Novel attenuated virus strain and its use as vaccine

Abstract

The present invention relates to an attenuated virus strain derived from a human metapneumovirus strain, which comprises a genome sequence shown in SEQ ID NO: 1. The attenuated strain comprises one or more genetic modifications of the sequence SEQ ID NO: 1, and the genetic modifications reduce the virulence of the strain.

Description

Technical Field

[0001] The present invention relates to genetically modified virus strains derived from novel human metapneumovirus strains.

[0002] The present invention relates to these modified strains with reduced virulence, their use for preventing or treating infections caused by viruses of the Pneumoviridae family, and vaccine compositions comprising the modified virus strains. Background Art

[0003] Acute infections of the lower respiratory tract are one of the main causes of morbidity and mortality worldwide, especially among children under 5 years of age, where the mortality rate is as high as more than 15% depending on the countries evaluated, and is therefore the second leading cause of death according to the World Health Organization.

[0004] Most acute infections of the lower respiratory tract are associated with a variety of pathologies (from simple colds to severe pneumonia) and are mainly caused by viruses, such as those belonging to the following families:

[0005] (i) Viruses of the family Orthomyxoviridae (mainly including influenza viruses);

[0006] (ii) viruses of the family Paramyxoviridae;

[0007] (iii) viruses of the family Adenoviridae;

[0008] (iv) viruses of the family Picornaviridae;

[0009] (v) viruses of the family Coronaviridae; and

[0010] (vi) Viruses of the family Pneumoviridae, mainly including human respiratory syncytial virus and human metapneumovirus.

[0011] Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are viruses that cause acute infections of the respiratory tract, such as bronchiolitis, bronchitis or pneumonia. They mainly affect populations at risk, namely young children under 5 years of age, the elderly and immunosuppressed people.

[0012] hRSV is a major cause of bronchiolitis and pneumonia in infants under 1 year of age, with an increased incidence in infants under 6 months of age. In adults, infection with hRSV is rare and benign, except in elderly subjects. Transmission is essentially by the respiratory route, with the virus replicating in the respiratory tract.

[0013] hRSV was isolated in 1957. It is a single-stranded RNA virus with negative polarity, an "enveloped" type, with a helically symmetrical capsid. It belongs to the genus Orthopneumovirus of the family Pneumoviridae.

[0014] hMPV is commonly found in infants with bronchiolitis and pneumonia, particularly severely affecting children aged 1 to 3 years.

[0015] The main symptoms of hMPV infection in children include runny nose, cough, respiratory distress or fever. hMPV may also cause infections of the upper respiratory tract and may be associated with ear infections, while non-respiratory symptoms (such as diarrhea, vomiting and erythema) rarely occur. hMPV preferentially targets ciliated cells of the human respiratory tree.

[0016] hMPV infection can cause histopathological changes in the lungs of host organisms, especially the following physiological effects:

[0017] - Damage at the level of the respiratory epithelium;

[0018] - Excessive mucus production, and

[0019] -Acute inflammation, especially at the level of the interstitial lung tissue, which is associated with a massive secretion of proinflammatory cytokines and chemokines.

[0020] In respiratory pathology in adults, hMPV has been found in 5% to 10% of adults or elderly people with acute respiratory infections and in 3% to 5% of adults with exacerbations of chronic lung disease or community-acquired pneumonia.

[0021] hMPV was first isolated and described in the Netherlands in 2001 (van den Hoogen et al., 2001). The following year, several strains of hMPV were isolated from patients in North America (Peret et al., 2002). hMPV is a virus with negative single-stranded RNA belonging to the genus Metapneumovirus of the family Pneumoviridae.

[0022] The average age of children hospitalized for complications of hMPV infection is 6 to 12 months, which is later than the average age of children aged 0 to 3 months, when complications due to hRSV occur primarily. However, the epidemiology of hMPV has many similarities to that of hRSV:

[0023] -It is present on all continents;

[0024] - Its annual distribution is mainly in winter and spring, overlapping with hRSV;

[0025] - Transmission may also occur through saliva droplets.

[0026] Despite active ongoing research, there are no effective and specific preventive or therapeutic approaches for hRSV and hMPV viral infections on the market today (Mazur et al., 2018).

[0027] For treatment, in severe cases of hMPV or hRSV infection, ribavirin (adverse effects cannot be excluded) or intravenous immunoglobulin (very expensive) can occasionally be used. Other types of treatment are currently being developed, such as the use of fusion inhibitor peptides, sulfated glycosaminoglycans, interfering RNA, and certain immunomodulators.

[0028] Today, the commonly used clinical approach is to place patients on respiratory assistance (providing oxygen or mechanical ventilation) and to administer bronchodilators, corticosteroids and / or antibiotics, in particular while treating the symptoms of infection, in order to prevent or treat bacterial superinfections.

[0029] Regarding vaccination, the groups particularly affected by hMPV are infants, young children, and the elderly, and it is important to have a rapid, safe, and effective vaccine to reduce severe respiratory episodes, which has a significant impact on these age groups.

[0030] Therefore, the development of vaccines against hRSV and hMPV represents not only a major health challenge but also a real socioeconomic issue with the goal of reducing the high costs of treatment and hospitalization associated with these infections and reducing the use of antibiotics in cases of bacterial superinfections, thereby limiting the emergence of resistance.

[0031] Prior art

[0032] Tables 1, 2 and 3 below list the different approaches under development for obtaining live attenuated vaccines from wild hMPV strains.

[0033] Viral strains are considered attenuated in vitro when they show reduced replication capacity and / or when the formation of infectious bursts caused by these virus strains is more limited compared to wild-type (WT) virus, especially syncytia (fusion with adjacent cells after virus infection). In vivo, attenuated virus strains replicate at a lower maximum load and / or induce less pathology (in terms of weight loss, or inflammatory conditions, or histopathological lesions) than wild-type virus strains.

[0034] Table 1. List of candidate live vaccines developed or in development that are attenuated based on hMPV strains with one or more mutations

[0035]

[0036] The term "Vero" refers to the African green monkey kidney cell line, which is widely used in cell culture to test various viruses.

[0037] The term "LLC-MK2" refers to a cell line derived from rhesus monkey kidney cells, which was used to test infection with various viruses.

[0038] The symbol √ indicates that the cells used are susceptible to virus infection.

[0039] The following abbreviations are used for in vivo study models: M, mouse; H, hamster; CR, cotton rat; CM, cynomolgus macaque; AGM, African green monkey; Ch, chimpanzee; Rh, rhesus monkey; SCID, severe combined immunodeficiency.

[0040] Table 2. List of candidate live attenuated vaccines that have been developed or are under development, containing at least one hMPV strain with a complete gene deletion

[0041]

[0042]

[0043] Δ: Complete deletion of the gene. Or more replicable.

[0044] All attenuated virus strains tested were derived from the wild-type strain CAN97-83 of the A2 subgroup.

[0045] Table 3. List of candidate live attenuated vaccines based on chimeric hMPV strains that have been developed or are under development

[0046]

[0047]

[0048] The following abbreviations are used: TM = transmembrane domain; PIV = parainfluenza virus; SeV = Sendai virus.

[0049] International application WO 2005 / 014626 relates to several hMPV strains, named after the following names: CAN 97-83, CAN 98-75 and HMPV 00-1. The application describes a recombinant hMPV strain, named CAN 97-83, which has been genetically modified to reduce its virulence. The proposed modification particularly relates to the complete deletion of the genes encoding the G and / or SH proteins. The modified virus strains can be used for human treatment to prevent or treat pneumonia virus infections. Administration of wild strains or these attenuated strains to hamsters makes it possible to protect them from subsequent CAN 97-83hMPV infections. However, WO 2005 / 014626 does not mention the complete protection characteristics, in particular, no weight monitoring of the treated animals is performed. The same experimental results are described in the scientific articles cited in Table 2.

[0050] Next, the same attenuated strain was used to obtain in vivo results in a monkey animal model; the recombinant virus strain lacking the SH, G, and M2-2 genes continued to replicate in the respiratory tract of monkeys; and, like the CAN97-83 wild-type virus strain, further induced the production of neutralizing antibodies.

[0051] Other isolated hMPV strains have been described in patent US 8,841,433, and their use in the preparation of vaccines has also been proposed.

[0052] Vaccines based on the use of live attenuated virus strains have many advantages.

[0053] On the one hand, vaccines produced from live attenuated viruses have the advantage of inducing a strong immune response to a virus whose reproductive capacity has been greatly reduced, making it possible to mimic natural infection while avoiding pathology. In the development of these attenuated strains, it is important to find the ideal balance between attenuation and immunogenicity.

[0054] On the other hand, these vaccines can be administered via the intranasal route and thus mimic the natural entry route of wild-type viruses, thereby inducing an immune response that closely resembles the physiological response to infection.

[0055] Additionally, this vaccination strategy does not produce the excessive inflammatory response that inactivated vaccines do. Finally, the addition of adjuvants is usually not required.

[0056] Therefore, this is the best vaccination strategy for at-risk groups, such as young children and infants. Summary of the invention

[0057] The subject of the present invention is an attenuated virus strain derived from a specific clinical strain of human metapneumovirus rC-85473, comprising a genome sequence represented by the sequence SEQ ID NO: 1, wherein the attenuated strain comprises one or more genetic modifications in the sequence SEQ ID NO: 1, wherein the genetic modifications reduce the virulence of the strain.

[0058] The attenuated virus strain according to the present invention is specifically a recombinant strain modified by inactivating or deleting at least one gene encoding one of the G and SH accessory proteins to produce recombinant viruses designated as ΔG rC-85473 and ΔSH rC-85473, respectively.

[0059] More specifically, the subject of the present invention is an attenuated virus strain derived from a clinical strain of human metapneumovirus, comprising a genome sequence represented by the sequence SEQ ID NO: 1, wherein the attenuated strain comprises at least one genetic modification selected from the following: inactivation of the gene encoding the auxiliary G protein and inactivation of the gene encoding the auxiliary SH protein.

[0060] These strains were characterized in in vitro (LLC-MK2 cell line) and ex vivo (reconstituted human respiratory epithelium, cultured at an air-liquid interface) cell models with respect to infectivity and viral replication, and in the hMPV murine infection model with respect to viral pathogenesis and its vaccine protective properties.

[0061] The results presented in the experimental part highlight that these strains ΔG rC-85473 and ΔSH rC-85473 have specific properties compared to those strains modified in a similar manner, also lacking the G or SH gene, but derived from another clinical strain of hMPV (CAN98-75).

