mae I 5 Sac I -

ApaL I - Dra I - Mae II 2 Sac II -

Ase I - Dra III - Mae III 4 Sal I -

Asp718 - Drd I - Mbo I 5 Sau3A I 5

Ava I 1 Dsa I 1 Mbo II 8 Sau96 I 3

Ava II 3 Eae I 4 Mlu I - Sea I -

Avr II - Eag I 1 Mme I 1 ScrF I 9

BamH I 1 Ear I 1 Mnl I 14 Sec I 5

Ban I 1 Eco47 III - Mse I 3 SfaN I 4

Ban II 1 Eco57 I - Mse I 6 Sfi I -

Bbe I 1 EcoN I 1 Msp I 2 Sma I -

Bbv I 7 ECOO109 I 2 Nae I - SnaB I -

Bbv II - EcoR I - Nar I 1 Spe I -

Bel I 3 EcoR II 8 Nci I 1 Sph I -

Ben I 1 EcoR V 1 Nco I 1 Spl I -

Bgl I - Esp I - Nde I 1 Ssp I 3

Bgl II - Fnu4H I 9 Nhe I - stu I 1

BsaA I - Fok I 5 Nla III 8 sty I 2

Bsm I - Fsp I - Nla iv 5 Taq I 1

BsmA I 4 Gdi II 1 Not I 1 Tthlll I -

Bspl286 I 2 GSU I 2 Nru I - Tthlll II 1 BspH I - Hae I 4 Nsi I - Xba I 1

BspM I 2 Hae II 1 Nsp7524 I 2 Xea I -

BspM II - Hae III 5 NspB II 1 Xho I 1

Bsr I 6 Hga I - NspH I 2 Xcm I -

BssH II - HgiA I 1 PaeR7 I 1 Xma I -

BstB I - HgiE II 1 PflM I - Xmn I 1

BstE II - Hha I 1

Enzyme Site Use Site position (Fragment length) Fragment order

ACC I gt/m ac 1 1 ( 494) 2 495 ( 1319) 1

Afl III a/crygt 1 1 ( 1273) 1 1274 ( 540) 2

Aha II gr/cgyc 1 1 ( 910) 1 911 ( 903) 2

AlwN I cagnnn/ctg 1 1 ( 420) 2 421 ( 1393) 1

Ava I c/ycgrg 1 1 ( 28) 2 29 ( 1785) 1

BamH I g/gatcc 1 1 ( 1142) 1 1143 ( 671) 2

Ban I g/gyrcc 1 1 ( 910) 1 911 ( 903) 2

Ban II grgcy/c 1 1 ( 25) 2 26( 1788) 1

Bbe I ggcgc/c 1 1 ( 910) 1 911 ( 903) 2

Ben I ccs/gg 1 1 ( 1033) 1 1034 ( 780) 2

BstY I r/gatcy 1 1 ( 1142) 1 1143 ( 671) 2

Bsu36 I cc/tnagg 1 1 ( 500) 2 501 ( 1313) 1

Dsa I c/crygg 1 1 ( 1369) 1 1370 ( 444) 2

Ξag I c/ggccg 1 1 ( 34) 2 35 ( 1779) 1

Ear I ctcttc 1/4 1 1 ( 182) 2 183 ( 1631) 1

EcoN I cctnn/nnnagg 1 1 ( 1642) 1 1643 ( 171) 2

EcoR V gat/ate 1 1 697) 2 698 ( 1116) 1

Gdi II yggccg -5/-1 1 1 34) 2 35 ( 1779) 1

Hae II rgcgc/y 1 1 910) 1 911 ( 903) 2

HgiA I gwgcw/c 1 1 859) 2 860 ( 954) 1

HgiE II accnnnnnnggt 1 1 1706) 1 1707 ( 107) 2

Hha I gcg/c 1 1 911) 1 912 ( 902) 2

HinD III a/agctt 1 1 672) 2 673 ( 1141) 1

HinP I g/cgc 1 1 911) 1 912 ( 902) 2

Hpa I gtt/aac 1 1 625) 2 626 ( 1188) 1

Mme I tccrac 20/18 1 1 889) 2 890 ( 924) 1

Nar I gg/cgcc 1 1 910) 1 911 ( 903) 2

Nci I cc/sgg 1 1 1033) 1 1034 ( 780) 2

Nco I c/catgg 1 1 1369) 1 1370 ( 444) 2

