EP1003895A2

EP1003895A2 - Cloning vectors for producing adenoviral minimal viruses

Info

Publication number: EP1003895A2
Application number: EP98944984A
Authority: EP
Inventors: Moritz Hillgenberg; Peter LÖSER; Frank Schnieders; Volker Sandig; Michael Strauss
Original assignee: HepaVec AG fur Gentherapie
Current assignee: Develogen AG
Priority date: 1997-07-10
Filing date: 1998-07-06
Publication date: 2000-05-31
Also published as: WO1999002647A2; WO1999002647A3; JP2001509375A

Abstract

The invention relates to cloning vectors for producing adenoviral minimal viruses, consisting of: a) two adenoviral, inverted terminal replication sequences (ITR, inverted terminal repeats) which are flanked by ab) two interfaces for a restriction endonuclease with a recognition sequence more than 8 Bp in length, and enclose ac) an adenoviral packaging signal, ad) a multiple cloning site for inserting therapeutic DNA fragments into which non-coding chromosomal mammal DNA is possibly cloned in addition, ae) possibly a recognition site for a recombinase situated between one of the inverted terminal replication sequences and the adenoviral packaging signal, and af) possibly a reporter gene cassette; b) a bacterial plasmid backbone with replication origin and bacterial resistance gene, into which ba) a packaging signal of a bacteriophage is cloned.

Description

Cloning vectors for the production of minimal adenoviral viruses

description

The invention relates to cloning gates for the production of adenoviral minimal viruses. Areas of application of the invention are medicine and the pharmaceutical industry.

Adenoviral vectors are efficient gene transfer vehicles for somatic gene therapy. The first generation vectors consist of the complete genome of the adenovirus serotype 5 (Ad5) with deletions in the E1 and E3 regions into which foreign genes can be inserted. These vectors are capable of independent propagation in suitable cell lines which provide the El functions. Due to the remaining viral coding sequences, such vectors have a maximum transport capacity of approx. 8 Kb Fre d-DNA. Residual expression of adenoviral genes appears to be largely responsible for the immunological elimination of transduced cells in vivo, and thus also for the temporal limitation of transgene expression and the corresponding therapeutic effects (Yang, Y. et al., 1994, Cellular immunity to viral antigens limits El- deleted for gene therapy, Proc. Natl. Acad. Sci. USA 91, 4401-4411).

A promising approach to increasing transport capacity while simultaneously reducing immunogenicity is the outsourcing of all coding adenoviral genome sections from the therapeutic vector (Chen, HH et al., 1997, Persistence in muscle of an adenoviral vector that lacks all viral genes, Proc. Natl. Acad.See. USA 94, 1645-50). For the multiplication and packaging of such so-called adenoviral minimal viruses which, in addition to the therapeutic foreign gene to be transported, only the short signal sequences for the viral replication machinery (inverted terminal replication sequences, English inverted terminal repeats, ITRs) and for packaging in viral envelopes (English packaging signal , PS), systems based on helper viruses have already been described (Parks. RJ et al., 1996, A helper-dependent adenovirus vector system: re oval of helper virus by cre-mediated excision of the viral packaging signal, Proc. Natl. Acad. Be. USA 93: 13565-13570) and are currently being further developed worldwide. Due to the size of the constructs, which places special demands on the cloning strategies, the cloning of minimal viruses is currently a complex and time-consuming process.

A vector for minimal virus production, from which minimal viruses can be produced after insertion of therapeutic expression cassettes, has already been described (Hardy et al., 1997, Construction of adenovirus vectors through cre-lox recombination, J. Virol. 71, 1842-1849). In exchange for the adenoviral coding sequences, however, DNA of the phage La bda was incorporated in it, which, according to current knowledge, has a destabilizing effect on the adenoviral minimal virus genome. Furthermore, the mechanism for virus generation selected there limits the selection of the transgenes. In addition, the minimal viruses generated with this system can only be used in vitro, since a strongly immunogenic reporter enzyme was used.

The object of the invention is to provide a cloning system with which the production of adenoviral minimal viruses is simplified and to insert new components which allow the use of these vectors in vitro and in vivo.

The task is accomplished according to the claims. According to the invention, two constructs (pMV and pMVX) are presented below, with which a significantly simplified and accelerated production of recombinant adenoviruses of the minimal virus type is possible.

The cloning of the adenoviral minimal viruses was simplified here by using cosmid cloning (see CI4.). The mechanism for virus formation created here does not restrict the choice of transgenes (see CI3.). In addition, a novel titer system was constructed, which allows the in vivo use of the adenoviral minimal viruses allowed (see CI5.2.). In addition, pMVX contains a fragment of chromosomal human DNA as a viral backbone to improve stability (see C.2.6.1.) And flexibility in the choice of the Fre d DNA to be incorporated (see C.2.6.2.). Both vectors are compatible with all helper systems described so far for the propagation and packaging of minimal viruses.

C. Components of the cloning vectors

C.l. Cloning vector pMV (see Fig. 1)

C.l.l. Bacterial backbone from pBluescript

The bacterial backbone of the plasmid comes from pBluescript and contains, in addition to the origin of replication, a beta-lactamase gene for mediating ampicillin resistance. The beta lacta asegen enables the use of conventional selection methods for the multiplication of pMV and its derivatives and their cloning. The origin of replication of pBluescript is subject to a multi-copy control in E. coli and thereby enables the preparative multiplication of the minimal virus vector with high yield in suitable E. coli strains.

C.l.2. Sequence signals for replication and packaging from adenovirus serotype 5 (Ad5)

The plas id contains the 5 ^' ITR with the packaging signal (the first 514 base pairs (bp) of the adenovirus genome) and the 3 ^' ITR (the last 162 bp of the adenovirus genome). These are the minimally required signal sequences for the recognition of the minimal virus genome by the replication machinery of the adenoviral helper virus as well as the packaging of the replicated minimal virus genomes in viral capsids.

The use of the ends of the adenovirus genome to insert the ITRs and the packaging signal into constructs Minimal virus production is a mandatory basis for the production of minimal viruses and is therefore also described by others.

C.l.3. The ITRs flanking restriction sites for the meganuclease (I-Scel) to release the recombinant virus genome

The release of the ITRs enables them to be effectively recognized by the replication apparatus. The 18 bp recognition sequence conveys the highest specificity for the release of the ends and at the same time maximum flexibility in the choice of the transgenic sequences. An 18 bp sequence statistically occurs only once in 6.8 x 10 ¹¹ base pairs, the human genome as the source of potential transgenes contains only approx. 3 x 10 ⁹ base pairs. It can therefore be practically ruled out that a recognition sequence occurs in a desired transgene which would interfere with the release mechanism.

In addition to the ITRs, restriction sites for rarely cutting restriction enzymes have also been introduced specifically by Hardy et al. (1997). Interfaces for the restriction endonuclease Sfil are used here to release the ITRs. However, this enzyme has a specific recognition sequence that is only 8 bp long. The occurrence of interfaces in the desired transgene cassette cannot be excluded with this system.

C.l.4. The phage La bda cos signal to facilitate cloning

Minimal virus genomes have the greatest stability if their genome size is as close as possible to that of wild-type adenovirus (approx. 36 kilobase pairs (Kb)). In this size range, the classic cloning processes are very inefficient and prone to errors.

The construct receives the properties of a cosmid vector through the cos signal. The use of cosmid cloning techniques enables a highly efficient and size-selected construction of recombinant minimal virus genomes. The size selection results from the packaging capacity of the lambda phage, which, after subtracting the bacterial part in the plasmid, corresponds exactly to the minimum virus size range of approx. 25-38 Kb that is considered stable (Mitani, K. et al., 1995, Rescue, propagation , and partial purification of a helper virus-dependent adenovirus vector, Proc. Natl. Acad. Sci. USA 92, 3854-858). So far, the cosmid cloning technique has mainly been used for the production of gene banks. Your application to simplify minimal virus cloning is new.

C.l.5. Reporter gene cassette for titration

Minimal viruses are not capable of independent replication, so titering cannot be carried out using the classic adenovirus titering method. Minimal viruses that were generated from pMV and its derivatives can be titered by reporter genes, which enable direct visualization of infected cells via enzymatic color reactions or immunological detection methods. There is a choice between two reporter systems, one of which is new:

C.l.5.1. Constitutive beta-galactosidase reporter gene cassette in pMV-BG for in vitro applications

A constitutive beta-galactoidase expression cassette is inserted in pMV-BG. It consists of the beta-galactosidase gene under the control of the RSV-3 ^' LTR (RSV promoter) and a downstream polyadenylation signal. Infected cells can be visualized by X-Gal staining.

The beta-galactosidase reporter system for visualizing viral infection and for titering viruses is a general procedure and has been used in gene transfer vectors various types, including adenoviral minimal viruses, already used.

C. _l .5.2. Regulated PLAP reporter gene cassette in pMV-PLAP for in vivo applications

Beta-galactosidase is a potent immunogen in vivo. In order to generate minimal viruses which are intended for in vivo use, an alternative titer system is offered in pMV-PLAP, which is based on a reporter gene system, and which does not appear as an immunogen in vivo. In addition to the polyadenylation signal, the regulated reporter gene cassette contains two components that ensure the lowest possible immunogenicity:

Reporter gene expression is controlled by the tet promoter, which is currently considered the most regulatable promoter in mammalian cells (Gossen, M. & Bujard, H., 1992, Tight control of gene expression in mam alian cells by tetracycline-responsive promoters, Proc. Natl. Acad. Sci. USA 89, 5547-551) The promoter is only active in the presence of the tet transactivator. In mammalian cells that do not naturally express the transactivator, the promoter is largely silent. For titering, we have generated a cell line that is easily infected by adenoviruses and that constitutively expresses the tet transactivator.

PLAP (placenta-like alkaline phosphatase) is inserted as a reporter gene, a well-characterized human reporter gene with established detection methods (Kumar-Singh, R. and Chamberlain, JS, 1996, Encapsidated adenovirus minichromosomes allow delivery and expression of a 14 kb dystrophin cDNA to muscle cells, Hum. Mol. Genet. 5, 913-921.), which has no known antigenic properties in humans.

In this titering system, both the two components (tet promoter and PLAP gene) and the underlying principle of a double strategy for avoiding an immune response (regulated promoter that is silent in the target organism and use of a reporter gene that connects the genome of the target organism originated) for titering gene therapy vectors generally novel.

C.l.6. Multiple cloning site for the insertion of therapeutic DNA fragments up to a size of approx. 30 Kb

pMV contains a multiple cloning site with unique interfaces for several restriction enzymes to facilitate the insertion of therapeutic DNA fragments. Taking into account the adenoviral packaging capacity, fragment sizes up to approx. 30 Kb can be inserted. Vectors for minimal virus production with a multiple cloning site have not yet been described.

C.2. Cloning vector pMVX (see Fig. 2)

The minimal virus vector pMVX has all components of pMV except the multiple cloning site, in whose place a piece of non-coding DNA from the human genome is inserted. This gives the vector new properties. PMVX is also offered with two different reporter gene cassettes.

C.2.1. see C.l.l,

C.2.2. see C.l.2

C.2.3, see C.l.3.

C.2.4. see C.l.4

C.2.5. Reporter gene cassette C.2.5.1. Constitutive beta-galactosidase reporter gene cassette in pMVX-BG see Cl5.1.

C.2.5.2. Regulated PLAP reporter gene cassette in pMVX-PLAP see

C.2.6. 27.4 Kb DNA fragment of non-coding human genomic DNA

pMVX contains a 27.4 Kb human DNA fragment X-chromosomal origin, the sequence of which is completely known and contains no coding regions. Undesired effects due to gene expression from this component can therefore largely be excluded. Compared to pMV, the following additional properties result:

C.2.6.1. Stabilization of the minimal virus genome

The chromosomal fragment forms the backbone of the minimal viruses that can be derived from pMVX. Existing systems use e.g. DNA of phage La bda as the backbone. The minimal viruses filled in with lambda DNA are, as known from congress reports, unstable, while the insertion of chromosomal fragments has a stabilizing effect. Although this is known, vectors for minimal virus generation, which exploit this systematically in that different transgene cassettes can be installed in a constant chromosomal context, have not been described to date.

C.2.6.2. Restriction endonuclease interfaces for easier insertion of therapeutic cassettes with great flexibility in terms of their size

With suitable interfaces for commercially available restriction endonucleases, cassettes in a size range of up to 18 Kb can be inserted in pMVX with a cloning step, and at the same time a minimal virus genome size can be maintained, the highest Stability of the derived minimal virus ensures. This flexibility with regard to the size of the cassette to be inserted is novel for minimal virus cloning vectors. All enzymes provide ends that are either already smooth or can be filled in with Klenow polymerase. Cloning over smooth DNA ends is preferred in the cosmid cloning used here.

In the middle of the backbone is a unique SnaBI interface (directly creates smooth DNA ends). Here, taking into account the adenoviral packaging capacity, expression cassettes up to a size of approximately 5 Kb can be inserted into the chromosomal environment. Interfaces organized in pairs are available for the insertion of larger cassettes. Depending on the size of the cassette to be inserted, cloning can be carried out using Xhol / Xhol (approx. 5 Kb increased absorption capacity), PmaCI / PmaCI or PshAI / PεhAI (approx. 10 Kb increased absorption capacity) or PmaCI / PshAI (approx. 18 Kb increased absorption capacity). The result is always an expression cassette framed by the chromoεo al spine or parts thereof in a minimal virus genome with the required genome size. By combining the interfaces described, even two and three vectors can be constructed without any problems.

C.2.6.3. More effective gene transfer

Embedding the therapeutic cassette in the chromosomal context leads to more effective gene transfer through various mechanisms. This is primarily due to the fact that the human genomic DNA used in the adenoviral minimal viruses stabilizes the vector after transfer to the target cell.

D. Potential areas of application for pMV and pMVX

D.I. General adenoviral gene transfer

According to the current state of knowledge, minimal viruses derived from pMV and pMVX are without restriction for the whole Spectrum of adenoviral gene transfer can be used. They meet the safety criteria for gene therapy vectors and are compatible with all currently described helper systems for minimal virus propagation.

D.2. Adenoviral transfer of genes in unabridged chromosomal form

Most of the minimal viruses constructed so far contain cDNA constructs as therapeutic expression cassettes, which are usually controlled by viral promoters. These constructs can cause unnaturally high expression of the transgene, which goes beyond the therapeutic scope and can damage the cell.

Complete genes in a natural chromosomal organization with a total size of up to approx. 30 Kb (pMV) or approx. 23 Kb (pMVX) including their physiological promoters can be used in the minimal virus vectors described here, the expression of which is then subject to the cell's own regulatory mechanisms. Damaging effects of overexposure can be avoided in this way.