[0062] Virus ΔG or ΔSH rC-85473 has in particular:

[0063] (i) The virus has attenuated characteristics in the mouse model, which is different from the recombinant virus ΔSH CAN98-75, which has a very strong toxic part;

[0064] (ii) it simultaneously retains its ability to replicate in a bound cell system (reference line LLC-MK2) and in suspension, unlike the recombinant virus CAN98-75, which has a much lower productivity;

[0065] (iii) Virus ΔG or ΔSH C-85473 advantageously possessed significant protective characteristics: 100% survival rate of infected mice, minimal weight loss, induction of neutralizing antibody responses against the homologous virus strain (C-85473, serotype A) and against at least one heterologous strain (CAN98-75, serotype B), and reduced pulmonary pathogenesis in infected animals, compared to infectious challenge with wild-type hMPV (strain rC-85473), which resulted in 50% mortality in the unimmunized control group.

[0066] This attenuated virus strain can be used as an expression vector of at least one foreign gene in vivo, in vitro and in vitro, specifically a gene encoding a viral antigen, wherein the viral antigen is derived from human respiratory syncytial virus (hRSV), especially its F fusion protein.

[0067] The present invention also relates to the attenuated virus strain and its use as a medicine, more specifically, for preventing and / or treating Pneumoviridae virus infection.

[0068] Finally, the present invention also relates to a vaccine composition comprising at least one attenuated virus strain as defined above, and optionally an adjuvant, in a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1. Recombinant hMPV generated from clinical strains C-85473 and CAN98-75.

[0070] [ Figure 1A ] Schematic diagram of the recombinant viral genome generated by reverse genetics from clinical strain C-85473, and the GFP (green fluorescent protein) coding sequence has been cloned at its 3' end. Virus rC-85473 contains the genome of wild-type strain C-85473, which also expresses the GFP gene. The genome of the recombinant virus is derived from strain C-85473 and lacks the SH (ΔSH-rC-85473) or G (ΔG-rC-85473) gene, as shown below. The genetically modified viruses are functional and replicative, as shown in images of LLC-MK2 cells infected with these viruses at a multiplicity of infection (MOI) of 0.01 and observed by fluorescence microscopy three days after infection.

[0071] [ Figure 1B] Schematic diagram of the recombinant viral genome generated by reverse genetics from the clinical strain CAN98-75, into which the GFP (green fluorescent protein) coding sequence has been cloned. The virus rCAN98-75 contains the genome of the wild-type strain CAN98-75, with the GFP gene additionally expressed at its 3' end. The genome of the recombinant virus is derived from strain CAN98-75 and lacks the SH (ΔSH-rCAN98-75) or G (ΔG-rCAN98-75) gene, as shown below. The genetically modified viruses are functional and replicative, as shown in the images of LLC-MK2 cells infected with these viruses at an MOI of 0.01 and observed by fluorescence microscopy three days after infection.

[0072] Figure 2. In vitro replication ability of virus strains rC-85473 and rCAN98-75 and their derivatives.

[0073] [ Figure 2A ] After LLC-MK2 cells were infected with recombinant viruses rCAN98-75 (black circles), ΔSH-rCAN98-75 (black squares) and ΔG-rCAN98-75 (black triangles) at a multiplicity of infection (MOI) of 0.01, the viral load was assessed using TCID50 / ml in the cell supernatants collected daily for 7 consecutive days.

[0074] [ Figure 2B ]After LLC-MK2 cells were infected with recombinant viruses rC-85473 (black circles), ΔSH-rC-85473 (black squares) and ΔG-rC-85473 (black triangles) at an infection multiplicity of 0.01, the viral load was assessed using TCID50 / ml in the cell supernatants collected daily for 7 consecutive days.

[0075] **p<0.01, and ***p<0.001 (two-way ANOVA statistical test).

[0076] Figure 3: In vivo characterization of recombinant hMPV (rCAN98-75 and rC-85473) by infection of BALB / c mice: body weight curves and lung viral loads 5 days after infection.

[0077] [ Figure 3A ] by intranasal instillation of OptiMem culture medium (“mock” negative control, black triangles), or 2.5×10 5 FFU recombinant viruses rCAN98-75 (black circles), ΔSH-rCAN98-75 (grey squares) and ΔG-rCAN98-75 (grey triangles) were used to infect BALB / c mice. The body weight and survival of mice were monitored daily for 14 days.

[0078] [ Figure 3B] Five days after infection, five mice in each group (except the negative control group) were euthanized to measure the lung infectious virus load in terms of TCID50 / g of lung.

[0079] [ Figure 3C ] by intranasal instillation of OptiMem culture medium (“mock” negative control, black diamonds), or 5×10 5 TCID50 recombinant viruses rC-85473 (black circles), ΔSH-rC-85473 (grey squares) and ΔG-rC-85473 (grey triangles) infected BALB / c mice. The body weight and survival of mice were monitored daily for 14 days.

[0080] [ Figure 3D ] Five days after infection, five mice in each group (except the negative control group) were euthanized to measure the lung infectious virus load in terms of TCID50 / g of lung.

[0081] Statistical test: Two-way ANOVA test comparing each virus to the "mock" condition. *p<0.05, **p<0.01, and ***p<0.001.

[0082] Figure 4: In vivo protective characteristics of recombinant hMPVΔG-rC-85473 and ΔSH-rC-85473 in the BALB / c mouse infectious challenge model.

[0083] [ Figure 4A ] Immunization scheme (top), followed by infectious challenge scheme (bottom). The lethal dose is equivalent to 1×10 6 TCID 50 .

[0084] [ Figure 4B ] Mice immunized with virus strains rC-85473 (black circles), ΔSH-rC-85473 (grey squares), or ΔG-rC-85473 (grey triangles), or unimmunized (“mock”, black triangles) mice were challenged with infection.

[0085] 21 days after immunization, a lethal dose (1×10 6 TCID 50 The mice were infected with wild-type virus rC-85473. Their body weight was monitored daily for 14 days.

[0086] [ Figure 4C ] Post-challenge survival curves of mice in different groups of immunized mice.

[0087] Only 50% of the "mock" unimmunized mice survived; the survival rate of mice immunized with recombinant virus rC-85473, ΔSH-rC-85473 or ΔG-rC-85473 was 100%.

[0088] [ Figure 4D ] Five days after infectious challenge (i.e., on day 26 after immunization), four mice in each group were euthanized to measure their lung infection virus load of TCID50 / g lung. "nd" means not detectable.

[0089] [ Figure 4E ] Five days after infectious challenge (i.e., on day 26 after immunization), four mice in each group were euthanized to measure their lung infection virus load by RTqPCR (N gene).

[0090] Statistical test: Two-way ANOVA test comparing each virus to mock condition. *p<0.05, **p<0.01, and ***p<0.001.

[0091] Figure 5: In vitro cell binding ability of recombinant viruses rC-85473 and rCAN98-75 and their deletion forms.

[0092] [ Figure 5A ] Percentage of LLC-MK2 cells infected as a function of binding time to the virus rCAN98-75 (circles) and its attenuated forms ΔSH-rCAN98-75 (squares) and ΔG-rCAN98-75 (triangles).

[0093] [ Figure 5B ] Percentage of LLC-MK2 cells infected as a function of binding time to the virus rC-85473 (circles) and its attenuated forms ΔSH-rC-85473 (squares) and ΔG-rC-85473 (triangles).

[0094] Figure 6: Ex vivo replication ability of recombinant viruses rC-85473 and rCAN98-75 when cultured at the air-liquid interface on a reconstructed 3D model of human airway epithelium.

[0095] [ Fig. 6A ]Human respiratory epithelial model in culture was infected with recombinant viruses rCAN98-75, ΔSH-rCAN98-75 and ΔG-rCAN98-75 (upper row of pictures) or recombinant viruses rC-85473 (white), ΔSH-rC-85473 (dark gray) and ΔG-rC-85473 (uncertain) (lower row of pictures).

[0096] [ Figure 6B] Quantification of viral progeny (by quantification of viral genomes) measured on the apical surface of epithelia infected with recombinant viruses rCAN98-75 (WT, white), ΔSH-rCAN98-75 (dark gray) and ΔG-rCAN98-75 (light gray) (left side of the dashed line) or recombinant viruses rC-85473 (WT), ΔSH-rC-85473 and ΔG-rC-85473 (right side of the dashed line).

[0097] [ Figure 6C ] Quantification of viral genomes present in epithelia infected with recombinant viruses rCAN98-75 (WT, white), ΔSH-rCAN98-75 and ΔG-rCAN98-75 (left side of the dashed line) or recombinant viruses rC-85473 (WT, white), ΔSH-rC-85473 and ΔG-rC-85473 (right side of the dashed line).

[0098] Figure 7. Secretion of cytokines and chemokines induced by infection with the recombinant virus rC-85473 in a reconstructed 3D model of human airway epithelium and cultured at the air-liquid interface.

[0099] Reconstruction of human airway epithelium ( HAE, Epithelix) were infected with recombinant viruses rC-85473, ΔSH-rC-85473 and ΔG-rC-85473 at an MOI of 0.1. Five days after infection, the amount of several cytokines and chemokines was measured from the basal medium of the infected epithelium by Luminex technology (BioPlex Pro kit method, BioRad):

[0100] [ Fig. 7A ] Chemokines MCP-1, IP-10, RANTES, IL-8, T lymphocytes, monocytes / macrophages, eosinophils and / or neutrophils that attract G-CSF.

[0101] [ Figure 7B ] Quantification of proinflammatory cytokines IFN-γ and TNF-α.

[0102] [ Figure 7C ] Quantification of the cytokine IL-7, which stimulates the differentiation of T and B lymphocytes.

[0103] [ Fig.7D ] Growth factor FGF, which is involved in tissue repair.

[0104] Statistical test: One-way ANOVA analysis comparing viruses ΔSH-rC-85473 and ΔG-rC-85473 with virus rC-85473. *p<0.05, **p<0.01 and ***p<0.001.

[0105] FIG. 8 . In vitro secretion of the virus-induced pro-inflammatory cytokines IL-1β and TNF-α in an immortalized murine macrophage model infected with recombinant hMPV rC-85473.

[0106] [ Fig. 8A ] Two days after the immortalized mouse macrophage cell line (BMDM line derived from wild mouse bone marrow) was infected with recombinant viruses rC-85473 (black), ΔSH-rC-85473 (dark gray) or ΔG-rC-85473 (light gray) at an MOI of 0.1, the viral load was assessed in FFU / ml in the cell supernatant collected daily.