Nde I ca/tatg 1 1 1767) 1 1768 ( 46) 2

Not I gc/ggccgc 1 1 33) 2 34 { 1780) 1

NspB II cmg/ckg 1 1 1628) 1 1629 ( 185) 2

PaeR7 I c/tcgag 1 1 28) 2 29 ( 1785) 1

Pvu II cag/ctg_ 1 1 1628) 1 1629( 185) 2

Stu I agg/cct 1 1 801) 2 802 ( 1012) 1

Taq I t/cga 1 1( 29) 2 30( 1784) 1

Tthlll II caarca 11/9 1 1( 678) 2 679 ( 1135) 1

Xba I t/ctaga 1 1( 2) 2 3( 1811) 1

Xho I c/tcgag 1 1( 28) 2 29 ( 1785) 1

Xmn I gaann/nnttc 1 1( 1673) 1 1674 ( 140) 2

Alw I ggatc 4/5 2 1( 1142) 1 1143 ( 1) 3 1144 ( 670) 2

Bspl286 I gdgch/c 2 1( 25) 3 26( 834) 2 860 ( 954) 1

BspM I acctgc 4/8 2 1( 611) 2 612 ( 308) 3 920 ( 894) 1

EcoO109 I rg/gnccy 2 1( 8) 3 9( 1159) 1 1168 ( 646) 2

Gsu I ctggag 16/14 2 1( 817) 2 818 ( 125) 3 943 ( 871) 1

HinC II gty/rac 2 1( 594) 2 595 ( 31) 3 626 { 1188) 1

Hpa II c/cgg 2 1( 1033) 1 1034 ( 399) 2 1433 ( 381) 3

Mae II a/cgt 2 1( 515) 2 516 ( 955) 1 1471 ( 343) 3

Msp I c/cgg 2 1( 1033) 1 1034 ( 399) 2 1433 ( 381) 3

Nsp7524 I r/catgy 2 11 338) 3 339( 935) 1 1274 ( 540) 2

NspH I rcatg/y 2 1( 338) 3 339( 935) 1 1274 ( 540) 2

PpuM I rg/gwccy 2 1( 8) 3 9( 1159) 1 1168 ( 646) 2

Pst I ctgca/g 2 1( 921) 1 922 ( 587) 2 1509 ( 305) 3

Rsa I gt/ac 2 1( 796) 1 797 ( 475) 3 1272 ( 542) 2

Sty I c/cwwgg 2 1( 1369) 1 1370 ( 278) 2 1648 ( 166) 3

Ava II g/gwcc 3 1( 9) 4 10 ( 160) 3 170 ( 999) 1 1169 ( 645) 2

Bel I t/gatca 3 1{ 277) 3 278 { 723) 1 1001 ( 614) 2 1615 ( 199) 4

BstX I ccannnnn/ntgg 3 1( 511) 2 512 ( 642) 1 1154 ( 470) 3 1624 ( 190) 4

Mse I tgg/cca 3 1( 545) 2 546 ( 758) 1 1304 ( 341) 3 1645 ( 169) 4

Sau96 I g/gncc 3 1( 9) 4 10 ( 160) 3 170 ( 999) 1 1169 ( 645) 2

Ssp I aat/att 3 1( 163) 3 164 ( 559) 2 723 ( 953) 1 16761 138) 4 BsmA I gtctc 1/5 4 11 127) 4 1281 804) 1 932( 14) 5 9461 534) 2 14801 334) 3

Eae I y/ggccr 4 11 34) 5 351 511) 2 5461 758) 1 13041 341) 3 16451 169) 4

Hae I wgg/ccw 4 11 545) 1 5461 256) 4 8021 502) 2 13041 341) 3 16451 169) 5

Mae III /gtnac 4 11 142) 4 1431 688) 1 8311 433) 3 12641 500) 2 17641 50) 5

Pie I gagtc 4/5 4 11 454) 2 4551 24) 5 4791 102) 4 5811 248) 3 8291 985) 1

SfaN I gcatc 5/9 4 11 223) 3 2241 524) 2 7481 104) 5 8521 201) 4 10531 761) 1

Dpn I ga/tc 5 11 69) 6 70( 209) 3 2791 723) 1 10021 142) 5 11441 472) 2 16161 198) 4