D.3. Manufacture of virus banks

In the production of minimal therapeutic viruses with the vectors described here, clonal work is carried out with homogeneous inert populations. However, if inhomogeneous preparations of DNA fragments (e.g. fragmented genomic DNA) are used in pMV or pMVX, cosmid banks are created. If these are used for minimal virus production, virus banks are created which can be used for the expression analysis of genes.

This could be used in genome analysis projects, since techniques for systematic functional analysis of the sequenced pieces of DNA are sought. Since DNA fragments which lie in the area of the adenovirus packaging capacity are frequently examined in the geno analysis, adenoviruses would be interesting expression vehicles for this systematic functional analysis. D.4. Generation of transgenic animals

First generation adenoviruses were used for the production of transgenic mice (Tεukui et al., 1996, Transgenesis by adenovirus-mediated gene transfer into mouεe zona-free eggε, Nature Biotechnology 14, 982-985). The construction of the corresponding homologous recombination constructs with the vectors described here provides the possibility of inserting longer homologous regions due to the considerably increased absorption capacity of minimal viruses for foreign DNA, which should lead to an increased recombination frequency. In addition, the generation of transgenic animals in species whose genetic manipulation via ES cell technology is currently not yet possible could be made possible by a viral strategy using minimal viruses.

E. embodiment

The invention will be explained in more detail below using the example of cloning the vector pMVX-BG:

E.l. Preparatory work

The ITRs and the packaging signal of adenovirus type 5 (Ad5) and the constitutive beta-galactosidase expression cassette were first cloned into the plasmid pSL1190 sold by Pharmacia.

Ell cloning of the 3 ^' ITR of Ad5 in pSL1190 (pSLITR is formed)

The 3 ^' -ITR was amplified with the starter oligonucleotides AD5ITR1 (contains an extension of a BglII site) and AD5ITR3 (contains an extension of an EcoRI site) and wt-Ad5-DNA as a template by polymerase chain reaction (PCR). The resulting 176 bp PCR product was digested with BglII and EcoRI and into the corresponding ones Restriction interfaces in the multiple cloning site of pSL1190 are inserted.

Sequence of the starter oligonucleotides used:

AD5ITR1 5 ^' - CGGAGATCTCATCATCAATAATATACCTTATTTTGG -3 ^'

AD5ITR3 5 ^' - CGCTGAATTCCATTTTAAGAAAACTACAATTCCC -3 ^'

E.1.2. Cloning of the 5 ^' ITR and the packaging signal from Ad5 in pSL1190 (pSLITRPS is created)

The 5 ^' ITR with the adjacent packaging signal of Ad5 was amplified by PCR with the starter oligonucleotides AD5ITR1 (contains an extension of a terminal BglII interface) and AD5ITR2 (contains an extension of a terminal Asp718 interface) and wt-Ad5 DNA as a template. The resulting 545 bp PCR product was digested with BglII and EcoRI and inserted into the corresponding restriction sites in the multiple cloning site of pSL1190.

Sequence of the starter oligonucleotides used:

Ad5ITRl 5'- CGGAGATCTCATCATCAATAATATACCTTATTTTGG -3 ^'

Ad5ITR2 5'- GTGACGGTACCAACTCTACTCGCTGGCACTCAAG -3 ^'

E.1.3. Cloning of the beta-galactosidase expression cassette in pSLITRPS (pSLITRPSBG is formed)

The constitutive beta-galactosidase cassette - consisting of the RSV promoter, the beta-galactosidase gene and the SV40 polyadenylation signal - was released from pRSVBGSV40 (cloned by Mr. Peter Löser) by double digestion with Nrul and Xbal as a 4230 bp fragment and inserted into the compatible Hpal and Avrll restriction interfaces in the multiple cloning site of pSLITRPS (cf. 1.2).

E.2. Cloning of the vector pMVX-BG The starting point for the construction of the Vector pMVX-BG was - in addition to the plasmids pSLITR described above (cf. 1.1) and pSLITR2BG (cf. 1.3) - the plasmid pBSKS - sold by the company Stratagene, on its bacterial backbone (replication origin and Ampicillin resistance genes) building up the individual components were inserted.

E.2.1. Insertion of the 3 ^' ITR from pSLITR (pBSKS- creates pBSITR)

The 3 ^' ITR was obtained by double digestion with EcoRI and Asp718

222 bp fragments released from pSLITR and inserted into the corresponding restriction sites of the polylinker by pBSKS.

E.2.2. Insertion of the 5 ^' ITR, the packaging signal and the constitutive beta-galactosidase expression cassette from pSLITRPSBG (from pBSITR pBSITR2BG is created)

Double digestion of the starting plasmid pSLITRPSBG with Pεtl and Bglll released a 4780 bp fragment, which contains the 5 ^' - ITR, the packaging signal and a directly adjoining constitutive beta-galactosidase expression cassette (consisting of RSV promoter, beta-galactoεidae Gene and SV40 polyadenylation signal) contains. This was inserted into the multiple cloning site of pBSITR via the corresponding restriction interfaces.

E.2.3. Insertion of ITR-flanking interfaces for the meganuclease I-Scel (pMV-BG.MH is created from pBSITR2BG)

In pMV-BG.PL there are unique interfaces for Bglll and Spei to accompany the ITRs. To insert interfaces for the meganuclease I-Scel, the plasmid was digested with BglII and Spei into a 2952 bp fragment (containing the bacterial backbone) and a 4965 bp fragment (the beta-galactosidase cassette as well as the ITRs and the PS) containing) split. The fragments were isolated separately and the 5 ^" overhanging ends of the 2952 bp fragment were filled in to blunt ends by Klenow polymerase. The directional insertion of the meganuclease interfaces was carried out by a four-fragment ligation of these two fragments with two synthetic DNA compound pieces which contain the recognition sequence of the meganuclease each contain a smooth end and an overhanging end (matching the smooth ends of the 2952 fragment or the overhanging Bglll and Spel ends of the 4965-bp fragment). These DNA connectors were also hybridized with the oligonucleotide IScel-Rev the oligonucleotide IScel-Bglll or IScel-Spel generated.

Sequence of the oligonucleotides used: IScel-Rev 5 ^' - AATATTACCCTGTTATCCCAT -3 ^' IScel-Bglll 5 ^' - GATCTAGGGATAACAGGGTAATATT -3 ^' IScel-Spel 5 ^' - CTAGTAGGGATAACAGGGTAATATT -3 ^'

E.2.4. Insertion of the cos recognition sequence of the bacteriophage lambda (pMV-BG.MH is pMV-BG.COS)

The CoS sequence motif was removed from the cosmid vector pWE15 by double digestion with Clal and AccI as a 2367 bp fragment and inserted into the unique Nhel site in the bacterial backbone of pMV-Bg.MH. Since the ends of the enzymes used are not compatible, all fragment ends were blunt-ended with Klenow polymerase and then ligated. This resulted in two products with different insertion orientations of the cos sequence. Since the sequence is independent of orientation, one of the two orientations was selected arbitrarily.

E.2.5. Insertion of the 27, 4 kB fragment of non-coding human genomic X-chromosomal DNA (pMV-BG.COS results in pMVX-BG)

The 27.4 kB fragment was extracted from a David Bauer, AG Genome Analysis, IMB Jena, provided Cos id (cos3H10) released by digestion with NgoMI. The human genomic sequence between the 5 ^" ITR / PS and the beta-galactosidase expression cassette was inserted by insertion into the Ago restriction restriction interface of pMV-BG.COS, which is compatible with NgoMI.