[0107] [ Figure 8B ] The amount of the proinflammatory cytokine TNF-α assessed by ELISA assay in cell supernatants collected daily 2 days after immortalized mouse macrophages were infected with recombinant viruses rC-85473 (black), ΔSH-rC-85473 (dark gray) or ΔG-rC-85473 (light gray) at an MOI of 0.1 compared to uninfected "mock" immortalized mouse macrophages (white).

[0108] [ Figure 8C ] The amount of the proinflammatory cytokine IL-1β assessed by ELISA assay in cell supernatants collected daily 2 days after immortalized mouse macrophages were infected with recombinant viruses rC-85473 (black), ΔSH-rC-85473 (dark gray) or ΔG-rC-85473 (light gray) at an MOI of 0.1 compared to uninfected "mock" immortalized mouse macrophages (white).

[0109] Statistical test: Two-way ANOVA analysis comparing viruses ΔSH-rC-85473 and ΔG-rC-85473 with virus rC-85473. *p<0.05 and **p<0.01.

[0110] Figure 9. Pulmonary inflammation and immune cell recruitment at the infection site in vivo 5 days after infection of BALB / c mice with recombinant hMPV rC-85473, ΔSH-rC-85473, and ΔG-rC-85473 (pulmonary inflammation specific to ΔG-rC-85473).

[0111] [ Fig.9A] Cumulative lung histopathology scores were calculated based on the assessment of the intensity of inflammation in bronchial / endobronchial, peribronchial, perivascular, interstitial, pleural, and intraalveolar tissues of the lungs of BALB / c mice, which were infected with the viruses rC-85473 (black), ΔSH-rC-85473 (dark gray), ΔG-rC-85473 (light gray), or "mock" uninfected, and the tissues were collected 5 days after virus infection and fixed with formaldehyde.

[0112] [ Fig. 9B ] Five days after infection, flow cytometry was performed to determine the phenotype of immune cells in lysates of lungs of BALB / c mice infected with virus rC-85473 and ΔSH-rC-85473 by immunolabeling (anti-CD45, anti-CD11b, anti-CD170, anti-Ly6C, anti-Ly6G, anti-CD11c, anti-CD115, anti-B220, anti-CD3ε, anti-CD4, anti-CD8a). The recruited immune cells were divided into four categories: CD8+T lymphocytes (CD8+T cells), CD4+T lymphocytes (CD4+T cells), monocytes / macrophages, and neutrophils.

[0113] Statistical test: One-way ANOVA analysis comparing viruses ΔSH-rC-85473 and ΔG-rC-85473 with virus rC-85473. **p<0.01 and ***p<0.001.

[0114] Figure 10. Characterization of the in vivo secondary immune response in a BALB / c mouse model immunized with recombinant hMPVΔG-C-85473 and ΔSH-C-85473 following infectious challenge.

[0115] like Figure 4A As shown, the amounts of several cytokines and chemokines were measured in lung lysates by Luminex technology (BioPlex Pro kit assay, BioRad) 1 day and / or 5 days after infectious challenge with the virus WT rC-85473 [ Figure 10A-10C ]; flow cytometry was performed to determine the phenotype of immune cells infiltrating lung tissue from lung lysates by immunolabeling (anti-CD45, anti-Ly6G, anti-CD11b, anti-CD170 / Siglec-F, anti-Ly6C, anti-CD11c, anti-F4 / 80, anti-B220, anti-CD3ε, anti-CD4, and anti-CD8a) [ Figure 10D-10F ]; and, cumulative lung histopathology scores were calculated based on the assessment of the intensity of inflammation in the bronchial / endobronchial, peribronchial, perivascular, interstitial, pleural, and intraalveolar tissues of formaldehyde-fixed rat lungs [ Figure 10G ].

[0116] exist FIG. 10A to FIG. 10GIn the statement:

[0117] - Black, group of BALB / c mice immunized with virus rC-85473;

[0118] - Dark gray, group immunized with virus ΔSH-C-85473;

[0119] - Light gray, group immunized with virus ΔG-C-85473;

[0120] - and (where available), white represents the so-called "mock" unimmunized group.

[0121] N = 2 or 3 mice / group for cytokine histopathology or secretion / cellular recruitment analysis, respectively.

[0122] Statistical test: Two-way ANOVA analysis to compare groups with each other, *p<0.05, **p<0.01 and ***p<0.001.

[0123] [ Fig. 10A ] The proinflammatory cytokines IL-6 and IFN-γ were quantified 1 and 5 days after infectious challenge.

[0124] [ Fig. 10B ] The anti-inflammatory cytokine IL-10 was quantified 1 and 5 days after infectious challenge.

[0125] [ Fig. 10C ] At 1 and 5 days after infectious challenge, the attracted chemokines G-CSF, MCP-1, MIP-1α, and MIP-1β were quantified and stimulated the activation and differentiation of immune cells.

[0126] [ Fig. 10D ] Quantification of CD8+ and CD4+ T lymphocytes infiltrating lung tissue of BALB / c mice 1 and 5 days after infectious challenge.

[0127] [ Fig. 10E ] Quantification of B lymphocytes infiltrating lung tissue of BALB / c mice 1 and 5 days after infectious challenge.

[0128] [ Fig.10F ] Quantification of macrophages and neutrophils infiltrating lung tissue of BALB / c mice 1 and 5 days after infectious challenge.

[0129] [ Figure 10G ] Cumulative lung histopathology scores 5 days after infectious challenge. DETAILED DESCRIPTION

[0130] Human metapneumovirus (hMPV) strains

[0131] In 2001, hMPV was identified as a member of the Pneumoviridae family.

[0132] The genomic structure of hMPV is similar to that of hRSV. It is subdivided into several ORFs (open reading frames) encoding N, P, M, F, M2 (M2-1 and M2-2), SH, G and L proteins. Figure 1A and Figure 1B The general genome structure of viruses rC-8543 and rCAN98-75 is shown in FIG.

[0133] Virus members of the Pneumoviridae family express three glycoproteins on their surface called F, G and SH proteins.

[0134] The F fusion glycoprotein is highly conserved between different hMPV subgroups and is involved in the penetration of the virus into target cells and the formation of syncytia, i.e. large multinucleated cells derived from the fusion of individual cells after infection with the virus. The F glycoprotein is also considered to be the major antigen of the viruses hRSV and hMPV, thereby inducing the production of neutralizing antibodies.

[0135] The G protein is a type II membrane protein with its C-terminus outside the cell. This protein is not essential for the assembly of viral components and for replication in vitro. For hRSV, it has been shown that the deletion of the gene encoding this protein reduces the virulence of the strain during respiratory infection in mice.

[0136] The SH protein is also a type II membrane protein whose function in the viral cycle remains unknown. In the case of hRSV, deletion of the gene encoding the SH protein produced a recombinant virus that was able to propagate in vitro and whose virulence was attenuated in the upper respiratory tract of mice, but not in the lower part of the respiratory tract.

[0137] Genetic analysis of clinical strains of hMPV, based primarily on sequence variability of the surface binding (G) and fusion (F) glycoproteins, enabled the definition of two major groups (serotypes A and B) and four "minor" subgroups (A1, A2, B1, and B2). It was subsequently shown that these groups could be further subdivided into sublineages, such as A2a and A2b (Huck et al., 2006).

[0138] Among the widely studied virus strains, there can be mentioned the NL00-1 strain belonging to serotype A1; the CAN97-83 strain belonging to serotype A2; and the CAN98-75 strain belonging to serotype B2.

[0139] The present invention is based on a strain of human metapneumovirus isolated from a Canadian patient sample, designated rC-85473, specifically mentioned in the article (Hamelin et al., 2010).

[0140] It has been proposed that the F protein of this strain be combined with the M protein as a vaccination strategy (Aerts et al., 2015a).

[0141] In the article (Palwithino et al., 2015), the use of the virus strain rC-85473 inactivated by formalin treatment as an inactivated live vaccine strain is described. A heat-inactivated version of the rC-85473 strain was also tested (Hamelin et al., 2007).

[0142] Recently, the strong fusion capacity of this strain was studied in detail based on the protein sequence of its F protein (Dubois et al., 2017).

[0143] The sequences of the F gene and the M gene of strain rC-85473 were cited in GenBank with accession numbers KM408076.1 and KM408077.1, respectively.

[0144] This article discloses for the first time the complete genome sequence of the virus strain rC-85473, which contains 13394 nucleotides: see SEQ ID NO: 1 in the attached sequence listing.

[0145] Table 4 below shows the percent identity of this strain rC-85473 with three other strains of human metapneumovirus: CAN 97-83, CAN 98-75 and NL 00-1.

[0146] Table 4 – Percentage of nucleotide sequence identity for each ORF between virus rC-85473 and other hMPVs (amino acid sequence identity in brackets)

[0147]

[0148] Regarding the high sequence homology observed with strain NL00-1, it can be concluded that strain rC-85473 is a member of subgroup A1.

[0149] The characteristics of strain rC-85473 are: It has a remarkable fusion capacity, which allows it to penetrate into target cells with high frequency / high infectivity. The remarkable fusion capacity of this strain makes it possible to produce particularly long syncytia. Syncytia are giant multinucleated cells. Virus rC-85473 has a high ability to induce cell fusion in vitro, resulting in the formation of very long syncytia composed of many nuclei.

[0150] It has now been shown that five amino acids in the HRA functional domain of the F protein, which are unique among other known hMPV strains, are partially responsible for this hyperfusion phenotype (Aerts et al., 2015b; Dubois et al., 2017).

[0151] According to a preferred embodiment of the present invention, the peptide sequence of the attenuated virus strain F protein derived from strain rC-85473 comprises these 5 amino acids of the HRA functional domain, as described, for example, in (Dubois et al., 2017).

[0152] According to a preferred embodiment of the present invention, the peptide sequence of the attenuated virus strain F protein derived from strain rC-85473 is not modified and is therefore identical to the peptide sequence of the F protein of strain rC-85473, as described in (Dubois et al., 2017).

[0153] Reverse genetics

[0154] Reverse genetics techniques can generate functional, encapsidated viruses from nucleic acid sequences (DNA or RNA) and from suitable host cells.

[0155] Thus, the recombinant strain of human metapneumovirus, rC-85473, was obtained from the clinical strain designated C-85473 (isolated from a patient).

[0156] The first reverse genetics system applied to hMPV was described in 2004 (Biachesi et al., 2004). This technique is also described in the article (Aerts et al., 2015b).

[0157] The principle of this technology is based on the use of a hamster kidney cell line (BHK-21) modified to constitutively express the RNA polymerase of the T7 phage (BHK-T7 or BSR-T7 / 5 cells), enabling the production of recombinant hMPV.