Fok I ggatg 9/13 5 11 352) 2 3531 11) 6 3641 742) 1 11061 302) 3 14081 175) 5 15831 231) 4

Hae III gg/cc 5 11 35) 6 361 511) 1 5471 256) 4 8031 502) 2 13051 341) 3 16461 168) 5

Mae I c/tag 5 11 3) 6 41 897) 1 9011 123) 5 10241 395) 2 14191 249) 3 16681 146) 4

Mbo I /gate 5 11 69) 6 701 209) 3 2791 723) ^~i 10021 142) 5 11441 472) 2 16161 198) 4

Nla IV ggn/ncc 5 11 9) 6 101 901) 1 9111 232) 3 11431 26) 5 11691 465) 2 16341 180) 4

Sau3A I /gate 5 11 69) 6 70( 209) 3 2791 723) 1 10021 142) ^'5 11441 472) 2 16161 198) 4

Sec I c/cnngg 5 11 445) 2 4461 588) 1 10341 336) 3 13701 272) 4 16421 6) 6 16481 166) 5

Bsr I actgg 1/-1 6 11 139) 6 1401 67) 7 2071 680) 1 887( 253) 3

11401 265) 2 14051 181) 5 15861 228) 4

Mse I t/taa 6 11 572) 1 5731 54) 5 6271 570) 2 11971 321) 3

15181 21) 7 15391 27) 6 15661 248) 4

Alu I ag/ct 7 11 673) 1 6741 142) 5 8161 6) 8 8221 227) 3

10491 372) 2 14211 209) 4 16301 95) 6 17251 89) 7

Bbv I gcagc 8/12 7 11 731) 1 7321 3) 8 7351 138) 4 8731 51) 6

9241 442) 2 13661 360) 3 17261 80) 5 18061 8) 7

Dde I c/tnag 7 11 121) 5 1221 297) 3 4191 83) 7 5021 688) 1

11901 63) 8 12531 99) 6 13521 133) 4 14851 329) 2

Hinf I g/antc 7 11 230) 3 2311 224) 4 4551 24) 8 4791 102) 6

5811 210) 5 7911 38) 7 8291 716) 1 15451 269) 2

BstN I cc/wgg 8 11 446) 1 447( 102) 7 5491 209) 4 7581 42) 9

8001 209) 5 10091 259) 3 12681 281) 2 15491 94) 8

16431 171) 6

EcoR II /ccwgg 8 11 446) 1 4471 102) 7 5491 209) 4 7581 42) 9

8001 209) 5 10091 259) 3 12681 281) 2 15491 94) 8 16431 171) 6

Mbo II gaaga 8/7 8 11 179) 4 1801 3) 9 1831 525) 1 7081 354) 3

10621 61) 7 11231 83) 5 12061 75) 6 12811 516) 2

17971 17) 8

Nla III catg/ 8 11 75) 8 76( 100) 6 1761 144) 4 3201 20) 9

3401 568) 1 9081 111) 5 10191 256) 3 12751 96) 7

13711 443) 2

Fnu4H I gc/ngc 9 11 33) 7 341 3) 9 37( 695) 1 7321 3)10

7351 138) 4 8731 51) 6 92 1 442) 2 13661 360) 3

17261 80) 5 18061 8) 8

ScrF I cc/ngg 9 11 446) 1 447( 102) 7 5491 209) 4 7581 42) 9

8001 209) 5 10091 25)10 10341 234) 3 12681 281) 2

15491 94) 8 16431 171) 6

Mnl I cctc 7/7 14 11 172) 4 1731 154) 5 327( 30)11 3571 22)13

3791 122) 6 5011 27)12 5281 412) 1 9401 103) 7

10431 214) 3 12571 100) 8 13571 39)10 13961 16)14

14121 72) 9 14841 12)15 14961 318) 2

256 sites found

No Sites found for the following Restriction Endonucleases

Aat II gacgt/c CfrlO I r/ccggy PflM I ccaπnnn/ntgg

Afl II c/ttaag Cla I at/cgat Pro! I cac/gtg Apa I gggcc/c Dra I ttt/aaa Pvu I cgat/cg ApaL I g/tgcac Dra III cacnnn/gtg Rsr II cg/gwccg Ase I at/taat Drd I gacnnnn/nngtc Sac I gagct/c Asp718 g/gtacc Eco47 III age/get Sac II ccgc/gg Avr II c/ctagg Eco57 I ctgaag 16/14 Sal I g/tcgac Bbv II gaagac 2/6 EcoR I g/aattc Sea I agt/act Bgl I gccnπnn/nggc Esp I gc/tnagc Sfi I ggccnnnn/nggcc Bgl II a/gatct Fsp I tgc/gca Sma I ccc/ggg BsaA I yac/gtr Hga I gacgc 5/10 SnaB I tac/gta Bsm I gaatgc 1/-1 Hph I ggtga 8/7 Spe I a/ctagt BspH I t/catga Kpn I ggtac/c Sph I gcatg/c BspM II t/ccgga Mlu I a/cgcgt Spl I c/gtacg BssH II g/cgcgc Nae I gcc/ggc Tthlll gacn/nngtc BstB I tt/cgaa Nhe I g/ctagc Xca I gta/tac BstE II g/gtnacc Nru I tcg/cga Xcm I ccannnnn/nnnntgg BstU I cg/cg Nsi I atgca/t Xma I c/ccggg