E. 3rd Sequence of the vector pMVX-BG (37808 bp)

1 CTAGTGGATC TCATCATCAA TAATATACCT TATTTTGGAT TGAAGCCAAT

51 ATGATAATGA GGGGGTGGAG TTTGTGACGT GGCGCGGGGC GTGGGAACGG

101 GGCGGGTGAC GTAGTAGTGT GGCGGAAGTG TGATGTTGCA AGTGTGGCGG

151 AACACATGTA AGCGACGGAT GTGGCAAAAG TGACGTTTTT GGTGTGCGCC

201 GGTGTACACA GGAAGTGACA ATTTTCGCGC GGTTTTAGGC GGATGTTGTA

251 GTAAATTTGG GCGTAACCGA GTAAGATTTG GCCATTTTCG CGGGAAAACT

301 GAATAAGAGG AAGTGAAATC TGAATAATTT TGTGTTACTC ATAGCGCGTA

351 ATATTTGTCT AGGGCCGCGG GGACTTTGAC CGTTTACGTG GAGACTCGCC

401 CAGGTGTTTT TCTCAGGTGT TTTCCGCGTT CCGGGTCAAA GTTGGCGTTT

451 TATTATTATA GTCAGCTGAC GTGTAGTGTA TTTATACCCG GTGAGTTCCT

501 CAAGAGGCCA CTCTTGAGTG CCAGCGAGTA GAGTTGGTAC CGGCCTAGAT

551 ATGAGGAGGA AT ATCTACT TTCTGATAAA TTATAATAAA CCCTATAGAT

601 TTGAATCTAT GACATATATT TAAAGAGGTG ATCAGAGAAA AGTAGACCGC

651 TTTGAATACT CATAACCATG ACAATGGGAG TA AAAAATG AATCAGTGAC

701 ATTTCTAGCT CAAGGGCTAG ACATAAAACA AAGTTAAAAG AAAGCACGAA

751 CAAGTAATAA TGTTAATATA TAAAGCAGAC ATTAATGAGG GAGAAACAGA

801 AATAGGAGAC ACAAATATTG AAGAGTGTCT TTTTAAATCA ATGAGGCAGA

851 TGAAGCAGTA GCTAACTGAA TCAAAGAAGC AGAGATAGTG TTTCTGTATA

901 TA ATAATAG GTACACAGAC AGGTAGATAG GTAGGCAGGT AGATAGATAG

951 CTAGATAGAT AGACAGGCAT AGAGATAGAT AGATAGATAG ATAGATAGAT

1001 AGATAGATAG ATAGATAGAT AGATAGATTA GATAGATAGA TAGATAGATG

1051 ATAGATAGAT GTTTGCACAG GAGACACACA ACTACCAGCA TACTTTGTTT

1101 GATTGTGCTT TATTGCACTT TGCAGATGCA GCTTGTTTTG GAAGGTTCCT

1151 AATGACCCTG ATTTGAATAA TCCGTGGGCA CCATTTTTCA CAGCATGTGC

1201 TCACCTCGTG TCTCTGTGTC ACATTTTGGT AATTCTTACA ACATCTCAAA

1251 TTTTCAGTAG GACTATATCT GTTTTGGTGA TCCGTGATCA GTGCTCTTTG

1301 ATTTTGCTAT TGTAATCACA TTGGGAAGCC ACAAAATGCA CCCTAGAAGA

1351 AGCAAGCTTA ATCAATCAAT GTTGTGTGTG TTTTGACTGC TCCACTGACT 1401 GGCCTCTCCC CATCTCTCTC ACTCTGCTCT GGTCTCTCTA CTCCCTGTGA

1451 CACGACAGAA TTGAAATTAG GCCAAATAAT AACCCTACGA TGGCCTCTAA

1501 AGGTTCAAGT GAAAGGAGGA ATTGCAGATC TCTCACTTTA AATCAAAAGG

1551 GAGAAATTAT TAAGCTCAGT GAGGAGGGCA CGTTGCAAGC TGAGATAGGC

1601 CCCAAACTAG ACCACCTGTG CCAAAGTCTT AGCCAGGTGG TGAAAGAAAT

1651 GAAAAGTGCC ACTCCGGCGA ACACATGAAT GATAAGGAAG CGAAACAGAT

1701 TTCGTGCTGA TATGCAGAGA GTTTTAGTGG ACATATCATC AAACCAGCCA

1751 CCAGATGCCC TTAAGCCAAG GCCTAATCCA GAGCACAGCC CTATCTTTCC

1801 TCACTTCTCT GAGGCCGAGA GAGATGAGGA AGCTGCAGAA AAAAGACAAA

1851 AAACAAAAAA CAAAACAAAA CAAGACAAAA AACAAAAACA AAAAAAAGGA

1901 GAAGCTAGCA GATACTGGTT CATGGAGTGT AAGGAGAGAG GCCATCTCCA

1951 TCACATAAAA GTGCAAGATG GATCATCAAG TGCTGATGGA GGCACTGCAG

2001 GAAGTTCTCC AGAAGATCTA GCTGAGATCA TGGGTGAAGG TGGCTGTGCT

2051 CACGACAGGT TTTCCTTCTA GATGAAACGG CCTTCTACTG GAAGAAGATA

2101 CCATCTAGGA CTTTTGTAGG TAGAGAGGAG AAGTCAATGC CTGGCTTCAA

2151 ACTTTCAAAG GACAGGCTGA CTCTCTTGTT AGGGGGCTAA TGCAGCTGGT

2201 GACTGTAAGT TGAAGCCGAT GCTAATTTAT CGCTTTGAAA ATCCTAGGGC

2251 CTTTAAGAAT GATGCTAAAG CTACTCTGCC TGTGCTCTAG AAATGAAACA

2301 ACAAAGCCTG GACGACAGCA CATCTGTTTA CATCGTGACT TACTGAAGAT

2351 TTTAAGCCCA CTCTTGAGAC CTACTGCTCA GAAATAAAAA GATTCCCTTC

2401 CAAATATTAC CACTCATTCA CAGTGTGTCT GGTCACTCAA GAGCTGTCAT

2451 GAAGATGTAC AAGGAGGTTT TTTTTTTTTA TTTTTATGCC TGCTAGCATG

2501 CATGAAACAT TCACTCTGCA GCCCATGGAT CAAGAAATGA TTTTAACTTT

2551 CAAGTCTTCC TGTTTAGCAA ACAGATTCCA TAAGGCTGTA GCTGCTATAG

2601 ATAGTGATTC CTCTGAAGGA TCTGAAAAAA GGAAATTGAA AACCTTCTGG

2651 AAATGATTCC CCTTTCTGCA TGCCACTAAG AACATTCGTG ATTCATGGGA

2701 GGAAGTCAAA ATATCAACAT TAAAGGATGA AGTTGATTCC AACCCTCACA

2751 AATCACTTTG AGGGGTTCAC AATGTCAGTG GGGGAAGTAC CTGCAGATGT

2801 GATGGAAATA GCAAGAGAAC CAGAAAGAGT AGTGGAGCCT GAAGAAGAAA

2851 CTGAATCACT GCAATCTCAT GATCAAACTG GAAGGGACAA GGAGTTGCTT lo

2901 CTTACGGAAG AACAAAGAAA GTGATTTCTT GAAATGGAAT GGAATCCATT

2951 CCTGGAGAAG ATGCTGTGAA CGTAGTAGTA GTTGAAATGA CAGTACGGGA

3001 TTTAGAGTAT TGCATAAACT TAGTTGATAC AGCAGTGGCA GGGTTTAAGA

3051 GGGTTCGTTA CAACTTTGAA GGATATTCAA CTATGGGTGA GTAAAATGCT

3101 ATAAACAGCA TCGTATGCTA TGGAGACAGG TTTCATGAAA AGAAGAGTCA

3151 ATCTATGTGT CAAACTTCAT TGATGTCTAA TTTTAAGAAA TTGCCACAGC

3201 CACCCCAACT TTCACCAACC ACCCCACCGA TCACTCAGCA GCCATGAACA

3251 TTGAGGCAAC ACTTCACACC AGCTAAGAGT GTATGACTCA CTGACGGCTC

3301 AGGTGATATT GAGCATTTGT TATAATAAAC TCTTTTTAAG TTAAGGCATG

3351 TATATATTTT TGCCATAGTG CTATTGCACA TCTAATAGAC TATAATATAC

3401 TGTAAACTTA ACTTTTACAT GCAAGAAACA CCAAAAAATT CATGTCACTT

3451 GCTTTATTGT GATGGTCTGG AACTAAACCC ACAATATCTT TGAGGTATGC

3501 CTGTAAATAA CAATAAAGAG GAAATTTTTA AAAATCAGAG GGGCCTCTTT

3551 ACACATCTCT ATGCCGATAA CCTTGTAAAA CTATATGAAC TATGTAATTT

3601 CTTGTGAAAA TACACTCACT ATATAATTGA CCCCAATAGC GCCCAAAAGG

3651 ATAAAAGAAC ATTTGTACAG AGGAAATAGC CTTACGAAGG AAATCTCCCA

3701 TATGAGACCA CCAGGCACAG ACTGTTTCAT AGGCACTTTC TCCAAACCAA

3751 AGCCTACTCT TGAGAGCTAC TGCTCAGATC AAAAGGCTCC ACGCCCACCA

3801 AAACATAACC AGGTAGGGGA TACAATTACA AATAAAACAA TCATTTACAA

3851 AAGCAACAAA AATGATGAAA ATATTTAGGC ATAAAATTAA GACATATTGA

3901 AAGCACTATA CGGTGCCTTT TAAAAAGACA CAAAAGCAGA CTTGGGCAAG 3951 TGGGTAGAAA TCGTTTGTTC AGTGTTAGGA CAT ACGGCA TCATCAAGAT 4001 GTTGGTTCTC CCTAAGTGAG TTTATAAGTT TAATACAATT CCATAAAAAT 4051 ACCAACACCA TTCCCTTTTT CCTGGAGAGA AGAAGATGGT ATCATAATGC 4101 CCATTTCAAA AGGCAAGAAT AGCTACAAAG TCACGGGAAA GGAAGAGCTA 4151 TATGGGAAAA CTGGCCCCAA ACACATTACA GTATACACCC AGCCTCCATA 4201 ATTAAAACAC TGTGGTATTC ACTGGCATGG GAATAGAGAG ACAGACCAAG 4251 GCAACATTCT AGAAAGTGAT AAGACGACCC AGTAATATTT GCAAATTGGG

4301 AGTATGATAA AGGTAGCATT TCAAATCAGC AGGAACAAAG AGGGACTTTG 4351 CAGTAAATAA GCTGATAATG GGGAAAGTGT ATGCTCTTCA ACTAATGAGG

4401 ACAAGAAAAC CGAATTTCTG CACGGGAAGG AAAAAAAGGA ATTGGTTCTC

4451 CACCTGACAT CAGGCACAAA AATCAATTGT AGGTAAGTCA GGAAAAATAA

4501 CTAATGGGTA CTAAGCTTAA TAAGTGGGTG ATGAAATAAT CTGTACAACA

4551 AAGCCCCATG ACACAAGTTT ACCCTATAAC AAACCTGCAC ATATACCTCT

4601 GAACTTAAAG GTTAAAAAGA ACATGAAAAA TTAAATCTCA CAATCTTTAT

4651 TAGAAAA AG AGGGGAAATT TTTATGACTT CAGAGAAAAG ATAAATTTAT

4701 TACAAAAGGC ACAGGTAAAC ACAGTTATAA AGCTTAGCCT CACAATATAT

4751 AAGAACTGGA AGAAAAACAT CT AT AAAG TGAAAAATGG TTAATTCACC

4801 AACATGGATA TTGTACTGTA AAAGGATATG CAAACAGCCG AGAAAAAGAA

4851 CCCAGTCTTA TTAGTAACTG GAGAAATGCA AGATCCCAAA TCCATGCCAT

4901 TTCACACCCA CTAGAATGAC AGCATTAAGC AAAAGGCCTA TTCCACCAGC

4951 GAGTCTGTGT AACAGTGGGA ACACACTCTT CTGGTGGGGG ATGCCTCTTG 5001 GAAACAATTG AGCACTGGGG AATATTCCCT GAGTCTATGA TCCAGGAATT 5051 CCACTCTCTG ATACCGTGTA AACCTTTGGC ACGTATGCCA GACATCTCAC 5101 TCTTCATAGC ATCCTTGCTT AGAAGGGACT GACACTGGAA AGAACTGACA 5151 TGTCCATAGA CAGAGAACTA AGCGGATTTT TTGTAGATTA GCCCCACAGA 5201 GGTAGGTGAT GGAATAGGGG AAATGAAAGC AGCATGTCTA CAGGTAACCA

5251 CAACATTTAA TCAAAGTCAG AGCGGCCCTC TTTCACAACA GCCCTAGCCA 5301 CAACAATAGA GCAGCCCTCT TTCACAACAG CCCTGCCCAG AGGTGAATGG 5351 ACAGGAAATG GACGCATGCG GGGTGGAGTG GCCCCACACG GGTACCTGAT 5401 GCAACAGGGG AGATGAGGGC AGGACGGCAC CTGCCACCCC ATAGGCACCC 5451 TAAGTGACTC AACGGTGAGC TAAGGAGGAA GGGGCAGAAT CCCACAGAGA 5501 TAAGGAAGCA GGTCTTGTGA TGTGGTGGGT GCACGGGACG CACAGCAGGA 5551 ATGCCTGGGA TGCAGCTGGG CGTGTTGGAA GCACACAGGG CATGGGAGGG 5601 AGCCTGGGCC CCACTTCCAG GGTGATGGCC ATTTCTCCCA AAGGAGAAGG 5651 GTGGCTGGAA GGAGTCCAGG TACAAGGTCC TAGGCCACAG CCACATGGCC 5701 TCCACCTCTC AGGGCAGGGT CAGTAGCTGC TCCCCACTCC ACTCCCAGGT 5751 CCTTCTGTCC TTCCCCAGGA TGAGGAGCTG GAGGACCGTA GGGGCGGAGAGGTAGGGGGAGAGGAG1G 5851 GGGGGGGGGG GGCTTTTCTC GGGCCCCACC TCCCACATGC ACTGAGCCAG