[0158] The genomic elements are distributed among five plasmid elements: one plasmid encoding the hMPV antigenome, and four "satellite" plasmids encoding the viral proteins of the transcription machinery (L, P, N, and M2-1).

[0159] In these cells, after co-transfection of the antigenomic plasmid hMPV and four "satellite" plasmids, the RNA strand corresponding to the viral genome strand (the negative RNA strand) is transcribed by T7 polymerase from its promoter.

[0160] The transfected host cells also express four proteins involved in the transcription of hMPV virus, thus constituting an active RNA-dependent RNA polymerase (RdRP) complex. Thus, this functional viral polymerase transcribes the genomic RNA into viral mRNA, which is then copied into new molecules of viral genomic RNA by transcription of the matrix strand.

[0161] Thus, translation and assembly of viral proteins with genomic RNA allowed infectious hMPV particles to bud from the plasma membrane of transfected BHK-T7 cells. Next, amplification of the recombinant virus was enabled by the addition of LLC-MK2 cells (ATCC CCL-7) to the co-culture to allow infection.

[0162] Therefore, since these modified cells are commercially available, recombinant viruses can be produced from the genomic sequence of a specific viral strain.

[0163] Attenuated virus strain derived from strain rC-85473

[0164] The present invention relates to an attenuated virus strain derived from a human metapneumovirus strain, which comprises a genome sequence shown in SEQ ID NO: 1. The attenuated strain comprises one or more genetic modifications of the sequence SEQ ID NO: 1, thereby reducing the virulence of the strain.

[0165] In this application, the terms "virus" and "viral strain" are used indiscriminately to refer to a specific viral strain, such as one previously identified.

[0166] In the sense of the present invention, a "derivative strain" refers to a recombinant virus strain obtained by introducing a genetic modification into the genome of a so-called "original strain". The original strain is advantageously a wild strain, such as a clinical isolate.

[0167] All genetic modifications introduced into the original strain were aimed at reducing the virulence of the original strain rather than changing the characteristics of its genome.

[0168] Specifically, these genetic modifications only involve genes encoding proteins that are not essential for viral replication, in other words, genes encoding "accessory proteins", such as SH and G proteins. In this genetically modified attenuated strain, the F protein peptide sequence of the original strain rC-85473 has not been modified, so it has the same peptide sequence as the original strain.

[0169] The virulence of a virus strain corresponds to the speed with which the virus multiplies in a given organism and therefore to its penetration rate. Thus, "reducing virulence" means reducing the penetration rate of a virus in an organism.

[0170] Such attenuation may take the form of a reduction in the ability of the viral strain to replicate, and / or a reduction in its ability to infect target cells, and / or a reduction in pathology caused by viral infection in an organism.

[0171] Therefore, in the sense of the present invention, an "attenuated virus strain" refers to a recombinant virus, the virulence of which is reduced compared to the original virus strain, that is to say, is less virulent than the original virus strain.

[0172] To measure the virulence of a viral strain, in vitro, ex vivo, or in vivo tests can be performed, such as in vitro replication capacity testing (measured by TCID50 / ml titration or quantitative PCR), monitoring the evolution of in vitro and ex vivo cytopathic effects by microscopic observation, or monitoring clinical signs of pathology in an in vivo infection model, lung tissue pathology observation, and measurement of lung viral load.

[0173] In order to compare the physiological effects of wild-type and attenuated strains, different physiological parameters altered at the level of the lung and / or other immune systems may also be measured / determined in the viral host organism.

[0174] As described in Examples 7, 8 and 9 of the present application, the following virus-induced effects can be evaluated to compare the physiological effects of attenuated virus strains with each other or between attenuated virus strains and wild-type virus strains:

[0175] - Secretion of pro-inflammatory cytokines (by epithelial cells and / or macrophages);

[0176] - Secretion of chemokines;

[0177] - Secretion of growth factors;

[0178] - the level of lung inflammation (a score is calculated by assessing the intensity of inflammation in the tissue); and

[0179] - Phenotype of immune cells recruited to infection sites in vivo.

[0180] These different measurements allow for a more precise understanding of the physiological role of each virus strain and, based on these characteristics, selection of those most suitable for the development of attenuated live virus vaccines.

[0181] Introducing genetic modifications into a virus strain to obtain its attenuated form

[0182] The present invention relates to an attenuated virus strain derived from a human metapneumovirus strain comprising a genome sequence shown in SEQ ID NO: 1, wherein the attenuated strain comprises one or more genetic modifications of the sequence SEQ ID NO: 1, thereby reducing the virulence of the strain.

[0183] "Genetic modification that reduces the virulence of the strain" refers to genetic modification of genes encoding proteins that are not essential for viral replication, which is well known to those skilled in the art. Among these proteins, the G and SH proteins as described above are well known.

[0184] In other words, the present application relates to an attenuated virus strain derived from a human metapneumovirus strain comprising a genomic sequence as shown in SEQ ID NO: 1, wherein the attenuated strain has been genetically modified to reduce its virulence, that is, the introduced genetic mutation has been uniquely introduced into a gene encoding a protein that is not essential for viral replication.

[0185] Various genetic modifications that can reduce the virulence of the virus strain are known to those skilled in the art. These modifications can be introduced into the genome of the original strain rC-85473.

[0186] In the sense of the present invention, genetic modification refers to all modifications to the original nucleotide sequence, such as the deletion of one or more nucleotides, the interpolation of one or more nucleotides and the replacement of one or more nucleotides. These modifications specifically include all modifications that can change the genetic reading frame, or introduce a terminator codon into the middle of the coding sequence, or lack all or part of one or more coding sequences.

[0187] Among the genetic modifications aimed at attenuating the virulence of a viral strain, it is known that the genetic modification may inactivate or even delete one or more genes encoding proteins that are not essential for viral replication in culture.

[0188] For example, attenuated strains of human metapneumovirus were obtained by inactivating, in particular by deletion, the genes encoding the SH, G and M2-2 accessory proteins.

[0189] However, the functional characteristics of the attenuated virus strain may be very different depending on the characteristics of the original virus strain from which it is derived, and the virulence of the strain depends on the original genome and the genetic modifications that have been performed.

[0190] According to a first aspect of the invention, the attenuated virus strain according to the invention is characterized in that the modification of the sequence SEQ ID NO: 1 comprises the inactivation of the gene encoding the SH protein.

[0191] More specifically, the attenuated virus strain according to the invention is characterized in that the genetic modification of the sequence SEQ ID NO: 1 which attenuates the virulence of the strain consists in the inactivation of the gene encoding the SH protein.

[0192] In the sense of the present invention, the inactivation of a gene means the fact that the gene is modified so that the product of the gene is no longer expressed or is expressed in an inactive form, in such a small amount that there is no activity of the protein. The inactivation of a gene can be carried out by all techniques well known to those skilled in the art.

[0193] Specifically, the inactivation of a gene can be obtained by introducing a point mutation into the gene, by partially or completely deleting the coding sequence of the gene, or by modifying the gene promoter. These different genetic modifications will be performed according to any molecular biological technique well known to those skilled in the art.

[0194] In the sense of the present invention, "gene deletion" refers to the removal of a significant part of the gene coding sequence from the genome of the virus strain, in particular:

[0195] - when it comes to partial deletions, at least 50%, 60%, 70%, 80%, 90% or 95% of the coding sequence;

[0196] - When a complete deletion is concerned, 100% of the coding sequence.

[0197] According to a preferred embodiment, in the attenuated virus strain according to the present invention, the gene encoding the SH protein is completely deleted, that is to say all coding sequences (100%) of the SH protein are removed from the original sequence SEQ ID NO:1.

[0198] Specifically, the virus strain comprises a nucleotide sequence, such as the nucleotide sequence shown in SEQ ID NO: 2. More specifically, the sequence of the virus strain consists of the nucleotide sequence shown in SEQ ID NO: 2.

[0199] As shown in the Examples, this attenuated strain is called ΔSH-rC-85473 and has the following characteristics:

[0200] - In vitro, infection and replication in cultured cells are normal (no differences from the characteristics of the original strain);

[0201] - In vivo, infection of mice with the recombinant virus ΔSH-rC-85473 caused only minimal weight loss, unlike infection with the original strain; the survival rate of infected mice was 100%, but the attenuated virus replicated in lung tissue;

[0202] - In vivo, mice previously immunized with this attenuated strain were 100% protected from challenge with a lethal dose of wild-type virus rC-85473. This protection was also demonstrated by the absence of infectious virus rC-85473 detected in the lungs on day 5 after challenge; this protection was also expressed in the serum at D+21 after infectious challenge, against the homologous virus strain (C-85473, serotype A) and against at least one heterologous strain (CAN98-75, serotype B);

[0203] - In vitro, the attenuated virus ΔSH-rC-85473 has a better ability to bind to and infect LLC-MK2 cells in culture (about 60% of infected cells) than the virus ΔSH-rCAN98-75 (about 40% of infected cells);

[0204] - In vitro, infection of the respiratory epithelial model with this attenuated strain induces secretion of cytokines and chemokines in amounts equal to or less than those induced by the wild-type strain; however, the inflammatory response is reduced compared to that induced by the wild-type strain ( Fig. 7A , Figure 7B );

[0205] - In vitro, infection of macrophages with this attenuated strain induces the secretion of proinflammatory cytokines, but with kinetics different from those induced by the wild-type strain ( Figure 8B , Figure 8C );

[0206] - In vivo, the virus-induced lung inflammation after infection with this attenuated strain was significantly lower than that of the wild-type strain ( Fig.9A ); however, the recruitment of immune cells can still be ensured ( Fig. 9B ).

[0207] According to a second aspect of the present invention, the attenuated virus strain according to the present invention is characterized in that the modification of the sequence SEQ ID NO: 1 comprises inactivating the gene encoding the G protein.

[0208] More specifically, the attenuated virus strain according to the present invention is characterized in that the genetic modification of the sequence SEQ ID NO: 1 which attenuates the virulence of the strain consists in inactivating the gene encoding the G protein.

[0209] According to a preferred embodiment, in the attenuated virus strain according to the present invention, the gene encoding the G protein is completely deleted, that is to say all coding sequences of the G protein are removed (100%) from the original sequence SEQ ID NO:1.

[0210] Specifically, the virus strain comprises a nucleotide sequence, such as the nucleotide sequence shown in SEQ ID NO: 3. More specifically, the sequence of the virus strain consists of the nucleotide sequence shown in SEQ ID NO: 3.