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Claims

1. A polynucleotide which is selected from the group consisting of : a) a polynucleotide comprising at least 8 consecutive nucleotides of SEQ ID N┬░1 ;

(b) a polynucleotide differing from the polynucleotide defined in (a) by mutation, insertion, deletion or substitution of one or more bases;

(c) a polynucleotide, the sequence of which is complementary to the sequence of anyone of polynucleotides defined in (a) and (b).

(d) a polynucleotide which hybridizes specifically with anyone of the polynucleotides defined in (a), (b) or (c).

2. A polynucleotide according to claim 1 which is selected from the group consisting of :

(a) a polynucleotide comprising at least 8 consecutive nucleotides of SEQ ID N┬░2;

(c) a polynucleotide, the sequence of which is complementary to the sequence of anyone of polynucleotides defined in (a) and (b) ;

3. A polynucleotide according to claim 1 which is selected from the group consisting of :

(a) a polynucleotide comprising at least 8 consecutive nucleotides of SEQ ID N┬░3;

4. A polynucleotide according to claim 1 which is selected from the group consisting of :

(a) a polynucleotide comprising at least 8 consecutive nucleotides of SEQ ID N┬░4;

5. A polynucleotide according to claim 1 which is selected from the group consisting of :

(a) a polynucleotide comprising at least 8 consecutive nucleotides of SEQ ID N┬░5;

6. A polynucleotide according to claim 1 which is selected from the group consisting of :

(a) a polynucleotide comprising at least 8 consecutive nucleotides of SEQ ID N┬░6;

7. An oligonucleotide useful as a primer or as a probe consisting of 8 to 300 consecutive nucleotides of the polynucleotide according to anyone of claims 1 to 6.

8. The oligonucleotide according to claim 7 which sequence is selected from the group consisting of SEQID N┬░ 11 to SEQ ID N┬░ 47.

9. A pair of oligonucleotides consisting of two oligonucleotides according to anyone of claims 7 and 8.

10. A pair of oligonucleotides according to claim 9 which is selected from the group consisting of the following pairs :

Pair l : SEQ ID N┬░ 11

SEQ ID N┬░ 12 this pair of oligonucleotides is used for amplifying the full length coding region of SEQ ID N┬░2.

Pair 2 : SEQ ID N┬░ 13

SEQ ID N┬░ 14 this pair of oligonucleotides is used as internal primers to amplify exons E and F of SEQ ID N┬░2.

Pair 3 : SEQ ID N┬░15

SEQ ID N┬░ 16 this pair of oligonucleotides is used to amplify exon z of SEQ ID N┬░2.

Pair 4 : SEQ ID N┬░17

SEQ ID N┬░18 this pair of oligonucleotides is used to amplify exon A of SEQ ID N┬░2.

Pair 5 : SEQ ID N┬░ 19

SEQ ID N┬░ 20 this pair of oligonucleotides is used to amplify exon B of SEQ ID N┬░2.

Pair 6 : SEQ ID N┬░ 21

SEQ ID N┬░ 22 this pair of oligonucleotides is used to amplify exon C of SEQ ID N┬░2.

Pair 7 : SEQ ID N┬░ 23

SEQ ID N┬░ 24 this pair of oligonucleotides is used to amplify exon D of SEQ ID N┬░2.

Pair 8 : SEQ ID N┬░ 25

SEQ ID N┬░ 26 this pair of oligonucleotides is used to amplify exons E and F of SEQ ID N┬░2.