5901 CCAATCAGGA AAGGGCACCC ACAGTCAGGC CAGCTGCTCT CGCGAGGACC

5951 AAGGACCCAG GCCCTCAGAG GAGCATCAAG GTGCCAGGTC CTGGAGGTCC

6001 TGTGAGGAGG AGAGGTCGAG GGTACCTCAG GAGAGGACCT GGCCGGAGGG

6051 GAGACTGGGA CCGTGTAGTC CGAGGTCCTC AGAGGACGAG AGTGTGGATC

6101 TTGGGGCAGC TCAGGCTGAC CTGTGAGGAA ATCTCCCCAG CGTCACCTGG

6151 GGCCTGGGTC AGGGAGAGTC CCCTAGGGGC AGGCTGGCAT GGGCCAGGGA

6201 AGGGAGATCG AGGGGTCCAG GTGGTCCCCA GTGCCCGGGA GCATCACTGG

6251 GTTCTGTGCA TGTGCTGGAG GCTGGGGGAG AGTCCGCGTG TGTCCCTCTT

6301 GCTCGGCCAC CCATCTGGAT GGGACCCTTG GTCACCGGGG GTCTGCTGGG

6351 CGTCCAGGCA GGTGAGAGGG CAGATGCCAG CACCTGACTG TCGAGGACGG

6401 GTACACAGAG GTTCATTCCC ATGCAGATGG CTGGGGAGGT CCTGTGTCAC

6451 CCTACAGCTC TCCCGACCAA CACGGGGTCA CTTGGGGACC CGGGGCAGGG

6501 GCAATCTCTG TCCCAAGGGC ACGGCAAGAA TTCAGCAGAG GACCTGGAGT

6551 TCAGGTGTGC GGAGCAGGTG TGAGCTCTGC AGGCAGCCCC AGGGAAGGGC

6601 TGGATGGGAC ATAACGGGAC AGGTGGGGAC GCTGGCCTCT TCTCCCTTGT

6651 CCTTTGCTCC TTCAGCAGTA GACACGGAGC ATGCCAGCCC CACTCTTCTT

6701 CAACCTGGGG ACCTCCCCGT TGGCTCTCAT TTAACACACC TGGGTTCCTT

6751 AGTGTGTCTT TGCACTCACA AGGTTCAGGA GTCGTCTCTA GCCTCCCAGG

6801 ACGGCCAGCC ATGGTGCGTA TGGACGGGAA GTGCGTCAGG GAGGCTGACA

6851 AGGCCCTGCT GTAAACTCAG TTTATGGTAG CCCAGGAAAC GGGGCATGGA

6901 CGTAAAACTG GAATGAAGGG TCAAGGAAAC AGGTGCCCGC CCAAGGGGAC

6951 GGTCTGTCTT ATCTCCTTGC ACCGGGTCGT GGGGAGCACA TGGGCAGCCC

7001 GTGGCACCCT CGTTACTTTG GTGCCCACCT TGCATATGCT AAGAAGCCCT

7051 AGGTTTCTAA AGCAGTTTCA AGGGCTTTAG GTTTCCCATG GGAAGCTCCT

7101 GCCCCACGGC AGAGTTACAG ATGCCCTTAG AGCTGCTCTT CTGGATGTGT

7151 GCAGTCCGTG ACGTCCAAGG AGTCTGATCC GCAGTGCATG GTTATCTATG

7201 ACTTCAGTCC TGGAGGGCTT GCAAAAATGT CTCTGTAAAA TGGTCAGGGC

7251 TCAAGGGACT CCATGTATAT AGGTCTGCTT TAAAGCTTTA ATTGGCAAAC 7301 AGCCATACAA ATCCACAAAT AGTACATTTC CATGTGAAAA AATGTCCACA

7351 ATAGCATCCT TCCTCTAAAA ACGTTTGCCT TTTCAAATTT TCAGTGCAGC

7401 ACGGAATTCA GGAATGCACA GCCATCCCGA AAGTAGTCAT TTTGCAGAAA

7451 CAGATGAGGA AGACATGTGG TAATGTGTTT AATGTAATTG TGCCTCCTAG

7501 TAAAGTAAGT TCAGAAAAAT CAACTTTGAC TCGTGGCACA TTTTGTGTTT

7551 AAAACACTGG GCATCAGGCA TGCCCTACAA CACAAGCGTG GTATCTTATT

7601 CACACCTAGT GAAAAAAAAA AAGTAATTTC TTCCAGGAAT GAATTTCAAA

7651 ATATGTGGGA AAGCTTTGTA GGTGTCTGGG AGATACTTGT AATTCAAGAA

7701 CATTGTTGTC AGCCATATAT CTTTTATAGC AAGAAATGCT AATGAGGGGG

7751 CTGCATTAGT GTTATTTAAT GCTTTGTTCT CCTATCCAGG ATGCCACATA

7801 AGGATTGAGT GTGATTCAGC TCCATCGATT TCCTTCCTTA CCCTTTTAAG

7851 CTGTTGAGTG AAGTGGATGG TACACACAGT GGTTAGGCAG AGTAATGCTT

7901 TTACTTTATT CACTGTGGAA TATTAGAAAA TAATTTACTA ATAATATATA

7951 TTCCACATTG TGTCAGATAG TAAGTGATAT ATCTGTTTTT CATATCCATT

8001 GCTGTTTTCT TATTCTAGTT ATTTTATAAG ACTTTCCCAT TTCTTTCTTG

8051 GGGTGGGCAG TGCAAACTGA GGCCTCTAAA ATAGCCTCTT ATTGTTATGT

8101 TTACATTAAT AACCTCTGTA GTGGAGAACT CAGACAACTT TGCTGGCATG

8151 TGAGAGACGT GTGATCACAA CCAAAAACAT CTAAGTTTTA AATGGATACC

8201 TCAGCTATCA AGCATCTAAG TTTTAAATGG ATACCTCAGC TATCAGTTAA

8251 ATTAACTATG TAAAAGGACA AACTCTTAAC CGTGTCATTC TCAAGTTTTA

8301 TCTCCTCTAT TTGGATCATA TATGTGAGCA GATACATGTT GAAAGCTTTG

8351 TTGTATTTTG TGGTGAAAAT GTGCTTGATG TGCGAGCTGT CTTΛTTAATT

8401 GGTTGTTATA GACGGTAAAC TGCATTTATA AAATATTTTT ATAGTAATAC

8451 AGCTCCCTCC TGAACACTGG AAAATAACCA CTGACGGTAG GTCCATAACA

8501 TAGAATCAAT CATATGGTTA GCAAGGAATT GTTTGAAATA CTTAAAAAAT

8551 GTACAGTGTT ACAGATTGTA AATTTTACAA TTTTGCAAAC TTACAATCTG

8601 CAGCAATTTG GGGAGAAATG ATATATGAAA ACCTGCATGA AAGTATCCAG

8651 GAAGTGTTCT TGAATAAATA TTAATTTCCT TAAACTATCT GTGGTCATTT

8701 TTCTTAGTCA AATTGATTTA AAAGCTTCCC GGGCTAAATT TTAAAAATAC

8751 TATTCTTAGT TTATCATTTA TTTATTTTTA TTCCTTTGAA AATGGAATAA 8801 TTGTAGTTGG TTAATATAAT GTGTTCTAAC CATGGTCGGT CTAAACATAT

8851 CCTTGAGTTA GAGTACATTT TATTAATTTA TTAAGAATTA TTTTTCAGAA

8901 AAGTTTTCTC ATAAAAAATT ATAGGCTCTC CTTGAGACCC TTTTTACAAG

8951 TTTTAGAAAA CAGACAGCTT TTCTCTTTAA CTCCTTATTA CATCTTCGAA

9001 ACAATGTTTT CCATATTGAG GTATCTGTCA CTGTAAGTTT TCACAGACGA

9051 AAATGAGGCA AATAATCTTT TATTTTGAAA TGATGCCAAT TTGTTCATGC

9101 TAGGTGTCTT TTTAATACTG CTGAACCAAC ATTACCTTTA TGGACGTGAA

9151 TATGTGGGAG CAAAATAATT TTAAACAAAA TTTTAATGAG TGTTTTCCAT

9201 ATAGGGCACT TTAAAAATGT TCCTAAAGCA TCGATAAAAC ATTTCAAAAT

9251 GAATATCAAT GCTTCCACGA AAATCATTCA GCAGGAAATT GAACTTGCTT

9301 GGCATACCCG GGAAGAACAA TGGTAAGATT GTTGGTTATG TTGGAGCCAG

9351 ATGCTTTGTT TCATTTCTTA GCATTCGTGG ATACAAACTA CTTTGGAAAA

9401 ATGCTATGAT CGTCCATGTG CTGGAAGTCC AATGAAAAAA TATATTTAGC

9451 TGGATTTTTC TCTAAAGAAA TAATTCCTTA ATTAAGAATT AATTAATACT

9501 TAATTTAAAT AATCTAAGTG CTATGATGCC ACATCATAAT GAAATGAGAT

9551 GTGTATCCTC GAGTTCACTA AACATGACCT GTTTTCTCTT TCAAATTACA

9601 GTCGATTTTG CTTATCATGA AAATTTGTAT TCATTCCATG AATGTGCTTG

9651 CTGAATTAAA GTTAAAAGTC TTAAACATTT AGTCTTGTAA TAGTTATTTG

9701 TATTCAATTA CACAGACAAA GAT AATGTT ATATTGTAAA TAATTTGTAA

9751 GCTTCACTGA ACTGGGTTCA GTATATTTTT TTTTGTACAG AATTCTTATT

9801 GTCAGAAAGT GAGGAAAATG AAATATTCCT GTTTGGAAAT CATAGATGAT

9851 AAAGGAGTGT AATATCCAAA TTAAGTCTAT TTGTGATATC TCAACTTATA

9901 TACTACTCAA AAAGAGAAAA TAAAAACTAA AACACTTGCA GAGAAAAATG

9951 CATTTTTTTC TGGAAAATTT ATAATACAAT AAATCTTCCT TAATTTATAT

10001 TAGCTAAGGC ATGTTAAGAT AGCTTCCGTC TTACATTTAC TAGCCTTCTT

10051 TTTTCACCCA TGCCTTGACT GTACCATGGT CACACATTTC TTATCCAAAA

10101 GCAAAGGGAT TGTTCATATA AAGAAATTAT GAGTGTTGGT TGATCAGCTC

10151 AGTTTTGGAA ATGTTAAAGT TGAGGTGCAT ATGAAGCACC TGAGGGCGGA

10201 TGTCAAGTGG GGATAΛGCAA TATACCCTGG ATATATGAGC CTCATGTTCA 10251 GGAGAGTGGT AGGAGCTGAG GACACCAGAG CGGGAATTAA TATCACACAG

10301 ATTGCGTTTA ATCAAGGAGA CTAGGAGAGA TCACCTAGGG AATGACTGTA

10351 AACAAAAAAG GGAAGTCTAA AGACTGATTC CGCGTTCCTT CAATACTTTG

10401 AGCTACCGCA GAACTGGGGC ATCAGTAATG AAGCCGAAGA ATGAGTGAAC

10451 AAATAAGTAA AAGAAAACCA GCTGAAACAC AGTAAAGAAG ACCTTTTAGA

10501 AGGAGAGAGG ACCACCTCAT CACATGCTGC TGATAGGCTG AAGGAGGGGG

10551 ACTTGGTATA TGGAGGCCCT TTGATATCCT TAG AAGAGT GGTTTGGTGG

10601 AATGGTGGGT GCAAAGGACT GATTGGAAAA GACAAGAGAC AACTGGGAAG

10651 AAAGAACTGG GGTAGTAACC TGAGGCAAAA AGGAGTAGCT AGAGGTTTTT

10701 GTGGTTTTCT TAGTGATTAC AGAAGGACCG GCATGTTGTA TGCTGATAGG

10751 AACAATA AG AGGGAAACCT TGGAAATGTT GGAAAGAGGA GGGGAAAGTG

10801 CTGAACGATG TTCTTGAGAA GCAGTGGGCT CTAGTGGACA GGGGAGCAAG

10851 TTGACTTCAG GAAGGAGTAG TAGTTCATGC ATTTAAACAG GAGAGAAGGT

10901 CGAGTGCGTA AGCACTGATG GACAAAGGTG GGTGAAGATG TGATGCTCGG

10951 ACGCTGAATG TGCCCTGTGG GTTGCTTCTG TCTCTGTAGT GAGATAAAAG

11001 ATCATCAGTG ACAAGTAGAG ATGGGGCAGG AGGGCAGAGA AGAAGGACTA

11051 TACTTTTTTT CCTGAGACTG GGCCAGTGAG TGGACTAGGG GAATATAATT

11101 GTCAAGCAGC ATTGGGAGTG CAAAT AGAT TAGTGTCATC ATAAACACGA

11151 CTTTGAGATC TGTTGGCATG GTTGTGT AG GGCAGCTGGA GAGTTGGATT

11201 TTTCTGGGTT GGCATTTGGC TGAGCACTGT GACAGAGGGA GAAGCTGGCA

11251 GGGCACATAC ACAAGGATGG AGTTATGGTG AGTGACCATG GAACTTAATC

11301 TGGGAACGAA GAAAGGGAGT GTTGGGCACT CCAGTGTTGG ACAGTGAAAG

11351 GTGCTC ATT CAATGGGCTT CAGTTCCTGA TAGACTCCTG AGCTGTAGTG

11401 CTTAGAGTGA CCTACAGTGT GAGGGCGTTA GTTGTTGCAT GTTGGATACA

11451 TGGGGAAATA GCATAGTGTA GTGGTCAAAA GCCCGGACTC AACCCCCACT

11501 GTTTAGAATC TAAGTCTGGT TCGACCATTG CTGCCTGGGA AACCATGAGC

11551 AAATTCTTTC TGTGTCCCAG CTTGCTCCAT TAATATTGTG GGCATAATGT

11601 GAGTACCTAC CTCACAGAGT TGTTTTGAGT GTTAAATGAA TTTTTC ATA

11651 ATAAGGTATT TAAAATGGTT CAGAACAGGA TCTGACATAT GGTTCC ATA

11701 GTACAACTTT GCTTTTATTA ATGTTATGTA GATATTGTAG ATTCTCAGGA 11751 ATGTCAAAAT CTAGGATGTG GCTATGTGAG TGTTTAATTT GCAGGAAATG

11801 TCAAGATCAT TGGTGGTGGA ACAGAAAGAC CAGTTT ATT GGAAGGATTA

11851 CGTGTGTGGG CTTTGAAAGC ATCAAGGACT AAAACA ATT TGTAGAATAT

11901 TATTAAAAAG GAACATTTCC ATAAAAAATG CATGATAGGA TTATTA ATA

11951 GTTTTTGTAT ATTATTGTTG GACACAAATT TCATAAACTT GGTCATAGTA

12001 TTTGTTAATA AGAAAAAATA TTTTCAAATG ATAATTTCAA AATTATTAGC

12051 TTTAAGTTAA CAAAGAATTG TTTTTTTGAT AAATTTTAAA TTTTACTCGA

12101 ATTGTTATCG AGTTTTCTTT GTATATTTGT AGAGCA ATT TCTCAACAGC

12151 AACAAAAGAC TTTTTCAAGA ATCTGAAATT GTTGAGGGCC AGAGAACAAA

12201 AGCATTTAAT GCATTATGTG AGGAATTCTT ACAGGTCAGT GGTATTTGGT

12251 TACACAACAT ATTCTCGTAA GACTTTACAT GTATATTGAA AATTTAAAAA

12301 ACTGTTTTCC TTTCCAAAAT AGTGTTAAAA ATACTTTGAT TATAAAATTC

12351 TTAAGCATTT TCCATGTTAA TGTCATTGTT TTAAAGTAAG AACTAAATGT

12401 ATTTTTACTT TTAGAATTCA TTAATTGTTT TTGGTGTAAT CCAAAACAGG

12451 AAACGGTAAC TTGGCAATTT CACCCAGAAA TATACATACC GTGTTGGCTA

12501 TTCACACTTT ATTTACATGA CTAAACACCA TGACTCTTTT GCTCGACAGT

12551 TTTTACTGGT GCAGCAATAG ATGAAAGCAA GAAGTTTCTA CATCATTTCT

12601 AGATGTGGTT TAATAGTCTT TCTAGTGCCA GTGTTTCTCA AAGAGAAGGC

12651 ACCCAATTGT AACAAATGGG TATTTTTTAT TTCAAATTTA TTTCCTGATT

12701 ATTTCATGAC ATATTTGCTT GCACTATTAT CGAAGTGACT ACAAATTGAT

12751 TTCTCTGCCA AGTTACTATC ACAAGACAGG GCCCAGATGC GTTCAGAATG

12801 AAAAAGCCAG GAAAGAATTA ACAGTTTGTG ACTGAGAATT GTCTCCCGTC

12851 ACATTTGTAC TTTTGCAAGT TGTCCTCGGT GATATTGACC TCTTAACCAT

12901 GCAGGTATAT ATCTATTTAG GTTTCTATGG AATGGTGTAC ATTCCTATTC

12951 ATTTTATAAA TTTGGAGTGT TTTTAAAACA ATCTTGTTCA AATTCTTGTT

13001 AAAAATTCAC TCTTCTAAAC TGAGGGGATT GTTTCAGGAA AACGAGTTTC

13051 AGGATAGTCA TTCTTTACAT TGTTTACTAT GATACATATA CTACATGTGT

13101 GTATACATAT ATGTAATATG TACACTCAGT TGTTATTCAA GTGTTTCTAA

13151 GTAGAGGATG TGTTGTTTAA TGGTCTCTGA GAACCATGTA CCAATTGTTA 13201 CACATGTTTG GGAAATAAGA TCTTCTCAAT GTTAGTGCTA AATGTCACCA

13251 CTTATGGTGG GAACTTCATG ATGATTTAGT CTTCTGCATT TCCTTTTCCT

13301 TCCCATCCAG CTATCTATCA CTAGGCCCTG CCATTTTTAC CACCTCAACG

13351 TTATTTTAAG TTTTTTGTTT AGATTCTCAC TGCTGCTGTC CTAGCTCAGG

13401 ACCTCATCCT TCCTCACTTA CGCTATTAAA ATTTCATCTG AGTTCTCTCT

13451 TCCTCTTTCC ATCCTTCATA GATCTGCAAT CTTATCTTCC TAAGACATCG

13501 CCCTTCCCTC TTCCCAACCT GTCACGAGCT TTCCATTCTA AATTCATGGA

13551 TATTCAGAGA CACCTCTGCC TTGACATGAC CTCCTTTTAA AACTTCATTT

13601 GCTTTTGTCC TTGCGATAGT TTGCTGAGAA TGATGGTTTC CAGCTTCAAA

13651 AAACCAAACA CTGCATGTTC TCACTCATAG GTGGGAATTG AACAATGAGA

13701 ACACATGGAC ACAGGAAGGG GAACATCACA CACCGGGGCCCC TGTTGTGGGG

13751 TGGGGGGAGG GGGGAGGGAT AGCATTAGGA GATATACCTA ATGTTAAATG

13801 AAGAGTTAAT GGGTGCAGCA CACCAACATG GCACATGTAT ACATACGTAA 13851 CAAACCTGCA GGTTGTGCAC ATGTACCCTA AAAACTTAAA GTATATTAAA 13901 AAAAAAACCA CTTCATTTGTC TACATTCTTT CCTTCCTAGTTCATCCCTCTCCTTAG1 TCCATCTCCTAGTTC1

14001 AGCCCTTCCA CCATAAAAAT TTTATCTCCT TTGGCTAACC TATCGAAATC 14051 TAATTCTTTT GCCAACTAAG TAGGCATTGA TCTCCATTGT CCACTGTACA 14101 GTGCCAGCAA GTTGCCCTTC CATGTCTATT GTAACATTTT TGTATGACTT 14151 CAGTTTACAT GTTTCCTGAA GTCACTGGAA TATGGAAAAG ATCATGTGTT 14201 CATCTTCTAA TGCCACCATA GCACATGACA TCCTAACTTT TCTGTAGCAG 14251 GCATTATTTC ACTGTTTGTT GAATGAAGAA TAAAGATTTA AAATTCAAAT 14301 TGTATTTTTC GTAACTCGAG ATCATACTAC TGAGCATTTT ATTCCCTCTA 14351 TCTATGGAAA CCATGTTACA TGCAATATCA GGGATAATAA ATGGTCGAAT 14401 ACCTGGTGTT TGGGAAGCTT ATGAATGAAA TTAGCGCATG GATTTATATG 14451 GAACTTCCAT TCTTTTTGCT ATATCTTCAC CTGAACATTT CAATATCCCA 14501 TTGTTTGGAG CAAATTGTGA TATTGTCGTG CCTCCTACTG TAATCATAAT 14551 TGACTAAATT CACACATTTC TATGTTTCTT AAGTGAGTAT TTTGGTCTTC 14601 GGGTAGCTCT TAGAGTGCTT TTTAAATCTG GAATAGATAA TAACCATCAC 14651 TGGAATTTTA AAGTGTTTTG CTTTTGAAAT GTAGTTATAC ACAGTTATAA 2.0