[0211] As shown in the Examples, this attenuated strain is called ΔG-rC-85473 and has the following characteristics:

[0212] - In vitro, in the LLC-MK2 cell model, infection and replication in cultured cells were normal (no differences from the characteristics of the original strain);

[0213] - In vivo, infection of mice with the recombinant virus ΔG-rC-85473 caused only minimal weight loss; the survival rate of infected mice was 100%; however, the virus replicated in lung tissue;

[0214] - In vivo, mice previously immunized with this attenuated strain were 100% protected from challenge with a lethal dose of wild-type virus rC-85473. This protection was also demonstrated by the absence of infectious virus and wild-type genome rC-85473 detected in the lungs on D+5 days after challenge; this protection was also indicated by high micro-neutralization loads in the serum against the homologous virus strain (C-85473, serotype A) and against at least one heterologous strain (CAN98-75, serotype B) on D+21 after infectious challenge;

[0215] - In vitro, the attenuated virus ΔG-rC-85473 has a better ability to bind to and infect LLC-MK2 cells in culture (about 60% of infected cells) than the virus ΔG-rCAN98-75 (less than 20% of infected cells).

[0216] - In vitro, infection of the respiratory epithelial model with this attenuated strain induces the secretion of cytokines and chemokines in amounts significantly lower than those induced by the wild-type strain ( FIG. 7 );

[0217] - In vitro, infection of macrophages with this attenuated strain induced the secretion of TNF-α in the same manner as the wild-type virus, but only very slightly induced the secretion of IL-1β ( Figure 8B , Figure 8C );

[0218] - In vivo, the virus-induced lung inflammation after infection with this attenuated strain was significantly lower than that of the wild-type strain or the attenuated strain ΔSH-rC-85473 ( Fig.9A ).

[0219] According to the third aspect of the present invention, the attenuated virus strain according to the present invention is characterized in that the modification of the sequence SEQ ID NO: 1 includes inactivating two genes encoding the G protein and the SH protein.

[0220] More specifically, the attenuated virus strain according to the invention is characterized in that the genetic modification of the sequence SEQ ID NO: 1 which renders said strain attenuated consists in the inactivation of two genes, one encoding the SH protein and the other encoding the G protein.

[0221] According to a specific embodiment, the inactivation of both genes corresponds to the complete deletion of one, or the other, or both genes encoding the G and SH proteins.

[0222] In addition, it is understood that any other genetic modification that can attenuate the virulence of strain rC-85437 can be introduced into the genome of the strain shown in the sequence of SEQ ID NO:1.

[0223] Introducing foreign genes into the genome of virus strain rC-85473

[0224] According to one aspect of the present invention, the nucleotide sequence of the attenuated virus strain according to the present invention may be genetically modified by introducing at least one exogenous gene.

[0225] Therefore, the attenuated virus strain according to the present invention has a genome sequence comprising at least one exogenous gene. Specifically, the exogenous gene can be a gene encoding a viral antigen.

[0226] In the sense of the present invention, "viral antigens" are understood to mean protein elements expressed by a virus or elements of another nature, which are recognized as foreign by the immune system of an individual and which provoke a response in said individual by producing specific antibodies and / or stimulating a cellular immune response.

[0227] Specifically, the viral antigen can be selected from: at least one antigen expressed by influenza virus, or at least one antigen expressed by a virus of the Pneumoviridae family (eg, hRSV), or at least one antigen expressed by a virus of the Paramyxoviridae family (eg, parainfluenza virus).

[0228] More specifically, the viral antigen may be selected from: all or part of the F protein of hRSV, and all or part of the hemagglutinin of influenza virus or parainfluenza virus.

[0229] This attenuated virus strain will be able to generate multiple immune responses against expressed viral antigens and against hMPV during administration to a patient.

[0230] Such strains are very advantageous as they enable a combined immune response against several viruses after a single administration.

[0231] In addition, such attenuated virus strains comprising at least one foreign gene can be used as expression vectors of at least one foreign gene in target cells of human metapneumovirus in vivo, ex vivo or in vitro.

[0232] "Target cells of human metapneumovirus" are respiratory epithelial cells of an individual susceptible to infection by the virus. The expression also means all cell lines enabling the virus to replicate in vitro.

[0233] Use of attenuated virus strains as medicines

[0234] The invention also relates to an attenuated virus strain as defined above for use as a medicament.

[0235] Indeed, this strain is particularly useful for treating or preventing viral infections.

[0236] The term "treatment" refers to the fact of combating a viral infection in a human or animal organism. Due to the administration of at least one composition according to the invention, the level of viral infection (infectious load) in the organism will be reduced, and preferably the virus will completely disappear from the organism. The term "treatment" also refers to the fact of alleviating symptoms associated with viral infection (respiratory syndrome, renal failure, fever, etc.).

[0237] In the sense of the present invention, the term "prevention" refers to the fact that the risk of infection in a human or animal organism is avoided (or at least reduced). Due to the administration of at least one attenuated virus strain according to the invention, the human or animal cells of said organism become less susceptible to infection and are therefore optimally protected from infection by said virus.

[0238] More specifically, the present invention relates to an attenuated virus strain, such as defined above, for use in the prevention and / or treatment of infections by viruses of the Pneumoviridae family.

[0239] It will be appreciated that the attenuated viral strain will preferably be incorporated into a vaccine composition comprising a pharmaceutically acceptable carrier suitable for suspending the strain and for administering it.

[0240] The vaccine composition comprises at least one attenuated virus strain according to the invention, making it possible to stimulate the immune system of a human or animal organism in a specific manner.

[0241] Thus, the vaccine composition comprises at least one live attenuated virus strain that functions as an antigen, ie a compound that induces a specific immune response in an organism, the memory of which will be retained.

[0242] Such vaccine compositions can be used as prophylactic vaccines, that is to say aimed at stimulating a specific immune response before the pathogenic virus infects the organism.

[0243] Such vaccine compositions may also be used as therapeutic vaccines, that is to say intended to stimulate a specific immune response simultaneously with the pathogenic viral infection of the organism.

[0244] The present invention also relates to a method for preventing and / or treating Pneumoviridae infections in humans, which method comprises administering at least one attenuated virus strain as described above to an individual susceptible to infection by such a virus.

[0245] The present invention also relates to a method for preventing and / or treating Pneumoviridae infections in humans, which method comprises administering to an individual susceptible to infection by such a virus at least one vaccine composition as described above comprising at least one attenuated virus strain.

[0246] According to a specific embodiment of the present invention, the infection is an infection with human metapneumovirus.

[0247] According to another specific embodiment of the invention, the infection is an infection of the Orthopneuviridae family, such as human respiratory syncytial virus (hRSV).

[0248] The present invention also relates to an attenuated virus strain, the sequence of which comprises at least one foreign gene encoding a viral antigen, which is used for preventing and / or treating a viral infection expressing at least one viral antigen expressed by the attenuated virus strain.

[0249] According to a specific embodiment, the attenuated virus strain according to the present invention, which comprises the F protein of hRSV as an exogenous viral antigen, can be used to prevent and / or treat hRSV infection.

[0250] According to another specific embodiment, the attenuated virus strain according to the present invention comprises hemagglutinin of influenza virus as an exogenous viral antigen and can be used for preventing and / or treating influenza virus infection.

[0251] Pharmaceutical composition comprising at least one attenuated virus strain

[0252] The present invention also relates to a vaccine composition comprising at least one attenuated virus strain according to the present invention in a pharmaceutically acceptable carrier, and optionally an adjuvant.

[0253] According to the present invention, the term "pharmaceutically acceptable carrier" refers to a carrier or excipient, i.e. a compound that is considered herein to have no specific effect on infection. These carriers or excipients are pharmaceutically acceptable, which means that they can be administered to an individual or animal without significant risk of adverse effects or inhibitory undesirable effects.

[0254] It is understood that the vaccine composition according to the invention comprises at least one attenuated virus strain in an effective amount. In the sense of the present invention, "effective amount" refers to the amount of attenuated virus strain sufficient to trigger an immune response in an organism to which it is administered.

[0255] The vaccine composition for use according to the present invention is suitable for oral, sublingual, inhalation, subcutaneous, intramuscular or intravenous administration.

[0256] According to a particular embodiment of the invention, the vaccine composition according to the invention is characterized in that it is in a pharmaceutical form intended for administration by inhalation.

[0257] Inhalation means absorption by the respiratory tract. In particular, this is a method used to absorb compounds of therapeutic interest, certain substances in the form of gases, droplets in suspension or powders.

[0258] Administration of pharmaceutical or veterinary compositions by inhalation, ie through the nasal and / or buccal passages, is well known to those skilled in the art.

[0259] There are two types of inhalation administration:

[0260] - when the composition is in powder form, by insufflation, and

[0261] - Administration by nebulization when the composition is in the form of an aerosol (suspension) or in the form of a solution (eg a pressurized aqueous solution). It would then be recommended to use a nebulizer or spray to administer the pharmaceutical or veterinary composition.

[0262] Thus, the pharmaceutical forms contemplated here are advantageously selected from: powders, drops, aqueous suspensions or pressurized solutions.

[0263] The population of individuals to be vaccinated is mainly the pediatric population, that is to say composed of individuals younger than 18 years of age, more specifically infants and babies younger than 5 years of age. In fact, the viruses of the Pneumoviridae family mainly infect these individuals, who tend to have weaker immunity than older individuals.

[0264] The present invention also relates to the vaccine composition as described above, and its use for preventing and / or treating Pneumoviridae virus infection.

[0265] Example

[0266] Example 1: Recombinant hMPV produced from clinical strains C-85473 and CAN98-75.1A

[0267] preparation Figure 1A and Figure 1B The gene constructs shown were used so that the virus could be detected by the expression of GFP (green fluorescent protein) and genetic modifications aimed at reducing its virulence were introduced into the recombinant reference strains rC-85473 and rCAN98-75:

[0268] - Deletion of the gene encoding SH protein (ΔSH);

[0269] - Deletion of the gene encoding the G protein (ΔG).

[0270] The complete sequences of these gene constructs are shown in SEQ ID NO: 4 (GFP rC-85473), SEQ ID NO: 5 (GFPΔSH-rC-85473), SEQ ID NO: 6 (GFPΔG-rC-85473) and SEQ ID NO: 8 (GFP rCAN98-75). The sequences of the virus strain derived from rCAN98-75 and the sequences coupled to GFP are not shown, but those skilled in the art can obtain these sequences based on the other genetic constructs shown.