Pair 10: SEQ ID N┬░ 27

SEQ ID N┬░ 28 this pair of oligonucleotides is used to amplify exon G of SEQ ID N┬░2.

Pair 11 : SEQ ID N┬░ 30

SEQ ID N┬░ 31 this pair of oligonucleotides is used to amplify exon R of SEQ ID N┬░2.

Pair 12 : SEQ ID N┬░ 32 SEQ ID N┬░ 33 this-pair of oligonucleotides is used to amplify exon S of SEQ ID N┬░2.

Pair 13 : SEQ ID N┬░ 34

SEQ ID N┬░ 35 this pair of oligonucleotides are used to amplify exon T of SEQ ID N┬░2

Pair 14 : SEQ ID N┬░ 36

SEQ ID N┬░ 37 this pair of oligonucleotides are used to amplify exon U of SEQ ID N┬░2.

Pair 15 : SEQ ID N┬░ 38

SEQ ID N┬░ 39 this pair of oligonucleotides are used to amplify exon V of SEQ ID N┬░2 A18

Pair 16 : SEQ ID N┬░ 40

SEQ ID N┬░ 41 this pair of oligonucleotides are used to amplify exon W of SEQ ID N┬░2

Pair 17 : SEQ ID N┬░ 42 SEQ ID N┬░ 43 this pair of ohgonucleotides are used to amplify exon X of SEQ ID N┬░2

Pair 18 : SEQ ID N┬░ 44

SEQ ID N┬░ 45 this pair of o gonucleotides are used to amplify exon Y of SEQ ID N┬░2

Pair 19 : SEQ ID N┬░ 46

SEQ ID N┬░ 47 this pair of oligonucleotides are used to amplify exon H of SEQ ID N┬░2

11 A recombinant vector containing a polynucleotide according to anyone of claims 1 to 6, which is a cloning vector, an insertion vector or an expression vector

12 The recombinant vector according to claim 11 which is an expression vector containing a nucleic acid necessary for the expression of the said polynucleotide

13 A recombinant cell host which contains a polynucleotide according to anyone of claims 1 to 6 or a recombinant vector according to anyone of claims 11 or 12

14 A recombinant polypeptide which is encoded by a polynucleotide according to anyone of claims 1 to 6

15 A polypeptide which is selected from the group consisting of

(a) a polypeptide comprising anyone of the aminoacid sequences SEQ ID N┬░ 7 SEQ ID N┬░8, SEQ ID N┬░9 or SEQ ID N┬░10,

(b) a peptide fragment of at least 10 aminoacids in length of the polypeptide defined in (a), the said peptide fragment being recognized by an antibody directed against the polypeptide defined in (a),

(c) a polypeptide comprising, as regards to the polypeptide defined in (a) or (b) at least one modification by addition or substitution of an aminoacid, the said modified polypeptide being recognized by an antibody directed against the polypeptide defined in (a)

16 A polyclonal or a monoclonal antibody which recognizes specifically a recombinant polypeptide according to claim 14 or a polypeptide according to claim 15

17 The antibody of claim 16 which is radioactively or non-radioactively labeled

18 A method for detecting specifically a polypeptide according to claim 15 in a biological sample comprising the steps of a) bringing into contact the biological sample with an anibody according to anyone of claims 16 and 17, b) detecting the antigen-antibody complex formed

19 A method for detecting a genetic abnormality linked to the BO or to the BOR syndrome in a biological sample containing DNA, comprising the steps of a) bringing the biological sample into contact with a pair of o gonucleotide primers according to anyone of claims 7 or 8, the DNA contained in the sample having been optionally made available to hybridization and under conditions permitting a hybridization of the primers with the DNA contained in the biological sample, b) amplifying the DNA c) revealing the amplification products, d) optionally detecting a mutation or a deletion by appropriate techniques

20 The method of claim 19 wherein in step d) a mutation or a deletion is detected by Single-strand conformation polymorphism (SSCP) or denaturing gradient gel electrophoresis (DGGE)

21 The method according to claim 19 wherein step c) consists in the detection of the amplified products with an ohgonucleotide probe according to anyone of claims 7 or 8