14701 GGTGATGAAG AGTGGTTGGT TATAΛCTGAA CTTGGTTTTT TGTTGTAGCA

14751 CGTGGAGGAC CTGGAGGΛGA ATCATACAGT TTGTCTTACT GGTGAACAAA

14801 GTGAACTTAG AAΛAGAAATG GGGCCGGGCG TGGTGGCTCΛ CGCCTGTAAT

14851 CTCAGTGGGC GGATCACCTG AGGTCAGGAG TTCAAGACCA GCCTGGCCAA

14901 CATGGCGAAA CCCCGTCTCT ACTAAAAATA CAAAAAAAAT TAGCGGGACG

14951 TGGTGGCGGG CACCTGTAAT CCCAGCTACT AGGGAGGCTG AGGCAGGAGA

15001 ATTGTTTGAA CCTGGGAGGC AGATGTTGCA GTGAGCTGAG ATTGCGCCAT

15051 TGCACTCCAG CCTGGGCGAC AAGAGTGAAA CTCTGTCTCA AAAAAACAAA

15101 ATGGAAAAAA AAAAGAAATA GCAGTGTTGT AAACAGAATT TATGAAGGAA

15151 ATTGGTAAAA TTGAAGATTG ATTTAAGCAG TAAAATATTT TGTTCGCTGA

15201 ATTTTTCTAA TATTTACCTA AATATTCAAT AAATGATTAA TATCACTTTT

15251 CATGTAACAG CATCTGCAAG AGGTGTCTGG TGTTCTCCTA TCACAGTTAT

15301 TCTGATGACA CTTCAAAGAA CAAAGCTAAA CCTAGGTGAT AC-CATACAGC

15351 CATAAAATTA GCTCCATGAA ACCGATGTGG CCTGAAATTC CTAGTTTAAA

15401 TTGTTGCATA CTGTTTGTTT TCATGGTGGA TCCCTTTTCT GCTAAAACCC

15451 ATGTAAATCC TAAACAGTTA TGTGAGCAGT AGTAGCCAAT CCAAAGTTGT

15501 ATGTAGCGGG CTTTCAGTCC TTTCCCTAAT AATGGTCACC TAGTCTCCCA

15551 AACATTGTCA ATGAACTTTT AGCAATAAAG ATGATCTGAT TTTACATTTG

15601 GCCTCAGTGG ATCTATATCA GTCTCAAATG GTAGAAATCA CAAGTATTTA

15651 AGCTTACATT GTTATTCCTA AGGAGAAAAA ACATAAGACA AAATCTATTT

15701 TATGAACTTA TTTTTGTGTA TCCTTACTAA ATTATGAAAC CAATTTTCAT

15751 GAAATAAAAT TTGAGAGGAΛ ATATGAAAAC TTAAAAGCAA CATAAGTTTG

15801 TACCCCAGAA TAAATTAAAC TGCTAAAAAC CATTAAGACC ATTGAACAAG

15851 ACATGTAAGT ATACATTGTG AGTCATGGAT TCATGAATAT GGCATTTTCA

15901 CAAACACATC AAAATGTCAT TTAATTGATT TGGTCCACTA TTAAATGATG

15951 CTTGTCTTTA GCACTGACTA GAAGTGTTTC CTAAATATAC AAGTATGCAT

16001 AATTATTTAT GGTTTACATG TTTCAAGGCT TTCTGTAGAG ATAAATTATC

16051 TACAGAATTT TTTTCAAGTG CAATATTCAG TTAGTATATG CAAAGTCATT

16101 TGTATATTTA TTTGAATATG AGAACAATTA ATGCCTATAT GTTATTTTAA 16151 TGCACTTAAA AAATATAATA ACACTTGAAA TCTTCCTTTG GAAACATTTG

16201 ATCTAACAAA TGAAATTGTT TCAAATTTGG TTAGAATAAG TCAGTTAAAT

16251 GTATTCTTTT GCTGCATTAC AGAAATTGGC TTCATGCCAT TCAAAATCAT

16301 CAATATTTCC TTTTGAAGGA GAGGGGAACT TTCTTCTGTA TTTGTGCAGC

16351 TTTGGAGAGG GAGATGTACG AAACGGAATT AGCTGTCTTG TAGTGATTGG

16401 AGCTGTGTGA TGAAAACGTA GTGATCGCTA TAGTAGTCTT TCCAGTTTGG

16451 TGCATGTTTG ACAAGTTTCA AGATCCCCCC ACTGTGTAGA ATTGTCAGAA

16501 ACTACTCCAG CCACATCGAC GGACCTGAGA ACATTATGCT TAAGTTAAAC

16551 AAGCAATTTC CAGAAGGACA AATGCTTCAG ATTCCACTTT TATGAGATAT

16601 CTACAATAGT GAAACTCACA GAAGCAGAAA GTAGCATGGT GATTGCCAGG

16651 GGCTAGGAGG AGGGGAAAAC GGGGAATTGG ATTGCAATCG GCATAGAGTT

16701 GTAGTCATAG AAGATGAGAA AGTTCTAAGG ATCCACTGTC AAACAATGGG

16751 CATATACTTC AT ATACTGT GTTATATGCC TAAACATTTG TTAAGAGTAT

16801 ATATGTCACG CACTGTGTTT TTACCACAAT TTAAATAATA CTCCTAATTC

16851 CCTTATTTCA TCTATTCAAC ACCTTAAATT TAAATAATAC TGCTAATTCC

16901 CTTATTCCAT CTATTCAACA CCTTAGAAAA ACTGTTAGTG GCCAAGCGTG

16951 GTGGCTCACA CCTGTAATCC CAGCAGTTTG GGAGGCTGAG GCGGGTGGAT

17001 CACCTGAGGT GGGGAGTTCG AGACCAGTCT GGCCAAGATG GAGAAACCCC

17051 ATCTCTACTA AAAATACAAA ATTAGCCAGG CCTGGTGGCG CATGCCTGTA

17101 ATCCCAGCTA CTTGGGAGTC TGAGGCAGGG GAACCGCTTG AACCCCTTTG

17151 TCAGTTTATA AAATGGATGC GGCATCCTTT CTCAACAATA CATAACAACA

17201 TGTATCTCAC TTTTCACAAT ACGTTCTATA AATCTAAAGT GGGCCTACAC

17251 CAAAATAAAT TAAACTATTT CTTTCTAAGG ACACATAGAT TGATGTATGG

17301 AAGGAAGGAA GGAAGGAAGG AAAGGAGGGA GGGAGGAAAG AAAGAAAGAA

17351 AGAAAAAAGA AAGAAAGAAA GAGAAAGAAA AAGAAAGGAA AGAAACGAAA

17401 GAAAGGAAAG TAAGAAAATA AAGGAAAGAA AGAAAAGAAA GGAAAAAGAA

17451 AAGAAAACCT GTTAGTACTT CACTGGCCTT GTCTGGCCGC ATGGTGTATG

17501 AGGGAATGTG TGGGCTCTGG AGTCAGACAA GCTCGGTACA ACTGCTGGGT

17551 TATCTCGAAC AGGTTATTAA CCTTGCTGGT CCTCAGTGTC ATCTTTTCTA

17601 AAATAAGGAT GAAATAAGAT GGGCCCATCT ATCTTCCAGC ACCATTGTAT 17651 GGATTCGTCC TGTGAGTTGC TCAGTCATGA CAGTCATTAT TATTTATTAT

17701 TATTATTATT ATTATTTTGA GACAGTCTCG CTTTGTCGCC CAGGCTGGAG

17751 TGCAGTGGCG CGATCTCGGC TCACTACAAA CTCCGCCTCC CAGGTTCACG

17801 CCATTCTCCT GCCTCAGCCT CCCGAGTAGC TGGGATTACT GGCGTGTGCC

17851 ACCACACCTG GCTAATTTTT TTGTATTTTT AGTAGAGATG GGGTTTCATC

17901 GTATTAGCCA GGATGGTCTT GATCTCCTGA CCTTGTGATC CGCCCGCCTC

17951 GACCTCCCAA AGTGCTGGGG TTACAGGCAT GAGCCACCAC GCCCGGCCTA

18001 TTATTTTTTG ATTAGCATCT GGCCTTTCAC CGGGATCATA GTAGTTATCC

18051 AGTAATTATT TGTTGAAGTA ATAATTTAAT TCAACCTTAA TGAATTTTTT

18101 TATTTTACCC AGAATTCACA CACTTCTCCA TTTTTTACTG CAACAATTCT

18151 TAAGTAATTA TGGGTTGTAT TCAAAGTTCT CAGTCTAGAT AAGATTGAAA

18201 ATTATGTATC AGCTTACTAG TCGAGCTCTT TTTGTTATTA CGCTTCCTGA

18251 AGGGTCTTCA ACTCTGTTTC TATTCTGACT GTGTGTACTC TTTCCCACTT

18301 TCCTGGACCT GTGCCTAGGA CCCTTCTCCA ACAGCCCTAC AAGTAGTTAA

18351 TCCCCAGCAC AACACTTTTC TGCCAACAGT AGTCTTCTAT TTCTTTCAGG

18401 AAAACATTGG CTAAAAGCTC ACATTTTCCA TAAAAATATC CCTGTATGAA

18451 TTGACCCTAC ATCGATCTTG CTCTCACTGA GGGTTTCTTC TGAGCTCATA

18501 CAAACCTAAA GCAGGTCATT TCACACTTGG CTATTGGGTC TATAGCAGAG

18551 TCACAGAAAT ATGACTCCTG TCCCTAATAT AAGGTAAGAT GTGGTCCCTA

18601 AAGCAGGGGA CTTTTCTTGA GTGTCTTTTC TCCCTTGCAC AGTATCTAAT

18651 ACAGTGCTGC TCACAGCACT GAGCAGCAGT CTTTAAGACG GTGGTTTGGA

18701 ATTTGATCTT TGCAGACTTG AGTTTTCATC CTGGCCTTGT AAACTACAGG

18751 CACTGTAAGA AAGTGTCCCA AACGCTCTGC CACATTTGTA CAGGGGAGAT

18801 ACTATGCATT TTATAGGGCG TGATGGTTAA TTTTAGGTAA CCAAAATGTG

18851 ACTGCGTTCA GGAATACATA GAGAACTGGT AAAGCAGTAT TTCAGGGTGA

18901 GCTTCTGTAG GCATTCCAGA TAAGATTGGG GTGAGTTGGT GGGCAGAGTG

18951 AGGAAGTTAC CTTCAATCTG GGCGGGCCAC ATCCAATTCG TTCAGGACCT

19001 TGATAGAAGA AAGAAGGCAA GGCAGATGAA ATGCACTTTG CTCCCTTTCT

19051 CTGCTGAAGC TGAGATTCTT CTCTTTCTTT TCAGATGGAG TCTTGCTCTG 19101 TCACCCAGGC TGGAGTGCAG TGGGACCATT TCGGCTCACT GCAATCTCTG

19151 CCTCCTGGGT TCAAGCCATT CTCCTGTCTC AGCCTCCCGA AGAGCTGGGA

19201 CTACAGGCGC ATGCCACCAA GCCAGCTAAC TTTTGTATTT TTAGTAGAGA

19251 TGGGGTTTCA CCATATTGGC CAGGCTGGTC TCAAACTCCT GACCTCGTGA

19301 TCCGCCTGCC TTGGCCTCCC AAAGTGCTGG GATTACAGGC GTGAGCCACT

19351 GCGCCTGGCC GCTGGGACTC TTATTATCTG GCCATTGGAC ACCAGAACTC

19401 TTGGCTCTTT GGCGTTCAGA CTCCGGGACT TACACGCCCA TTCCCTGGCC

19451 TGCCCAGGTT CTCAGGCCTT TGGCCTTATA TTGAGAGTTA TAGCATTGGC

19501 TTCTCTGGTA CTGAGGCTTT TGGATGTGGA CTGAGTCACA TTCCTGGCCT

19551 CCCAGGGTCT TCAGCTTGCA GACAGCCTGA CCTTAGACGT TTCACCCTCC

19601 AACTCCCCGA ATCAATCCCC TCTGATCTGT CTATGTATCT TTGTCTATAT

19651 CCTGTTGATG GTGTCTCTTA AGAGTCCTGA CTATTCCACA GGGGTAATGT

19701 GGGGGAGAAA TGAGGTCATG CATGCAGAGC ATTTCACACC CTGCCTGACA

19751 CATGCCCTTG CCCAGTAGAC TGCAGCTACA GTTATTCTAG AGCTTACAAT

19801 CAGTGTTCAA AACAGTGAAA GGCCTTCTAT CTGGTACCTA GCCATCACAT

19851 TGAGCTTCTC CTGTCATTTA TCAATTTCAG ACTTGTTTTG AGAGCAAGTC

19901 CCTGTTCTAT GCAGCACTGA TGTGGGGCTA AAACATTTTT AGGCATGAGA

19951 TACTCAAGCT GGGAATGAAC AAAGGCCAGA ATGTTCTTCA GAGTTGGGAG

20001 CAGAGGAAGC TACATGGTCA AGAGACATGG ACATGAGCCT TCCTGCAATA

20051 TGTTTTATTT CCAGGGGTCT AATGAGAAAG ACATGCACAT ATGTATATAT

20101 CATCTCATGC TTAGCTCCTT AATATGCCCA AGGTGTTTTC ATATTTTTTC

20151 AGCAATTGTC TACCACAGCT GAGAGTCACA TGATTACCTG TTGTTGGTGG

20201 CTGTTGGCAA GGGGGTTTTT TCTTTAGGGA CACAGGAAGA TTTGCCTAAA

20251 AAGGTCAGAA AGCTTTGACA TCCATTATTT TATTATAAAG GTCCTCTACA

20301 GCTTTGGTTG TA AATATTT CGGGCCTTTA CTTTTCTCAT CCCACTCTTT

20351 ATTTTAAGCC TGAGCTTGTG GGTCCTTGGA GAAGGAGAGA AAATATTTAA

20401 ACTCTGATCA ACTAATTTAA CTATGTATAT AACATGTAGA ATAAATAAAG

20451 ACTTAAATGA AAGCATAGCA TTTACGAAAG CTCCAACAAA TTAAAAGGTA

20501 ATTTAAACGA TTGATTCCTG GTTTTAGTGA CTTGTTGTTA ACAAATTAAA

20551 GCAGTTTGTG AAAATAAACT CAGTTACATT TTTTGCTTGT ATAAGTTCAA 20601 AATCGTAATT CCTTTCCAAA GCTGAACATG CCTGAAATGG GTCTGCCAAT

20651 CATCACAATA AAACAGGAAG CCCGAATGCA AGAGCTAGTT AAGATGGGTT

20701 CCCAAGCAAA TAGTAGTTGA AGCTCCAACG GCCGTAAAAT GATGAAAGAA

20751 ACATGGTTTA AGAGCCATGG ATAATTTTTT TGAATGCCAA AAGAGTTATC

20801 ATAGACTGAT TTGACATAGA GATTTTAATG GAAATGTCAT GGCATCAAAA

20851 TAAGGTGCCA GTAAGCAAAT CAGTTCTTGA ATGTAATTTT GAAACACACT

20901 GTGCAAAATT GCTCAGTGCA ATGATGTACA CTGATCCAAA AAGTGACCAC

20951 TGACAAGACA TGGTTTATTT CGGTTTTTAA TCTATTATGG AAAAAACGAA

21001 TTTCTCTTTG GAAAGAGCTA TACAACCATG TCCAACGTGA TTTCCTAAAA

21051 AGACAGCAGG ATGTTTTCCT TCTGACCAGC TTCCCACCCA TAAATGCAAA

21101 TATTATTGTT CAGGGTTGAT GGAGCATTTG CAAATCAAGA GGCTTTGATT

21151 TACATCCAGC ATGCTCATTC TTCGATGGTA GATCCA ATA AGAACTACAA

21201 AAGACATAGA AGATACTAGA ATACATTGTT GGAGTTCAGG ATGTGGGACA

21251 ATGCCCTGTG TCTGTTGTAC AGTGTCCCAG ATTTTTCAGG ATTTCATAGG

21301 AGGCAGCTAC AACGTACACA TGCAAAGCCC TGAAATGCCT TCCTTAGCTA

21351 ATGAAGTGGA GCCCTGACGT CACCTAGGAA AGGGATGCAA CAGAACTCAT

21401 TTATAGTTTC TCAAATGATG CTCCCATTTC TTCAAAGCCA CCATTAATCT

21451 TAACACCTTT AAAGCTATAA TAACAATCTA TGTAACAAGT GGAAAATGCT

21501 TATAGAATCA GTGCTTTCCA TCACAAACCC TTATTGTATG GAAATTCACA

21551 TTTCCATAAT CACATAAAGG TAAAAAATGT TATTCATTAT GAAGCTAAAA

21601 GGTATGAATT TCTCTGTCAT AATACTTACT GAAAACTGGA AACTGAATTG

21651 TGAAAATTTT GTACTTTATT CTTCTTAAAA TGATAGTTTC CTTTCTCTGT

21701 TTGCCTGTGA AGCACGCTTC AACAATAACA CCTTACCTTC CAATACAAAT

21751 GATGGTACAC AAAACACACA CAGGCACACA CACACACACA CACACACACA

21801 CACACACACA TATATATATA CACACATATA TACATATATA TGCATAG AG

21851 AAAAGACCTA TGTGTTTCTA ATGTATGGAA ATGGGAAATG TACATCCAGT

21901 CTTCTCAGTT GCAAAAATAA AGCAGTTTTT AAGTAGA AG TTTTGGCTTT

21951 ACTTTGAATA GATTTGTATC AACTTTGAAT TTTTTTCTTA ATTATATTAG

22001 ATTGTAGGGA GGGGACTCTG TAAGGAAGGA TGATGCAAAG GATTCTTCCA 22051 CCAGTTTATA TGAACCAAAA GGTTTTGTGA CAGTGAAACA TAAGTAGTGT