[0271] The photograph of LLC-MK2 cells infected with a multiplicity of infection (MOI) of 0.01 shows that the virus is visible due to GFP and the following recombinant viruses are produced: ΔSH-rCAN98-75, ΔG-rCAN98-75, ΔSH-rC-85473 and ΔG-rC-85473 are functional and replication-competent. Cells were observed by fluorescence microscopy three days after infection.

[0272] These images show that the recombinant virus rCAN98-75 appears to be less infectious and less replicative than the recombinant virus rC-85473: the fluorescence intensity is lower and the size of the syncytial zone is smaller.

[0273] Example 2: In vitro replication ability of recombinant viruses rC-85473 and rCAN98-75

[0274] LLC-MK2 cells were infected with the following recombinant viruses at a multiplicity of infection of 0.01:

[0275] -rCAN98-75, ΔSH-rCAN98-75 and ΔG-rCAN98-75( Figure 2A );

[0276] -rC-85473, ΔSH-rC-85473 and ΔG-rC-85473 ( Figure 2B ).

[0277] Cell supernatants were collected daily in triplicate for 7 days and their viral loads were then assessed as TCID50 / ml. A two-way ANOVA statistical test was performed to compare each deletion recombinant virus (ΔSH and ΔG) with the wild-type recombinant virus.

[0278] Figure 2A and Figure 2B The results obtained are shown: for the attenuated recombinant viruses, the replication capacity and the production capacity differ depending on the properties of the original strains CAN98-75 or C-85473.

[0279] The recombinant viruses rC-85473, ΔSH-rC-85473 and ΔG-rC-85473 had better replication and production capacity than the viruses rCAN98-75, ΔSH-rCAN98-75 and ΔG-rCAN98-75. The viruses ΔSH-rCAN98-75 and ΔG-rCAN98-75 had the lowest efficiency, while the viruses ΔSH-rC-85473 and ΔG-rC-85473 had the best production capacity when cultured on LLC-MK2 cells.

[0280] The table below lists the average load of the virus stocks that were produced and concentrated for each of the recombinant viruses rCAN98-75, ΔSH-rCAN98-75 and ΔG-rCAN98-75 (Table 5) and rC-85473, ΔSH-rC-85473 and ΔG-rC-85473 (Table 6).

[0281] Viral load is counted in "TCID50" units, which represents the final virus dilution at which 50% of the cellular tissue is destroyed by infection, or shows a significant cytopathic effect (50% tissue culture infectious dose).

[0282] Table 5

[0283] Recombinant virus Average loading of concentrated stock solution (TCID50 / ml) rCAN98-75 4.37E+07 ΔSH-rCAN98-75 2.77E+07 ΔG-rCAN98-75 8.65E+06

[0284] Table 6

[0285] Recombinant virus Average loading of concentrated stock solution (TCID50 / ml) rC-85473 3.92E+07 ΔSH-rC-85473 1.81E+08 ΔG-rC-85473 2.04E+08

[0286] These viral loads confirmed the aforementioned results, namely that the attenuated virus strain derived from C-85437 had satisfactory replication capacity and was not diminished compared to the replication capacity of the original strain.

[0287] Example 3: In vivo characterization of recombinant hMPV rCAN98-75 and rC-85473 in a BALB / c mouse virus infection model

[0288] Infect BALB / c mice by intranasal instillation:

[0289] -OptiMem culture medium (mock) or

[0290] -2.5×10 5 Fluorescent focus units (FFU) of recombinant viruses expressing GFP: rCAN98-75, ΔSH-rCAN98-75, and ΔG-rCAN98-75, or

[0291] -5×10 5 TCID50 of viruses not expressing GFP: rC-85473, ΔSH-rC-85473, and ΔG-rC-85473.

[0292] The doses of the two viruses used were identical (the wild-type virus produced the same effects in mice after infection).

[0293] The body weight and survival rate of mice were monitored daily for 14 days (mean body weight ± SEM of 10 mice).

[0294] In addition, 5 mice per group were euthanized 5 days after infection to measure the lung infection virus load in terms of TCID50.

[0295] Statistical tests (two-way ANOVA) were performed to compare each virus to mock conditions. *p<0.05, **p<0.01 and ***p<0.001.

[0296] Figure 3A and Figure 3B Results obtained with viruses rCAN98-75, ΔSH-rCAN98-75, and ΔG-rCAN98-75 are shown; Figure 3C and Figure 3D Results obtained with viruses rC-85473, ΔSH-rC-85473 and ΔG-rC-85473 are shown.

[0297] These results indicate that the recombinant viruses ΔSH-rC-85473 or ΔG-rC-85473 caused only a small amount of weight loss; in fact, the behavior of mice in these groups was similar to that of mice in the uninfected group.

[0298] In addition, lung viral load ( Figure 3D ) were similar to those observed in the rC-85473-infected group, indicating that the deletion-attenuated virus replicated in the lungs.

[0299] The results were different from those of the recombinant viruses ΔSH-rCAN98-75 and ΔG-rCAN98-75: the virus ΔSH-rCAN98-75 caused a significant weight loss ( Figure 3A ); while virus ΔG-rCAN98-75 only caused very slight weight loss ( Figure 3A ), which is due to its low replication capacity in the lung ( Figure 3B ).

[0300] Example 4: In vivo protective characteristics of recombinant hMPVΔG-rC-85473 and ΔSH-rC-85473 in a BALB / c mouse model after infection with wild-type virus rC-85473

[0301] Immunization and infectious challenge protocols Figure 4A shown.

[0302] The cells were instilled intranasally with OptiMem culture medium (“mock” negative control) or 5 × 10 5 TCID 50 BALB / c mice were immunized with recombinant viruses rC-85473, ΔSH-rC-85473 or ΔG-rC-85473.

[0303] 21 days after immunization, a lethal dose (1×10 6 TCID 50) wild-type virus rC-85473 was used to infect mice (infectious challenge).

[0304] The body weight and survival rate of mice were monitored daily for 14 days (mean body weight ± SEM of 10 mice, Figure 4B ).

[0305] 14 days after the challenge, the survival rate of mice in different groups was monitored every day (the survival rate of the mock group was 50%, and the survival rate of mice immunized with recombinant virus rC-85473, ΔSH-rC-85473 or ΔG-rC-85473 was 100%). The results are shown in Figure 4C middle.

[0306] Five days after the infectious challenge (i.e., on day 26 after immunization), four mice in each group were euthanized to measure the TCID50 ( Figure 4D ) and RTqPCR (N gene, Figure 4E ) of lung infection viral load.

[0307] Statistical test: Two-way ANOVA, comparing each virus to mock condition. *p<0.05, **p<0.01 and ***p<0.001.

[0308] These results confirm that the recombinant virus ΔSH-rC-85473 or ΔG-rC-85473 caused only minimal weight loss (the behavior of the mouse group was similar to that of the uninfected group) and that under these experimental conditions, mice challenged with a lethal dose of wild-type virus rC-85473 were 100% protected. In mice previously immunized with the recombinant viruses ΔSH-rC-85473 and ΔG-rC-85473, no infectious virus and wild-type genome rC-85473 (nd) were detected in the lungs at D+5 after challenge, and the high micro-neutralization load (>160) in the serum at D+21 after challenge also demonstrated this protective effect.

[0309] Table 7 below shows the results of micro-neutralization tests performed on sera taken from different groups of mice on D+20 (1 day before infectious challenge) and D+42 (21 days after infectious challenge).

[0310] After inactivation at 56° C. for 30 minutes and dilution of the sera, they were incubated with 100 TCID50 (i.e. 100 infectious units, such as determined by TCDI50 / ml) of the virus rC-85473 or the virus rCAN98-75 for 2 hours at 37° C. The mixture was then applied to LLC-MK2 cells to determine the neutralization load.

[0311] The neutralizing antibody load was determined by the maximum dilution of serum at which LLC-MK2 cells incubated with the serum+virus mixture showed no cytopathic effect, ie were considered negative for infection (based on tests performed in duplicate).

[0312] Table 7. Micro-neutralization test

[0313]

[0314] The graph represents the dilution of the tested sera at which the specific antibody load effectively neutralized the viral infection (micro-neutralizing load).

[0315] A result of 5 or 10 represents a primary immune response (initial exposure to the virus). A result of 160 or greater is characteristic of a maximal immune response, indicating that the organism has been previously exposed to the virus and is therefore immune.

[0316] After prior immunization with the attenuated strains, virus strains derived from the original strain rC-85473 were able to achieve a satisfactory neutralizing humoral response against hMPV C-85473 (serotype A) and at least against the virus CAN98-75 (serotype B).

[0317] Example 5: In vitro cell binding ability of recombinant viruses rC-85473 and rCAN98-75

[0318] LLC-MK2 cells (t=0) were infected with:

[0319] - Virus rCAN98-75 and its attenuated forms ΔSH-rCAN98-75 and ΔG-rCAN98-75 ( Figure 5A );

[0320] - Virus rC-85473 and its attenuated forms ΔSH-rC-85473 and ΔG-rC-85473 ( Figure 5B );

[0321] To vary the time that the virus was bound to the cells, cells were infected on ice for 0.5, 1, 2, or 3 h, then washed and incubated at 37 °C for 24 h.

[0322] The percentage of infected cells measured at 24 h post-infection represents the amount of virus that was able to bind to the cells for the indicated time (0.5, 1, 2, or 3 h).

[0323] The results showed that the ability of the recombinant viruses to bind to LLC-MK2 cells in culture (and therefore their infectivity) varied depending on the source of the recombinant viral genome, CAN98-75 or C-85473.

[0324] The results showed that the attenuated viruses ΔSH-rC-85473 and ΔG-rC-85473 (about 60% of infected cells) had better cell binding and cell infection abilities compared to the viruses ΔSH-rCAN98-75 (40%) and ΔG-rCAN98-75 (less than 20% of infected cells).

[0325] Example 6: In vitro replication ability of recombinant viruses rC-85473 and rCAN98-75 in a reconstructed 3D model of human respiratory epithelium and cultured at the air-liquid interface

[0326] According to the instructions of the supplier Epithelix, reconstructed human airway epithelium ( HAE, Epithelix) were maintained in culture at an air-liquid interface for these in vitro experiments.

[0327] Fig. 6A The photographs shown in show that the recombinant virus rCAN98-75 has lower infectivity and replication (lower fluorescence intensity and smaller size of infection sites) than the recombinant virus rC-85473.