22 A method for detecting a genetic abnormality linked to the BO or to the BOR syndrome in a biological sample containing DNA, comprising the steps of a) bringing the biological sample into contact with an oligonucleotide probe according to anyone of claims 7 or 8, the DNA contained in the sample having been optionally made available to hybridization and under conditions permitting a hybridization of the primers with the DNA contained in the biological sample b) detecting the hybrid formed between the ohgonucleotide probe and the DNA contained in the biological sample

23 A method for detecting a genetic abnormality linked to the BO or to the BOR syndrome in a biological sample containing DNA, comprising the steps of a) bringing into contact a first ohgonucleotide probe according to anyone of claims 7 or 8 that has been immobilized on a support, the DNA contained in the sample having been optionally made available to hybridization and under conditions permitting a hybridization of the primers with the DNA contained in the biological sample, b) bringing into contact the hybrid formed between the immobilized first ohgonucleotide probe and the DNA contained in the biological sample with a second ohgonucleotide probe according to anyone of claims 7 or 8, which second probe hybridizes with a sequence different from the sequence to which the immobilized first probe hybridizes, optionally after having removed the DNA contained in the biological sample which has not hybridized with the immobilized first ohgonucleotide probe

24 The method according to anyone of claims 21 to 23, wherein, before step a), the DNA contained in the biological sample, or the cDNA obtained after reverse transcription of the RNA contained in the biological sample, is amplified using a pair of primers according to anyone of claims 9 or 10

25 A method for detecting a genetic abnormality linked to the BO or to the BOR syndrome in a biological sample containing DNA, by the detection of the presence and of the position of base substitutions or base deletions in a nucleotide sequence included in a double stranded DNA preparation to be tested, the said method comprising the steps of a) amplifying specifically the region containing, on one hand, the nucleotide sequence of the DNA to be tested and on the other hand the nucleotide sequence of a DNA of known sequence, the DNA of known sequence being a polynucleotide according to anyone of claims 1 to 6, b) labeling the sense and antisense strands of these DNA with diferent fluorescent or other non-isotopic labels, c) hybridizing the amplified DNAs, d) revealing the heteroduplex formed between the DNA of known sequence and he DNA to be tested by cleavage of the mismatched parts of the DNA strands

26 A method for detecting a genetic abnormality linked to the BO or to the BOR syndrome in a biological sample containing DNA, by the detection of the presence and of the position of base substitutions or base deletions in a nucleotide sequence included in a double stranded heterozygous or heterogenous DNA preparation to be tested, the said method comprising the steps of a) amplifying specifically the region containing the nucleotide sequence of the DNA to be tested, b) labeling the sense and antisense strands of these DNA with different fluorescent or other non-isotopic labels, c) hybridizing the amplified DNAs, d) revealing the heteroduplex formed between the DNA of known sequence and the DNA to be tested by cleavage of the mismatched parts of the DNA strands.

27. A therapeutic composition containing an active principle which is selected from the group consisting of : a) a purified EYA1 orEYA1-B protein or one of their biologically active derivatives ; b) a polynucleotide encoding the EYA1 or EYA1 -B protein or one of their biologically active derivatives ; c) an antisense polynucleotide hybridizing specifically with the genomic DNA of the EYA1 gene or hybridizing specifically with the mRNA encoding the EYA1 or EYA1-B protein ; d) a polyclonal or monoclonal antibody that specifically binds to the EYA1 or EYA1-B protein.

28. A method for modulating the expression or the biological activity of the EYA1 or the EYA1-B protein or one of their biologically active derivatives comprising the steps of : a) preparing a therapeutic composition according to claim 27; b) administering the composition of step a) to the patient.

29. A method for screening ligands that bind to the EYA1 , EYA1 -B, EYA2 or EYA3 protein.

Such a screening method, in one embodiment, comprises the steps of : a) Preparing a complex between the EYA1 , EYA1 -B, EYA2 or EYA3 protein and a ligand that binds to the said protein by a method selected among the followings :

ΓÇó preparing a tissue extract containing the EYA1 , EYA1-B, EYA2 or EYA3 protein putatively bound to a natural ligand;

ΓÇó bringing into contact the purified EYA1 , EYA1-B, EYA2 or EYA3 protein with a solution containing a molecule to be tested as a ligand binding to the said protein; b) visualizing the complex formed between the EYA1 , EYA1 -B, EYA2 or EYA3 protein from the tissue extract and the natural ligand of the EYA1 , EYA1 -B, EYA2 or EYA3 protein or the complex formed between the purified EYA1 , EYA1 -B, EYA2 or EYA3 protein and the molecule to be tested.