22101 ATTAGAGTTC TCTAGAGGGA CAGAACTAGT AAGATAAATG TGTGTGTGTG

22151 TGTGTGTGTG TGTGTGTGTG TGTATACATT TCATATATCT ATACATTTCA

22201 TATATCTATA GATTTCATAG ATATATGTAT ATATATGAAT GTATAGATAT

22251 TTTATTTATA TATATATATA TATATATATA TATATGAGAA AGGGAATTTA

22301 TTAGGGATAA TTGACTCACA GGATGACAAC ATAAAGTCCC ACCATAAGCT

22351 GTCTGCAAGT TGAGGAGCAA GGAAGCCAGT AGTGGATCAG TCCAAGTCCC

22401 AAAACCTCAA AACCTCAAAA GTAGGGAAGC CAACAGTGCA GCCTTCAGTC

22451 TGTGGTGGAA GGCCCGAGAG CCCCAGGAAA ACCACTAGCA TAAGACCAAG

22501 AGTCCAAAAG CCAAAGCAGT TGGAGTCTAA TGTTGGAGAG CÄGGAAGCAT

22551 CCÄGCATGGG GAAAGATGAA GGCCAGAAGA CGCAGCAAGT CTGCTCATTC

22601 CACCTTCTTC TGCCTGCTTT TGTCTAGCTG TGTTGGCAGG CAAGGGGATG

22651 GTGCCCACCC AGATTAAGGG TTGGTCTGCC TCTCACAGTC CACTGGCTCA

22701 AATGTTAATC TCCTCTAGCA ACACCCTCGC AGACACATCT AGAAACCACA

22751 CTTTGCATCC TTCAATCCAA TCÄAGTTGAC ACTTAÄTATT ATCCATCACA

22801 AGTAGGTAGA ATCCAGTTCT TGGTACTTGA TTTAÄGCACC ATCAAGATCC

22851 CATAAAGGCG GAAACCTTAC AAGAAGCCAA GTCATCCTGT TAAGGGGAAA

22901 TTTGTCTCCA GTGCTCAÄTC CTGACCTAAT CCTCATCCTC CATGTCTGGT

22951 TAACTGÄGGA CTTGTCCATC TGÄCCCCAAC CCCGCAACAC CCACCTTCCT

23001 CCCTTCCCAC ACACCACGTC TTCCCTCCTT CCTCTCTTCC CACCATCCGA

23051 GAAGGAGCAG ACCCACACAT CATTTTACAA CAAGTTCATT TTATTTTCAT

23101 CACCATGGGG CATACCCTGT GGGATCTAAA ATGTACTCTA GACCCCATTG

23151 ACTATTTATC TGGGGCCAGA TCTCTGGAGÄ CTTCTCAAÄT AGATCTGAGG

23201 TCTCTGGCCT TGCCCTGAAÄ TTAACGGCTG CCCAGGGAGA AGCTTCAGGT

23251 GGTGGAGGTC TCCTGGCTCT TTTGGTGTTG CTGTGGCTCA TTTTGATTGA

23301 GTTTCTCATT CAGCTGGTCA CAGTGTCTGG ATTGCATTGG CCCTCTCTCA

23351 TGCTCAGCTT TTCTTGAAGT GGTGATCATT CTGGAGAAGC TTTTTCCCTG

23401 GAACTCCCTC GAAGTCTTCC TTTTCCCTTT CAGATTGTCT TGGGACCCAG

23451 AAGGACAGCA GAAAGATCAC AGAAAGGCAC TGGTTTTGGG GAÄGGGGATA

23501 GAGGATGAGT GGGÄCCAGGG GTTTCATGTG AAGGTTACAG GCTGGGTTGG 23551 GCGGTTGTGC CAGGCAAGGA CAGGGTAGGA GTCTTGGGAG GGAGGAGTCA

23601 AGGGGAAGAC AAGGAGGAGC TTGGGTGTGT CACTCACCTT GATAGGGCTT

23651 CTGGGCCTGG GTTAAGAGGG GGCCTTTGTC TTCCTTGACT CTTTGGTGTT

23701 GGTGGGTGGG GGTTCTACAA CTGGTCTGAT TGCTTGGTTT CCTGGTTATC

23751 TTCACCACCT CGGGGTCTCT CAGTGGTCTA CTCCAGGCCT CCTGGCTTCC

23801 CTACATATAC CCTTCCAGGA CAGGGCGGGG CCACACCACT GAGCTTTGAT

23851 TGGTCAGCAA ACCACTACCC AGCCAATAGT GGCTCTGCTG GGGTGTGAGG

23901 GCCTTAGATA GCAGAAACCA TCATCAGAGC ATTGTCATGG TGGGAGGAGA

23951 GAAGGAGAGT GGGGAGGGTG ATCTGACGAG GAAGCCAGTA TGCTCTGGTT

24001 GAAGAATGGG GGACATTTGT TTGGCATGCC TAAAGAGCCT TCCCAAACTG

24051 ATTAGTGCTG CTCCTTTCCT CCAATGTTCA GCTTTGGGGG GGTCACATAG

24101 CAGAAGAGAG TGTATTTCCT CCAAGCCTTT CCCCAGAGTC ACCCTATTTC

24151 AGTGCTTCCT TCTGTTTTCT CCCACCACCC ACCATTATCT AGTTTTCCCA

24201 CACCTTACCA GAAAGCTCTG CAAGGTAATC TGGCTGTAAT TAAGTTTATA

24251 TGGAGGTGTG AATCATCCTA CTTCCCCCCT AAAAATTTCT CTTATGCATC

24301 TTGAAGCCCA CTCAGTTCGC TACTATCCAG GAATTTTTCT ACCCTACTTC

24351 CTGTTCCCAG TGTCCTCAGA AGTCTCCTGG TGTTGCGGGA AGTCAGGGAC

24401 CCTGAACGGA GGGACCAGTT GAAGCCATGG CAGAAGAACG TGGATTGTGA

24451 AGATTTCATG GACATTTATT AGTTCCCCAA ATTAATACTT TTATAATTTC

24501 TTATGCCTGT CTTTACTGCA GTCTCTGAAC ATAAATTGTG AAGACTTCAT

24551 GGACACTTAT CACTTCCCCA ATCAATACCC CTGTGATTTC CTATGCCTGT

24601 CTTTACTTTA ATCTCTTAAT CCCATCACCT TCCTAAACTG AGGAGGATGT

24651 ATGTCGCCTC AGGACCCTGT GATGATTGCG TTAACTGCAC AAATTGTTTG

24701 TAGAGCAAGT GTGTTTGAAC AATATGAAAT CTGGGCACCT TGAGAAAAGA

24751 ACAGGATAAC AGCAATGTTC AGGGAACAAG AGAGATAACC TTTAACTCAG

24801 ACCGCTGGTG AGCCGGGCGG AACAGAGCCA TATTTCTCTT CTTTCAAAAG

24851 CAAATGGGAG AAATATCGCT GAATTCTTTT TCTCAGCAAG GAACATCCCT

24901 GAGAAAGAGA GTGCATCCCT GAGGGTAGGC CTCTGAAATG GCCACTTCGG

24951 GGAGTGGCCA TCTTTTATGG TGGAAACTGT AGGGATGAAA TAAGCCCCAG 25001 TCTCCCATAG TGCTCCCAGG CTTATTAGGA TGAAGAAATT CCCGCCTAAT

25051 CAATTTTGGT CAGACCAGTT GTCTGCTCTC AAATCCTGTC TCCTGATAAG

25101 ATGTTATCAA CCATAATGCA TGCCCAAAAC TTCATTAGCA ATTTTAATTT

25151 CGCCCGGGTC CTGTGGTCCT GTGGTCCTGT GATCTCACCC TGCCTCCATT

25201 TGCCTTGTGA TATTCTATTA CCTTGTGAAG CACGTGATCT CTGTGACCCA

25251 CACCCTATTG GTACACTCCC TCCCCTTTTG AAAATCACTA ATAAAAACTT

25301 GCTGGTTTTG CAGCTTGTGG GGCATCACGG AACCTGCTGA CATGTGATGT

25351 CTCCCCCGGA CACCCAACTT TAAAATTTCT CTCTTTGGAC TCTGTCCCTT

25401 CATTTCTCAG ACCGGCTGAC ACTTAGGGAA AACAGAAAAC AGAAAATTTT

25451 TGCCCGATAT CTGGCTGAAT TTCGCCCAAC ATCTGGCTGA ATTTCCCCTG

25501 ATATCCTGGG GTTTCAGTTT TCCCCTGTCT GAAATCACTG CTTTCAAGCT

25551 GTCAAATTTC AGTGAATACA TTTGCTCCAC TGGATATCAC ATGAGTATTC

25601 CATCAACTGT GAGGTCATGA CCCTCTCAGT AGGTGTAGTA CCCATACTCA

25651 GTAGGTGTAG TACCGGGGCT TGGATTTTAG CAGTGTAGGG AGGATGAGGC

25701 AGATTTTCTC ATGCCAGAAA TCACGCAGGA TGTTTACATG TAGGAAACTA

25751 CAACCTAACA CTGATGAGTT CATGGAGCAG GGTGGGATTG TGTAGGATAG

25801 TTCATCACTG TGCCTGAGGT TCTCTCCAGT GTAACAAAGT TTTTTCAAAA

25851 GACCTTACAA TTGGAAAGGA ACAAATACAA AAAAACCTCC TCATTTACAT

25901 AATATAAGAG TATATGCAGA CAATCCTATC AATGTAAAAA TGCTTCTAGA

25951 ATGAATACAA CTTTAGCAAT GTTGTTGGAT TTAAGACCAT TATACTTGTA

26001 TTTTAATAAT AGCAATAAAC AATTGGCAAT TGAGACAAAC ATAGTACAAT

26051 TTAAATTAAC ATTAGAAAAT ATAAATTGAA AGGGATAAAT ATGACAGAAG

26101 ATGTAGAAGA TCTGTCCATT GAAAACTATA AAGCATTACA AAGATAAATT

26151 CAATGAGGAT ATATGTAAAT AGAGATACAA ACTTTATTCA TGAGTCAGAA

26201 GACTGAATAT TAAGATGTCA TTTATTTTTC CTCAACTTTT TTGATAGATT

26251 CAATGCAATC AAAATCCAAA TTCCAGATTT TTTAAAATAG AAATTGGGAG

26301 GCTAGCACTA ACATTGATAT AGAAATGTAA AGGTCCTAGA ATAGCCAAAG

26351 AAAACATTAA AAAATAAACG TTAGACCTAA AACCATAAAA ACCCTAGAAG

26401 AAAACCTAGG CATTACCATT CAGGACATAG GCATGGGCAA GGACTTCATG

26451 TCTAAAACAC CAAAAGCAAT GGCAACAAAA GCCAAAATTG ACAAATGGGA 26501 TCTAATTAAA CTAAAGAGCT TCTGCACAGC AAAAGAAACT ACCATCAGAG

26551 TGAACAAGCA ACCCACAAAA TGGGAGAAAA TTTTTGCAAC CTACTCATCT

26601 GACAAAGGGC TAATATCCAG AATCTACAAT GAACTCAAAC AAATTTACAA

26651 GAAAAAAACA AACAACCCCA TCAAAAAGTG GGCAAAGGAC ATGAACAGAC

26701 ACTTCTCAAA AGAAGACATT TATGCAGCCA AAAAACATAT GAAAAAATGC

26751 TCACCATCAC TGGCCATCAG AGAAATGCAA ATCAAAACCA CAATGAGATA

26801 CCATTTCACA CCAGTTAGAA TGGCAATCAT TAAAAAGTCA GGAAACAACA

26851 GGTGCTGGAG AGGATGTGGA GAAATAGGAA CACTTTTACA CTGTTGGTGG

26901 GACTGTAAAC TAGTTCAACC ATTGTGGAAG TCAGTGTGGC TATTCCTCAG

26951 GGATCTAGAA CTAGAAATAT CATTTGACCC AGCCATCCCA TTACTGGGTA

27001 TATACCCAAA GGACTATAAA TCATGCTGCT ATAAAGACAC ATGCACATGT

27051 ATGTTTATTG CGGCATTATT CACAATAGCA AAGACTTGGA ACCAAACCAA

27101 ATGTCCAACA GTGGATAGAC TGGATTAAGA AAATGTGGCA CATATACACC

27151 ATGGAATACT ATGCAGCCAT AAAAAATGAT GAGTTCATGT CCTTTGTAGG

27201 GACATGGATG AAATTGGAAA TCATCATTCT CAGTAAACTA TCGCAAGAAC

27251 AAAAAACCAA ACACCGCATA TTCTCACTCA TAGGTGGGAA TTGAACAATG

27301 AGAACACATC ATGGACACAG GAAGGGGAAC ATCACACTCT GGGGACTGTT

27351 GTGGGGTGGG GGAAGGGGGG AGGGATAGCA TTGGGAGATA TACCTAATGC

27401 TAGATGACGA GTTAGTGGGT GCAGCGCACC AGCATGGCAC ATGTATACAT

27451 ATGTAACTAA CGTGCACATT GTGCACATGT ACCCTAAAAC TTAAAGTATA

27501 ATAATAATAA ATAAATAAAT AAATAAATAA ATAAATAAAA AA AGAACAA

27551 ATGTGGTGGG GTTACCCTAC TTCATTTCAA GGCTTATAAA CTAACAATGA

27601 TCAGTGTAGT CGGAATTATC ATTATTATAA GAATATATCA ATGGAAGGGA

27651 ATAAAGCCCA ATAGTATATA CACATGTATA TGCATA ATA GACAACTAAT

27701 TATTAATAAA GATGCAAATG AAGGTGAGAG GAGAGAAATG GCAGTGTATT

27751 CAACAATTGG GTCTGGAACA ATTGGATAGC TCATATGGTT TGGCTCTGTG

27801 TCCCCACCCA AATCTCACCT GGAATTATAA TTCCCATAAT CTCCGTGTGT

27851 CAAGGGCAGG ACAAGCTGAA GGTAATTGGA TCATGAGGGC AGTTTCCCCA

27901 GGGCTGTTCT CATGACAGTG AGTGAGTTTC ACGAGATCTG ATGGTTTTAT 27951 AAGCATCTGG CATTTCCCTT GCTTGCACTC ACTCCATCCT GCCACCCTGT