[0328] Quantification of viral genomes in the epithelium ( Figure 6C ) confirmed the replication ability of the recombinant viruses rC-85473, ΔSH-rC-85473 and ΔG-rC-85473, as well as the recombinant viruses rCAN98-75 and ΔSH-rCAN98-75, which was different from the virus ΔG-rCAN98-75 with extremely weak replication ability.

[0329] Quantification of viral genomes on the apical surface of the epithelium ( Figure 6B ) shows the production of recombinant viruses rCAN98-75, ΔSH-rCAN98-75, ΔG-rCAN98-75, rC-85473 and ΔSH-rC-85473.

[0330] On the other hand, the genome of virus ΔG-rC-85473 was not detected, indicating that no viral progeny were produced. One hypothesis is that in this complex multi-level in vitro system, virus ΔG-rC-85473 may have lost the ability to bud on the surface of infected cells.

[0331] These differences may be of a nature that further supports the inventive features of our invention, focusing on the different characteristics between recombinant viruses of different origins for the protection of our live attenuated vaccine candidates ΔSH-rC-85473 and ΔG-rC-85473 against hMPV strains.

[0332] Example 7: Secretion of cytokines and chemokines induced by infection with the recombinant virus rC-85473 when cultured at an air-liquid interface on a reconstructed 3D model of human respiratory epithelium

[0333] According to the instructions of the supplier Epithelix, reconstructed human airway epithelium ( HAE, Epithelix) were maintained in culture at an air-liquid interface for these in vitro experiments.

[0334] Quantification of selected secreted cytokines and chemokines in the basal medium of infected epithelia reflected the induced cellular responses, which varied depending on the recombinant virus rC-85473 evaluated (Figure 7).

[0335] a) Compared to wild-type rC-85473, virus ΔG-rC-85473 induced significantly lower secretion of different tested cytokines and chemokines, with the exception of the proinflammatory cytokine TNF-α. These results are consistent with those shown in Figure 6, which showed that the infectivity of the virus in reconstructed human respiratory epithelium was significantly reduced compared to the infectivity of the wild-type strain.

[0336] b) Although the virus ΔSH-rC-85473 had improved infection and replication capabilities in the reconstituted human respiratory epithelial model compared to the wild-type recombinant virus rC-85473 ( Figure 6 ), the secretion of the assessed cytokine and chemokine profiles induced by the virus was the same or less than that induced by the wild-type virus.

[0337] The levels of secreted chemokines MCP-1, IP-10, RANTES, IL-8, G-CSF and proinflammatory cytokines IFN-γ and TNF-α appear to indicate that epithelial cells infected with this virus ΔSH-rC-85473 are able to recruit T lymphocytes, monocytes / macrophages, eosinophils and / or neutrophils immune cells, which are required for the host's innate and adaptive immune responses.

[0338] In addition, the virus ΔSH-rC-85473 caused the secretion of IL-7 (a cytokine that can induce inactive lymphocytes to differentiate into active T and B lymphocytes) and FGF (a growth factor involved in tissue repair in the body), and the amount of secretion was similar to that of wild virus rC-85473.

[0339] Thus, infection with the virus ΔSH-rC-85473 enabled epithelial cells to mount a primary defense response to infection, which was however associated with a more reduced host inflammatory response compared to that induced by the wild-type virus.

[0340] Therefore, these differences will facilitate the use of recombinant viruses derived from strain rC-85473 as attenuated live vaccines, and preferably recombinant viruses from attenuated strain ΔSH-rC-85473.

[0341] Example 8: In vitro viral replication of recombinant hMPV rC-85473 in an immortalized mouse macrophage model and secretion of proinflammatory cytokines IL-1β and TNF-α

[0342] Immortalized mouse macrophage cell lines were used to evaluate the replication and pro-inflammatory cytokine induction abilities of the recombinant viruses rC-85473, ΔSH-rC-85473, and ΔG-rC-85473.

[0343] Macrophages form part of the first cells of the immune system recruited at the site of infection (in this case, the respiratory epithelium). Macrophages contribute to the inflammatory response, the innate response, and stimulate the adaptive response of the host.

[0344] Macrophages were infected with recombinant virus rC-85473, ΔSH-rC-85473 or ΔG-rC-85473, and the cell supernatant was collected every day for 2 days. The results are shown in FIG8 .

[0345] a) Viral load measured in FFU / ml showed that virus ΔSH-rC-85473 had reduced replication capacity on macrophages compared to wild-type recombinant virus, whereas virus ΔG-rC-85473 showed similar viral replication to wild-type virus ( Fig. 8A A two-way ANOVA statistical test was performed to compare the replication kinetics of the deleted virus ΔSH-rC-85473 and the wild-type recombinant virus.

[0346] b) Then, the pro-inflammatory cytokine TNF-α ( Figure 8B ) and IL-1β( Figure 8C ) secretion.

[0347] The cytokine profile produced by virus ΔSH-rC-85473 was significantly different from that of wild-type virus rC-85473, with a secretion peak occurring earlier, at 1 day after infection, compared to wild-type virus, which had a secretion peak involving both cytokines at 2 days after infection.

[0348] Virus ΔG-rC-85473 induced the secretion of the cytokine TNF-α at a level comparable to that of the wild-type virus, but did not induce the secretion of the cytokine IL-1β during the first two days of infection.

[0349] These results suggest that despite altered kinetics, virus ΔSH-rC-85473 retains the ability to infect murine macrophages and, in particular, to cause the secretion of proinflammatory cytokines.

[0350] As observed in a reconstituted human airway epithelial model, virus ΔG-rC-85473 efficiently infects murine macrophages but induces weaker secretion of proinflammatory cytokines.

[0351] Example 9: Infection site lung inflammation and immune cell recruitment induced in vivo by recombinant hMPV rC-85473, ΔG-rC-85473 and ΔSH-rC-85473 in BALB / c mice for 5 days

[0352] Infect BALB / c mice by intranasal instillation:

[0353] -OptiMem (mock) culture medium;

[0354] -5×10 5 TCID 50 recombinant viruses rC-85473, ΔSH-rC-85473 or ΔG-rC-85473.

[0355] Five days after infection, five mice in each group were euthanized, and their lungs were subjected to histopathological analysis and infection-induced inflammation scores were measured ( Fig.9A ).

[0356] Statistical tests (two-way ANOVA) were performed to compare each condition with each other: *p<0.05, **p<0.01 and ***p<0.001.

[0357] Fig.9A It was shown that the wild virus rC-85473 caused strong lung inflammation in infected mice, while the inflammation caused by the viruses ΔSH-rC-85473 and ΔG-rC-85473 was significantly lower than that of the wild virus. This result is consistent with the reduced pathological severity of the attenuated strain, which is demonstrated in the aforementioned Figure 3.

[0358] In addition, 5 days after infection, 5 mice in each group were euthanized and their lungs were harvested for absolute quantification of CD4+, CD8+ lymphocytes, neutrophils, and macrophages immune cells infiltrating lung tissue after infection ( Fig. 9B ).

[0359] Statistical tests (one-way ANOVA) were performed to compare condition ΔSH-rC-85473 and condition rC-85473: **p<0.01.

[0360] Fig. 9BIt was shown that 5 days after inoculation with the viruses rC-85473 and ΔSH-rC-85473, different immune cell populations (CD4+T, CD8+T, neutrophils and macrophages) were recruited to the infection site; but the amounts were lower for the attenuated strains, especially for CD4+ lymphocytes.

[0361] Thus, it appears that virus ΔSH-rC-85473 leads to a reduction in lung inflammation, which is consistent with the attenuation of virus-induced pathology in the murine model (Figure 3) and the reduction of cytokine secretion by infected epithelial cells (Figure 7), while ensuring the effective recruitment of different immune cells at the site of infection.

[0362] These different results (Fig. 7 to Fig. 9) show different characteristics between wild-type recombinant rC-85473 and the deleted virus in terms of physiological effects after infection.

[0363] The attenuated strain ΔSH-rC-85473 appears particularly promising for the development of live attenuated vaccines from the following properties: (i) secretion of inflammatory cytokines and chemokines by epithelial and macrophage cells, (ii) induction of lung inflammation in vivo, and (iii) infiltration of immune cells into infected organs in vivo. Indeed, infection of the host with this attenuated strain was associated with both an effective primary immune response (equivalent to that observed with wild-type virus) and a reduction in the inflammatory response.

[0364] Example 10: Cytokine / chemokine secretion, recruitment of immune cells at the infection site in vivo, and lung inflammation after infectious challenge in a BALB / c mouse model immunized with recombinant hMPV rΔG-C-85473 and ΔSH-C-85473

[0365] After viral infection, due to the secretion of cytokines and chemokines by the infected epithelial cells (see Figure 7) and resident immune cells (Figure 8), complex inflammation and immune response occurred at the site of infection. First, the effector cells of the intrinsic response (non-specific) are recruited and activated subsequently, and secondly the effector cells of the adaptive response (specificity) are activated. When the infected individual is immunized or vaccinated in advance, the adaptive response is more rapid, stronger and more specific, and the memory response is more effective. This is the feature of this immune response, called secondary response, which is described in the present embodiment.

[0366] The cells were instilled intranasally with OptiMem culture medium (“mock” negative control) or 5 × 10 5 TCID 50 BALB / c mice were immunized with recombinant viruses rC-85473, ΔSH-C-85473 or ΔG-C-85473.

[0367] 21 days after immunization, a lethal dose (1×10 6 TCID 50 ) wild-type virus rC-85473 infected mice (infectious challenge). Immunization and infectious challenge protocols were as follows Figure 4A shown.

[0368] One or five days after infectious challenge (i.e., day 22 or day 26 after immunization, respectively), three mice per group were euthanized to quantify secreted selected cytokines and chemokines in lung tissues: the results are shown in Fig. 10A , Fig. 10B and Fig. 10C These results indicate that the induction of local immune responses differed depending on the recombinant virus rC-85473 used to immunize BALB / c mice:

[0369] - Groups immunized with viruses ΔG-C-85473 and ΔSH-C-85473 showed stronger secretion of the proinflammatory cytokine IL-6 and the chemokine MCP-1 compared to the group immunized with virus WT rC-85473, as early as 1 day after infectious challenge.

[0370] - In addition, the group immunized with virus ΔSH-C-85473 also showed a significant increase in the secretion of the anti-inflammatory cytokine IL-10 and the chemokines G-CSF and MIP-1β compared with the groups immunized with virus WT and / or virus ΔG-C-85473.

[0371] After 5 days, all tested cytokines / chemokines dropped dramatically compared to the levels measured on day 1 post-challenge, indicating an uptake of cytokines and inflammatory responses induced after challenge.