30. A method for screening molecules that modulate the expression of the EYA1 , EYA1-B, EYA2 or EYA3 protein said screening method comprising the steps of : a) cultivating a prokaryotic or an eukaryotic cell that has been transfected with a nucleotide sequence encoding the EYA1 , EYA1-B, EYA2 or EYA3 protein, placed under the control of its own promoter; b) bringing into contact the cultivated cell with a molecule to be tested; c) quantifying the expression of the EYA1 , EYA1 -B, EYA2 or EYA3 protein.

31. Plasmid selected from the following group :

- plasmid HSEYA1aNEYAK7 contained in the E. coli DH5-alpha which has been deposited at the CNCM under the accession number 1-1824 ;

- plasmid HSEYA1a6K7F4 contained in the E. coli DH5-alpha which has been deposited at the CNCM under the accession number 1-1836 ;

- plasmid HSEYA13'5' contained in the E. coli DH5-alpha which has been deposited at the CNCM under the accession number 1-1832 ;

- plasmid HSEYA1 Puc3 contained in the E. coli DH5-alpha which has been deposited at the CNCM under the accession number 1-1830 ;

- plasmid HSEYA1 b5p35 contained in the E. coli DH5-alpha which has been deposited at the CNCM under the accession number 1-1835 ;

- plasmid HSEYA1c14K7F6 contained in the E. coli DH5-alpha which has been deposited at the CNCM under the accession number 1-1822 ;

- plasmid HSEYA2-20R50.3 contained in the E. coli DH5-alpha which has been deposited at the CNCM under the accession number 1-1834 ;

- plasmid HSEYA2-20int8 contained in the E. coli DH5-alpha which has been deposited at the CNCM under the accession number 1-1829 ;

- plasmid HSEYA2-20F250.10 contained in the E. coli DH5-al╧üha which has been deposited at the CNCM under the accession number 1-1833 ;

- plasmid HSEYA3-z39F11 contained in the E. coli DH5-alpha which has been deposited at the CNCM under the accession number 1-1823 ;

- plasmid HSEYA3-z39int contained in the E. coli DH5-alpha which has been deposited at the CNCM under the accession number 1-1825 ;

- plasmid HSEYA-z39.23 contained in the E. coli DH5-alpha which has been deposited at the CNCM under the accession number 1-1831 ;

- plasmid HSEYA1 S1 C6 contained in the E. coli DH5-alpha which has been deposited at the CNCM under the accession number 1-1828 ;

- plasmid HSEYA1 S3B12 contained in the E. coli DH5-alpha which has been deposited at the CNCM under the accession number 1-1827 ;

- plasmid HSEYA1J4A10 contained in the E. coli DH5-alpha which has been deposited at the CNCM under the accession number 1-1826.

32. Recombinant lambda phage vector HSEYA1 B4CI.34 which has been deposited at the CNCM under the accession number 1-1837.

33. A kit for the detection of a genetic abnormality linked to the BOR syndrome in a biological sample, comprising the following elements : a) a pair of oligonucleotides according to anyone of claims 7 to 10; b) reagents necessary for carrying out a DNA amplification; c)a component which makes it possible to determine the length of the amplified fragments or to detect a mutation.

34. A method for the producing a polypeptide of the invention, and especialy a polypeptide of SEQ ID N┬░ 7, SEQ ID N┬░8, SEQ ID N┬░9 or SEQ ID N┬░10. the said method comprising the steps of : a) amplifying the nucleic acid coding for the desired polypeptide using a pair of oligonucleotides according to anyone of claims 7 to 10 if necessary ; b) inserting the nucleic acid of interest in an appropriate vector; c) growing, in an appropriate culture medium, a cell host previously transformed or transfected with the recombinant vector of step b); d) harvesting the culture medium thus conditioned or lyse the cell host , e) separating or purifying, from the said culture medium, or from the pellet of the resultant host cell lysate the thus produced polypeptide of interest ; and f) characterizing the produced polypeptide of interest.

35. A method for screening a ligand molecule capable to bind to a polynucleotide according to anyone of claims 1 to 6 comprising the steps of : a) bringing into contact a polynucleotide according to anyone of claims 1 to 6 with a ligand molecule to be tested; and b) detecting the complexes formed between the said polynucleotide and the ligand molecule.