28001 AAAGCTGCCG GTTGTTCGAT GTACGGGCCA GATATACGCG TATCTGAGGG

28051 GACTAGGGTG TGTTTAGGCG CCCAGCGGGG CTTCGGTTGT ACGCGGTTAG

28101 GAGTCCCCTC AGGATATAGT AGTTTCGCTT TTGCATAGGG AGGGGGAAAT

28151 GTAGTCTTAT GCAATACACT TGTAGTCTTG CAACATGGTA ACGATGAGTT

28201 AGCAACATGC CTTACAAGGA GAGAAAAAGC ACCGTGCATG CCGATTGGTG

28251 GAAGTAAGGT GGTACGATCG TGCCTTATTA GGAAGGCAAC AGACAGGTCT

28301 GACATGGATT GGACGAACCA CTGAATTCCG CATTGCAGAG ATAATTGTAT

28351 TTAAGTGCCT AGCTCGATAC AATAAACGCC ATTTGACCAT TCACCACATT

28401 GGTGTGCACC TCCAAGCTGG GTACCGAGCT CGAATTGATC TCGACGGCCT

28451 CTGAGCTATT CCAGAAGTAG TGAGGAGGCT TTTTTGGAGG CCTAGGCTTT

28501 TGCAAAAAGC TTTGCAAAGA TGGATAAAGT TTTCCGGAAT TCCGCAAAAA

28551 AGAAGAGAAA GGTAGAAGAC CCCAAGGACT TTCCTTCAGA ATTGCTAAGT

28601 TTTTTGAGTC CAAGCTTNNN NATGACCATG ATTACGGATT CACTGGCCGT

28651 CGTTTTACAA CGTCGTGACT GGGAAAACCC TGGCGTTACC CAACTTAATC

28701 GCCTTGCAGC ACATCCCCCT TTCGCCAGCT GGCGTAATAG CGAAGAGGCC

28751 CGCACCGATC GCCCTTCCCA ACAGTTGCGC AGCCTGAATG GCGAATGGCG

28801 CTTTGCCTGG TTTCCGGCAC CAGAAGCGGT GCCGGAAAGC TGGCTGGAGT

28851 GCGATCTTCC TGAGGCCGAT ACTGTCGTCG TCCCCTCAAA CTGGCAGATG

28901 CACGGTTACG ATGCGCCCAT CTACACCAAC GTAACCTATC CCATTACGGT

28951 CAATCCGCCG TTTGTTCCCA CGGAGAATCC GACGGGTTGT TACTCGCTCA

29001 CATTTAATGT TGATGAAAGC TGGCTACAGG AAGGCCAGAC GCGAATTATT

29051 TTTGATGGCG TTAACTCGGC GTTTCATCTG TGGTGCAACG GGCGCTGGGT

29101 CGGTTACGGC CAGGACAGTC GTTTGCCGTC TGAATTTGAC CTGAGCGCAT

29151 TTTTACGCGC CGGAGAAAAC CGCCTCGCGG TGATGGTGCT GCGTTGGAGT

29201 GACGGCAGTT ATCTGGAAGA TCAGGATATG TGGCGGATGA GCGGCATTTT

29251 CCGTGACGTC TCGTTGCTGC ATAAACCGAC TACACAAATC AGCGATTTCC

29301 ATGTTGCCAC TCGCTTTAAT GATGATTTCA GCCGCGCTGT ACTGGAGGCT

29351 GAAGTTCAGA TGTGCGGCGA GTTGCGTGAC TACCTACGGG TAACAGTTTC

29401 TTTATGGCAG GGTGAAACGC AGGTCGCCAG CGGCACCGCG CCTTTCGGCG 29451 GTGAAATTAT CGATGAGCGT GGTGGTTATG CCGATCGCGT CACACTACGT

29501 CTGAACGTCG AAAACCCGAA ACTGTGGAGC GCCGAAATCC CGAATCTCTA

29551 TCGTGCGGTG GTTGAACTGC ACACCGCCGA CGGCACGCTG ATTGAAGCAG

29601 AAGCCTGCGA TGTCGGTTTC CGCGAGGTGC GGATTGAAAA TGGTCTGCTG

29651 CTGCTGAACG GCAAGCCGTT GCTGATTCGA GGCGTTAACC GTCACGAGCA

29701 TCATCCTCTG CATGGTCAGG TCATGGATGA GCAGACGATG GTGCAGGATA

29751 TCCTGCTGAT GAAGCAGAAC AACTTTAACG CCGTGCGCTG TTCGCATTAT

29801 CCGAACCATC CGCTGTGGTA CACGCTGTGC GACCGCTACG GCCTGTATGT

29851 GGTGGATGAA GCCAATATTG AAACCCACGG CATGGTGCCA ATGAATCGTC

29901 TGACCGATGA TCCGCGCTGG CTACCGGCGA TGAGCGAACG CGTAACGCGA

29951 ATGGTGCAGC GCGATCGTAA TCACCCGAGT GTGATCATCT GGTCGCTGGG

30001 GAATGAATCA GGCCACGGCG CTAATCACGA CGCGCTGTAT CGCTGGATCA

30051 AATCTGTCGA TCCTTCCCGC CCGGTGCAGT ATGAAGGCGG CGGAGCCGAC

30101 ACCACGGCCA CCGATATTAT TTGCCCGÄTG TACGCGCGCG TGGATGAAGA

30151 CCAGCCCTTC CCGGCTGTGC CGAAATGGTC CATCAAAAAA TGGCTTTCGC

30201 TACCTGGAGA GACGCGCCCG CTGATCCTTT GCGAATACGC CCACGCGATG

30251 GGTAACAGTC TTGGCGGTTT CGCTAAATAC TGGCAGGCGT TTCGTCAGTA

30301 TCCCCGTTTA CAGGGCGGCT TCGTCTGGGA CTGGGTGGAT CAGTCGCTGA

30351 TTAAATATGA TGAAAACGGC AACCCGTGGT CGGCTTACGG CGGTGATTTT

30401 GGCGATACGC CGAACGATCG CCAGTTCTGT ATGAACGGTC TGGTCTTTGC

30451 CGACCGCACG CCGCATCCAG CGCTGACGGA AGCAAAACAC CAGCAGCAGT

30501 TTTTCCAGTT CCGTTTATCC GGGCAAACCA TCGAAGTGAC CAGCGAATAC

30551 CTGTTCCGTC ATAGCGATAA CGAGCTCCTG CACTGGATGG TGGCGCTGGA

30601 TGGTAAGCCG CTGGCAAGCG GTGAAGTGCC TCTGGATGTC GCTCCACAAG

30651 GTAAACAGTT GATTGAACTG CCTGAACTAC CGCAGCCGGA GAGCGCCGGG

30701 CAACTCTGGC TCACAGTACG CGTAGTGCAA CCGAACGCGA CCGCATGGTC

30751 AGAAGCCGGG CACATCAGCG CCTGGCAGCA GTGGCGTCTG GCGGAAAACC

30801 TCAGTGTGAC GCTCCCCGCC GCGTCCCACG CCATCCCGCA TCTGACCACC

30851 AGCGAAATGG ATTTTTGCAT CGAGCTGGGT AATAAGCGTT GGCAATTTAA 30901 CCGCCAGTCA GGCTTTCTTT CACAGATGTG GATTGGCGAT AAAAAACAAC

30951 TGCTGACGCC GCTGCGCGAT CAGTTCACCC GTGCACCGCT GGATAACGAC

31001 ATTGGCGTAA GTGAAGCGAC CCGCATTGAC CCTAACGCCT GGGTCGAACG

31051 CTGGAAGGCG GCGGGCCATT ACCAGGCCGA AGCAGCGTTG TTGCAGTGCA

31101 CGGCAGATAC ACTTGCTGAT GCGGTGCTGA TTACGACCGC TCACGCGTGG

31151 CAGCATCAGG GGAAAACCTT ATTTATCAGC CGGAAAACCT ACCGGATTGA

31201 TGGTAGTGGT CAAATGGCGA TTACCGTTGA TGTTGAAGTG GCGAGCGATA

31251 CACCGCATCC GGCGCGGATT GGCCTGAACT GCCAGCTGGC GCAGGTAGCA

31301 GAGCGGGTAA ACTGGCTCGG ATTAGGGCCG CAAGAAAACT ATCCCGACCG

31351 CCTTACTGCC GCCTGTTTTG ACCGCTGGGA TCTGCCATTG TCAGACATGT

31401 ATACCCCGTA CGTCTTCCCG AGCGAAAACG GTCTGCGCTG CGGGACGCGC

31451 GAATTGAATT ATGGCCCACA CCAGTGGCGC GGCGACTTCC AGTTCAACAT

31501 CAGCCGCTAC AGTCAACAGC AACTGATGGA AACCAGCCAT CGCCATCTGC

31551 TGCACGCGGA AGAAGGCACA TGGCTGAATA TCGACGGTTT CCATATGGGG

31601 ATTGGTGGCG ACGACTCCTG GAGCCCGTCA GTATCGGCGG AATTCCAGCT

31651 GAGCGCCGGT CGCTACCATT ACCAGTTGGT CTGGTGTCAA AAATAATAAT

31701 AANNNNAACT TGTTTATTGC AGCTTATAAT GGTTACAAAT AAAGCAATAG

31751 CATCÄCAAAT TTCACAAATA AAGCATTTTT TTCACTGCAT TCTAGTTGTG

31801 GTTTGTCCAA ACTCATCAAT GTATCTTATC ATGTCTGGAT CCAGACATGA

31851 TAAGATACAT TGATGAGTTT GGACAAACCA CAACTAGAAT GCAGTGAAAA

31901 AAATGCTTTA TTTGTGAAAT TTGTGATGCT ATTGCTTTAT TTGTAACCAT

31951 TATAAGCTGC AATAAACAAG TTAACAACAA CAATTGCATT CATTTTATGT

32001 TTCAGGTTCA GGGGGAGGTG GGGAGGTTTT TTAAAGCAAG TAAAACCTCT

32051 ACAAATGTGG TATGGCTGAT TATGATCCGG CTGCCTCGCG CGTTTCGGTG

32101 ATGACGGTGA AAACCTCTGA CACATGCAGC TCCCGGAGAC GGTCACAGCT

32151 TGTCTGTAAG CGGATGCCGG GAGCAGACÄA GCCCGTCAGG GCGCGTCAGC

32201 GGGTGTTGGC GGGTGTCGGG GCGCAGCCAT GACCGGTCGA CTCTAGGATG

32251 CATATGGCGG CCGCCTGCAG GAATTCCATT TTAAGAAAAC TACAATTCCC

32301 AACACATACA AGTTACTCCG CCCTAAAACC TACGTCACCC GCCCCGTTCC

32351 CACGCCCCGC GCCACGTCAC AAACTCCACC CCCTCATTAT CATATTGGCT 32401 TCAATCCAAA ATAAGGTATA TTATTGATGA TGAGATC AG GGATAACAGG

32451 GTAATATTCT AGTTCTAGAG CGGCCGCCAC CGCGGTGGAG CTCCAATTCG

32501 CCCTATAGTG AGTCGTATTA CAATTCACTG GCCGTCGTTT TACAACGTCG

32551 TGACTGGGAA AACCCTGGCG TTACCCAACT TAATCGCCTT GCAGCACATC

32601 CCCCTTTCGC CAGCTGGCGT AATAGCGAAG AGGCCCGCAC CGATCGCCCT

32651 TCCCAACAGT TGCGCAGCCT GAATGGCGAA TGGCGCACGC GCCCTGTAGC

32701 GGCGCATTAA GCGCGGCGGG TGTGGTGGTT ACGCGCAGCG TGACCGCTAC

32751 ACTTGCCAGC GCCCTAGCGC CCGCTCCTTT CGCTTTCTTC CCTTCCTTTC

32801 TCGCCACGTT CGCCGGCTTT CCCCGTCAAG CTCTAAATCG GGGGCTCCCT

32851 TTAGGGTTCC GATTTAGTGC TTTACGGCAC CTCGACCCCA AAAAACTTGA

32901 TTAGGGTGAT GGTTCACGTA GTGGGCCATC GCCCTGATAG ACGGTTTTTC

32951 GCCCTTTGAC GTTGGAGTCC ACGTTCTTTA ATAGTGGACT CTTGTTCCAA

33001 ACTGGAACAA CACTCAACCC TATCTCGGTC TATTCTTTTG ATTTATAAGG

33051 GATTTTGCCG ATTTCGGCCT ATTGGTTAAA AAATGAGCTG ATTTAACAAA

33101 AATTTAACGC GAATTTTAAC AAAATATTAA CGTTTACAAT TTCGTCAGGT

33151 GGCACTTTTC GGGGAAATGT GCGCGGAACC CCTATTTGTT TATTTTTCTA

33201 AATACATTCA AATATGTATC CGCTCATGAG ACAATAACCC TGATAAATGC

33251 TTCAATAATA TTGAAAAAGG AAGAGTATGA GTATTCAACA TTTCCGTGTC

33301 GCCCTTATTC CCTTTTTTGC GGCATTTTGC CTTCCTGTTT TTGCTCACCC

33351 AGAAACGCTG GTGAAAGTAA AAGATGCTGA AGATCAGTTG GGTGCACGAG

33401 TGGGTTACAT CGAACTGGAT CTCAACAGCG GTAAGATCCT TGAGAGTTTT

33451 CGCCCCGAAG AACGTTTTCC AATGATGAGC ACTTTTAAAG TTCTGCTATG

33501 TGGCGCGGTA TTATCCCGTA TTGACGCCGG GCAAGAGCAA CTCGGTCGCC

33551 GCATACACTA TTCTCAGAAT GACTTGGTTG AGTACTCACC AGTCACAGAA

33601 AAGCATCTTA CGGATGGCAT GACAGTAAGA GAATTATGCA GTGCTGCCAT

33651 AACCATGAGT GATAACACTG CGGCCAACTT ACTTCTGACA ACGATCGGAG

33701 GACCGAAGGA GCTAACCGCT TTTTTGCACA ACATGGGGGA TCATGTAACT

33751 CGCCTTGATC GTTGGGAACC GGAGCTGAAT GAAGCCATAC CAAACGACGA

33801 GCGTGACACC ACGATGCCTG TAGCAATGGC AACAACGTTG CGCAAACTAT 33851 TAACTGGCGA ACTACTTACT CTAGCTTCCC GGCAACAATT AATAGACTGG

33901 ATGGAGGCGG ATAAAGTTGC AGGACCACTT CTGCGCTCGG CCCTTCCGGC

33951 TGGCTGGTTT ATTGCTGATA AATCTGGAGC CGGTGAGCGT GGGTCTCGCG

34001 GTATCATTGC AGCACTGGGG CCAGATGGTA AGCCCTCCCG TATCGTAGTT

34051 ATCTACACGA CGGGGAGTCA GGCAACTATG GATGAACGAA ATAGACAGAT

34101 CGCTGAGATA GGTGCCTCAC TGATTAAGCA TTGGTAACTG TCAGACCAAG

34151 TTTACTCATA TATACTTTAG ATTGATTTAA AACTTCATTT TTAATTTAAA

34201 AGGATCTAGG TGAAGATCCT TTTTGATAAT CTCATGACCA AAATCCCTTA

34251 ACGTGAGTTT TCGTTCCACT GAGCGTCAGA CCCCGTAGAA AAGATCAAAG

34301 GATCTTCTTG AGATCCTTTT TTTCTGCGCG TAATCTGCTG CTTGCAAACA

34351 AAAAAACCAC CGCTACCAGC GGTGGTTTGT TTGCCGGATC AAGAGCTACC

34401 AACTCTTTTT CCGAAGGTAA CTGGCTTCAG CAGAGCGCAG ATACCAAATA

34451 CTGTCCTTCT AGTGTAGCCG TAGTTAGGCC ACCACTTCAA GAACTCTGTA

34501 GCACCGCCTA CATACCTCGC TCTGCTAATC CTGTTACCAG TGGCTGCTGC

34551 CAGTGGCGAT AAGTCGTGTC TTACCGGGTT GGACTCAAGA CGATAGTTAC

34601 CGGATAAGGC GCAGCGGTCG GGCTGAACGG GGGGTTCGTG CACACAGCCC

34651 AGCTTGGAGC GAACGACCTA CACCGAACTG AGATACCTAC AGCGTGAGCA

34701 TTGAGAAAGC GCCACGCTTC CCGAAGGGAG AAAGGCGGAC AGGTATCCGG

34751 TAAGCGGCAG GGTCGGAACA GGAGAGCGCA CGAGGGAGCT TCCAGGGGGA

34801 AACGCCTGGT ATCTTTATAG TCCTGTCGGG TTTCGCCACC TCTGACTTGA

34851 GCGTCGATTT TTGTGATGCT CGTCAGGGGG GCGGAGCCTA TGGAAAAACG

34901 CCAGCAACGC GGCCTTTTTA CGGTTCCTGG CCTTTTGCTG GCCTTTTGCT

34951 CACATGTTCT TTCCTGCGTT ATCCCCTGAT TCTGTGGATA ACCGTATTAC

35001 CGCCTTTGAG TGAGCTGATA CCGCTCGCCG CAGCCGAACG ACCGAGCGCA

35051 GCGAGTCAGT GAGCGAGGAA GCGGAAGAGC GCCCAATACG CAAACCGCCT

35101 CTCCCCGCGC GTTGGCCGAT TCATTAATGC AGCTGGCACG ACAGGTTTCC

35151 CGACTGGAAA GCGGGCAGTG AGCGCAACGC AATTAATGTG AGTTAGCTCA

35201 CTCATTAGGC ACCCCAGGCT TTACACTTTA TGCTTCCGGC TCGTATGTTG

35251 TGTGGAATTG TGAGCGGATA ACAATTTCAC ACAGGAAACA GCTATGACCA

35301 TGATTACGCC AAGCTCGGAA TTAACCCTCA CTAAAGGGAA CAAAAGCTGG 35351 GTACCTCTAG AACTATAGCT AGCGATAAGC TTTAATGCGG TAGTTTATCA

35401 CAGTTAAATT GCTAACGCAG TCAGGCACCG TGTATGAAAT CTAACAATGC

35451 GCTCATCGTC ATCCTCGGCA CCGTCACCCT GGATGCTGTA GGCATAGGCT

35501 TGGTTATGCC GGTACTGCCG GGCCTCTTGC GGGATATCGT CCATTCCGAC

35551 AGCATCGCCA GTCACTATGG CGTGCTGCTA GCGCTATATG CGTTGATGCA

35601 ATTTCTATGC GCACCCGTTC TCGGAGCACT GTCCGACCGC TTTGGCCGCC

35651 GCCCAGTCCT GCTCGCTTCG CTACTTGGAG CCACTATCGA CTACGCGATC

35701 ATGGCGACCA CACCCGTCCT GTGGATCTGC CTCGCTGGCC TGCCGCAGTT

35751 CTTCAACCTC CCGGCGCAGC TTTTCGTTCT CAATTTCAGC ATCCCTTTCG

35801 GCATACCATT TTATGACGGC GGCAGAGTCA TAAAGCACCT CATTACCCTT

35851 GCCACCGCCT CGCAGAACGG GCATTCCCTG TTCCTGCCAG TTCTGAATGG

35901 TACGGATACT CGCACCGAAA ATGTCAGCCA GCTGCTTTTT GTTGACTTCC

35951 ATTGTTCATT CCACGGACAA AAACAGAGAA AGGAAACGAC ÄGAGGCCAAA

36001 AAGCTCGCTT TCAGCACCTG TCGTTTCCTT TCTTTTCAGA GGGTATTTTA

36051 AATAAAAACA TTAAGTTATG ACGAAGAAGA ACGGAAACGC CTTAAACCGG

36101 AAAATTTTCA TAAATAGCGA AAACCCGCGA GGTCGCCGCC CCGTAACAAG

36151 GCGGATCGCC GGAAAGGACC CGCAAATGAT AATAATTATC AATTGCATAC

36201 TATCGACGGC ACTGCTGCCA GATAACACCA CCGGGGAAAC ATTCCATCAT

36251 GATGGCCGTG CGGACATAGG AAGCCAGTTC ATCCATCGCT TTCTTGTCTG

36301 CTGCCATTTG CTTTGTGACA TCCAGCGCCG CACATTCAGC AGCGTTTTTC

36351 AGCGCGTTTT CGATCAACGT TTCAATGTTG GTATCAACAC CAGGTTTAAC

36401 TTTGAACTTA TCGGCACTGA CGGTTACCTT GTTCTGCGCT GGCTCATCAC

36451 GCTGGATACC AAGGCTGATG TTGTAGATAT TGGTCACCGG CTGAGGTGTT

36501 TCGATTGCCG CTGCGTGGAT AGCACCATTT GCGATAGCGG CGTCCTTGAT

36551 GAATGACACT CCATTGCGAA TAAGTTCGAA GGAGACGGTG TCACGAATGC

36601 GCTGGTCCAG CTCGTCGATT GCCTTTTGTG CAGCAGAGGT ATCAATCTCA

36651 ACGCCAAGCG TCATCGAAGC GCAATATTGC TGCTCACCAA AACGCGTATT

36701 GACCAGGTGT TCAACGGCAA ATTTCTGCCC TTCTGATGTC AGAAAGGTAA

36751 AGTGATTTTC TTTCTGGTAT TCAGTTGCTG TGTGTCTGGT TTCAGCAAAA 36801 CCAAGCTCGC GCAATTCGGC TGTGCCAGAT TTAGAAGGCA GATCACCAGA

36851 CAGCAACGCG CCACGGAAAA ACAGCGCATA CAGAACATCC GTCGCCGCGC

36901 CGGACAACGT GATAATTTTA TGACCCATGA TTTATTTCCT TTTAGACGTG

36951 AGCCTGTCGC ACAGCAAAGC CGCCGAAAGT TAACGGTTTG CCCAGGCTCA

37001 CAACTGAAAG ACTTTCTACG GTGTGCGCGT GCGATGCGCG TAGAAGACTG

37051 ATTTATCAAC CTGTCTTTAT ATCAGGATTC ATTACCTGAC TATTTGTGGG

37101 TAAAGTTCGT AGTGCGCTGA TCGTGCAAAA TGATTTTAGT TGGGAACAGT

37151 TCGCAACTCT GTCCCATAAA AATCAGCATA TTCCCATCTA TCCCATATCC

37201 AGCGCATTGA CCATCGGGAT ACTGAAGGGA GATTCCATCA TCTCTTAGAA

37251 AGATCACCAT CTCTTTTGTT TCAATTTGCA TA AGCTACC TGGAGGATTT

37301 ATGAATACAA GGATTTTCAT GGACTATTAC CATGAGATTG ATTTTCCATC

37351 TTTATTCGCG AGAGCAGTGG AAAGCGATGA CGATGTGGGT ACTACATTGC

37401 GCATTCACCT ACTTTGTGAG CGCATGGTCG AAGCATGGAT ATGCGCATGC

37451 TGTGACTGCC AAGATCCTCT ACGCCGGACG CATCGTGGCC GGCATCACCG

37501 GCGCCACAGG TGCGGTTGCT GGCGCCTATA TCGCCGACAT CACCGATGGG

37551 GAAGATCGGG CTCGCCACTT CGGGCTCATG AGCGCTTGTT TCGGCGTGGG

37601 TATGGTGGCA GGCCCCGTGG CCGGGGGACT GTTGGGCGCC ATCTCCTTGC

37651 ATGCACCATT CCTTGCGGCG GCGGTGCTCA ACGGCCTCAA CCTACTACTG

37701 GGCTGCTTCC TAATGCAGGA GTCGCATAAG GGAGAGCGTC GCTAGCATGC

37751 GCAAATTTAA AGCGCTGATA TCGATCGCGC GCAGATCAAT ATTACCCTGT

37801 TATCCCTA

Legend for Figures 3a-d

3. pMV vector family:

a. pMV-Bg

b. pMVI-Bg: like pMV-Bg, but (1) additionally loxP

Detection point between 5 'ITR and adenoviral packaging signal

c. pMV: like pMV-Bg, but without Beta-Galaktoεidaεe-

Expression cassette

d. pMVI: like pMV-Bg, but (1) additionally loxP

Detection point between 5 'ITR and adenoviral packaging signal and (2) without beta-galactosidase expression cassette

Legend to Figures 4a-d

4. pMVX vector family:

a. pMVX-Bg

b. pMVIX-Bg: like pMV-Bg, but (1) additionally loxP

Detection point between 5 'ITR and adenoviral packaging signal

c. pMVX: like pMV-Bg, but without beta-galactosidase

Expression cassette

d. pMVIX: like pMV-Bg, but (1) additionally loxP

Detection point between 5 'ITR and adenoviral packaging signal and (2) without beta-galactoidase expression cassette

Claims

claims

1. Cloning vector for the production of adenoviral minimal viruses consisting of

a) two adenoviral terminal inverted replication sequences (ITR, inverted terminal repeats), which by

ab) two interfaces for a restriction endonuclease are flanked with a recognition sequence longer than 8 bp, and

ac) an adenoviral packaging signal,

ad) a multiple cloning site for the insertion of therapeutic DNA fragments, into which non-coding chromosomal mammalian DNA may also be cloned, and

ae) if appropriate, a recognition site for a recombinase which lies between one of the inverted terminal replication sequences and the adenoviral packaging signal,

af) frame a reporter gene cassette if necessary, and

b) a bacterial plasmid backbone with origin of replication and bacterial resistance gene, in which

ba) a packaging signal is cloned into a bacteriophage.

2. Cloning vector according to claim 1, characterized in that the terminal inverted Replikationsεequenzen in a), and daε packaging signal in ac) originate from the human adenovirus type 5 (Ad5).

3. Cloning vector according to claim 1, characterized in that the recognition sequences in ab) recognition sequences for the meganuclease I-Sce I εind.

4. Cloning vector according to claim 1, characterized in that the chromosomal mammalian DNA in ad) has a size of 15 to 35 Kb.

5. Cloning vector according to claim 1 and 4, characterized in that chromosomal DNA is used which comes from the genome of the organism in which the adenoviral minimal viruses formed by the cloning vector are used.

6. Cloning vector according to claim 1 and 4, characterized in that fragments of human chromosomal DNA are used.

7. Cloning vector according to claim 1, 4 and 6, characterized in that a fragment of human X-chromosomal DNA with at least two dual restriction sites is used.

8. Cloning vector according to claim 1, characterized in that in ae) there is a recognition site for the Cre recombinant (loxP).

9. Cloning vector according to claim 1, characterized in that the reporter gene cassette in af) is a constitutively expressed E.coli LacZ (beta-galactosidase) reporter gene cassette.

10. Cloning vector according to claim 1, characterized in that the reporter gene cassette in af) is a regulated expressed reporter gene cassette.

11. Cloning vector according to claim 1 and 10, characterized in that a promoter with tetracycline operator is used for regulation.

12. Cloning vector according to claims 1 and 10, characterized in that a reporter gene is used, the product of which is not detectably immunogenic in the target organism.

13. Klonierungεvektor according to claim 1, 10 and 12, characterized in that a reporter gene is used which comes from the genome of the organism in which the adenoviral minimal viruses formed by the cloning vector are used.

14. Cloning vector according to claim 1, 10 and 12, characterized in that the human placental alkaline phosphatase is used as the reporter gene.

15. Cloning vector according to claim 1, characterized in that a coε signal from the phage lambda is used as packaging signal in ba).

16. Cloning vector pMVn according to one of claims 1 to 15 according to Figure 1 a), which is part of the claim.

17. Cloning vector pMV-Bg according to one of claims 1 to 15 according to Figure 1 b), which is the part of the claim.

18. Cloning vector pMVln according to one of Claims 1 to 15 according to Figure 1 c), which is part of the claim.

19. Cloning vector pMVl-Bg according to one of claims 1 to 15 according to Figure 1 d), which is part of the claim.

20. Cloning vector pMVXn according to one of claims 1 to 15 according to Figure 2 a), which is part of the claim.

21. Cloning vector pMVX-Bg according to one of claims 1 to 15 according to Figure 2 b), which is part of the claim.

22. Cloning vector pMVIXn according to one of claims 1 to 15 according to Figure 2 c), which is part of the claim.

23. Cloning vector pMVIX-Bg according to one of Claims 1 to 15 according to Figure 2 d), which is part of Claim εt.