[0372] These results indicate that immunization with the attenuated virus ΔG-C-85473 (more specifically ΔSH-C-85473) results in a local cytokine response that is different from that induced by immunization with the corresponding non-attenuated virus WT.

[0373] To assess the recruitment of immune cells to the site of infection, the number of major immune cell populations that infiltrated the lung tissue to fight infection was measured; the results were Fig. 10D , Fig. 10E and Fig.10F shown.

[0374] Thus, 5 days after infectious challenge, the recruitment of T lymphocytes (CD8+ cytotoxic and CD4+ helper cells) and B lymphocytes was significantly increased after immunization with the virus ΔSH-C-85473 compared with the group immunized with the virus WT rC-85473, as well as compared with the group immunized with the virus ΔG-C-85473 (especially for B lymphocytes, which was significantly different, Fig. 10E ).

[0375] The populations of recruited macrophages and neutrophils showed no significant differences between the different viruses tested ( Fig.10F ).

[0376] The cell recruitment profile shown by the group of mice immunized with virus ΔG-C-85473 was similar to that of the group immunized with virus WT, indicating that virus ΔG-C-85473 is as effective as wild-type virus (WT) in inducing immune responses. In contrast, when mice were previously immunized with virus ΔSH-C-85473, the adaptive immune response in response to infectious challenge was stronger while maintaining a balance, while the innate response did not seem to be affected, indicating that virus ΔSH-C-85473 does not induce a more effective memory immune response.

[0377] Finally, 5 days after challenge, 2 mice per group were euthanized to measure the lung inflammation scores caused by challenge in each immunized group and in the “mock” unimmunized group; the results are shown in Figure 10G shown.

[0378] As expected with the lethal inoculation of virus WT rC-85473, this corresponded to a mortality of 50% of the population [see Figure 4C ], the cumulative inflammation score in the mock-immunized group was very high. As mentioned before, the inflammation scores in the groups immunized with viral WT rC-85473 or ΔG-C-85473 were quite comparable to each other and also slightly decreased compared with the mock-immunized group, even though the mice in these groups did not show any mortality [ Figure 4C ].

[0379] The results in Figure 10 show that mice immunized with virus ΔSH-C-85473 had a much reduced inflammatory response after infectious challenge, indicating that this group of mice would have a reduced lung attack. Likewise, lung function would be preserved during infection.

[0380] Therefore, in response to secondary infection, the attenuated virus ΔSH-C-85473 better recruits immune cells in the lungs in terms of quantity and quality, thereby helping to limit physiopathology and lung inflammation in infected hosts. This more effective response by the host may also reflect a better memory immune response against the hMPV virus.

[0381] In summary, this attenuated virus ΔSH-C-85473 has the following characteristics:

[0382] (i) it induces greater amounts of pro-inflammatory and anti-inflammatory cytokines and chemokines than other tested viruses;

[0383] (ii) A stronger but balanced recruitment of immune cells (CD8+ / CD4+ T lymphocytes and B lymphocytes) at the site of infection,

[0384] (iii) It also produces limited lung inflammation.

[0385] Table 8. Summary of sequences shown in the sequence listing

[0386] describe SEQ ID NO: 1 Complete genome sequence of hMPV C-85473 wild-type strain SEQ ID NO: 2 The complete sequence of recombinant hMPVΔSH-rC-85473 SEQ ID NO: 3 The complete sequence of recombinant hMPVΔG-rC-85473 SEQ ID NO: 4 The complete sequence of wild-type recombinant hMPV v rC-85473 coupled with GFP sequence SEQ ID NO: 5 The complete sequence of the recombinant hMPVΔSH-rC-85473 coupled with the GFP sequence SEQ ID NO: 6 The complete sequence of recombinant hMPVΔG-rC-85473 coupled with GFP sequence SEQ ID NO: 7 Complete sequence of wild-type HMPV CAN98-75 SEQ ID NO: 8 The complete sequence of wild-type recombinant hMPV CAN98-75 coupled with GFP sequence

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[0415] Hamelin ME, Couture C, Sackett MK, Boivin G. Enhanced lung disease and Th2 response following human metapneumovirus infection in mice immunized with the inactivated virus. J Gen Virol. 2007 Dec;88(Pt 12):3391-400. PubMed PMID: 18024909. 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agtcataaaa gatctcaatc tattatagga 1380 gaaaaagtga acactgtatc agaaacattg gaattaccta ctatcagtag acctgcaaaa 1440 ccaaccatac tgtcagaacc aaagttagca tggactgata aaggtggggc aatcaaact 1500 gaaataagc aagcaatca agtcatcat cctattgagg aagaaggtc tactgagag 1560 aaggtgctgc cctccagtga tgggaaacc cctgcagaaa agaactgaa accatcact 1620 aacaccaaaa agaagtttc gtttacacca atgaaccag gaaaatac aaagttggaa 1680 aagatgctc tagattgct ctcagataat gagagaag atgcagaatc ttcaatctta 1740 acctttgaag aagagatac ttcatcgtta agcattgagg ccagattgga atcaatagag 1800 gagaaattaa gcatgatatt agggctatta agacactca acatgctac agcaggaccc 1860 accgagcaa gagatgggat acagagatgca atgattggcg tagagagga attatagca 1920s gaataata aggaagccaa aggaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa hasga havea 1980s cgatcaaaaa taggaaacgg tagtgtaaaaaaacagaaaagcaaaga gcttaaaaaa 2040 attgttgag atgaagcac aagtgagaa tctgagaag aaagaacc aaagacata 2100 cagacaata gtcacaga tgacattac cagttaatta tgtagtttta taaaataa 2160 caatgggaca agtaaaaatg gagtcctacc tagtagcac ttatcaaggc attccttaca cagcagctgt tcaagttgat ctaatagaaa aggacctgtt acctgcaagc ctaacaatat ggttcccttt gtttcaggcc aacacaccac cagcagtgct gctcgatcag ttgaaaaccc 2400. 2400. 2400. 2400. 2400. 2400. 2400. 2400. 2400. 2400 cagcccaagg tgcagcaatg tctgtacttc ccaaaaaatt tgaagtcaat gcgactgtag cactcgatga atatagcaaa ttggaattg acaaactcac agtctgtga gtaaaaacag tttacttaac aaccatgaaa ccatacggga tggtatcaaa atttgtgagc tcagccaaat cagttggcaa aaaaacacat gatctaatcg cactgtgtga ttttatggat ctagaaaaga acacacctgt tacaatacca gcattcatca aatcagtttc aatcaaagag agtgagtcag ctactgttga agctgctata cagaccaagc tctaacacag gccaaaattg caccttatgc gggattgatt atgatcatga ctatgaacaa tcccaaaggc atattcaaaa agcttggagc tgggactcaa gttatagtag aactaggagc atatgtccag gctgaaagca taagtaaaat atgcaagct tggagccatc aagggacaag atatgtgttg aagtccagat 2940 aacagccaag caccttggcc aagagctact aactctatct catagattat aaagtcacca 3000 ttctagttat aaaaatca agttagaaca agaatttaat caatcaagaa tgggacaat 3060 aaaaatgtct tggaagtgg tgatcatttt ttcattgtta atacaccctc aacacggtct 3120 taaagagagc tattagag agtcatgtag cactactc gaaggatatc tcagtgttct 3180 gaggacaggt tggtatacca acgttttac actggaggta ggtgatgtag agaaccttac 3240 atgtgctgat ggacctagct tataaaaac agaattagac ctgaccaaaa gtgcactaag 3300 agaactcaga acagttctctg ctgatcaact ggcagagag gagcaattg agaatcccag 3360 acatctga ttgttctag gagcaatagc actcggtgtt gcaacagcag ctgcagttac 3420 agcaggtgtt gcaatgcca aaaccatccg gcttgaagt gaagtacag cattagaa 3480 tgccctcaa aagaccaatg aagcagtatc tacattgggg aatggagttc gagtgttggc 3540 aactgcagtg agggagctgg aagattttgt gagcaagaat ctacacgtg caatcaaca 3600 aaacaagtgc gacattgctg acctgaaaat ggccgttagc ttcagtcaat tcaacagaag gtttctaat gttgtgcggc aatttcga caatgctgga ataacaccag caatatcctt ggacttaatg acagatgctg aactagccag agctgtttcc aacatgccaa catctgcagg acaaataaaa ctgatgttgg agaccgtgc aatggtaaga agaaaggggt tcggaatcct gataggagtt tacggagct ccgtaattta catggtgcaa ctgccaatct ttggagttat agacacgcct tgctggatag taaaagcggc cccttcttgc tcagaaaaaa agggaaacta tgcttgcctt ttaagagaag atcaaggatg gtattgtcag aatgcagggt caactgttta ctacccaaat gaaaaagact gcgaaacaag aggagaccat gtcttttgcg acacagcagc aggaatcaat gttgctgagc agtcaaagga gtgcaacatc aacatatcca ctactaatta cccatgcaaa gttagcacag gaagacaccc tatcagtatg gttgcactgt ctcctcttgg ggctttggtt gcttgctaca agggagtgag ctgttccatt ggcagcaaca gagtaggat 4260 catcaagcaa ctgaacaaag gctgctctta tataaccaac caagacgcag acacagtgac atagacaac actgtatacc agctaagcaa agttgaggcc gaacaccatg tttaaaagg 4380 aaggccagtg tcaagcagct ttgatccagt caatttcct gagatcaat tcaatgttgc 4440 acttgaccaa gtttcgaaa gcattgagaa cagtcaggcc tggtggatc atcaacag 4500 aatcctaagc agtgcagaga aaggaacac tggcttcatc attgtaataa ttctaattgc 4560 tgtccttggc tctaccatga tcctagtgag tgtttttac atataaaga aaacaaagaa 4620 acccacagga gcacctccag agctgagtgg tgtcacaaac aatggcttca taccacataa 4680 Tttagttatt aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 4740 aaaaaata aaatttggg aaatcata atgtctcgca aggctccatg caatatgaa 4800 gtgcggggca aatgcaatag aggaagtgag tgcaagttta accaatta ctggagttgg 4860 ccagatagat acttactaat aagatcaat tattatta atcaactttt aaggaacact 4920 gagagctg atggcttatc ataatatca ggchaggca gagagatag gachagat 4980 ttgtcctag gttccaccaa tgtggttcaa ggttatattg atgataacca aagcataaca 5040 aaagctgcag cctgttacag tctacataat atatcaac aactacaaga agttgaagtt 5100 aggcaggcta gagataacaa accatctgac agcaaacatg tggcacttca caacttagtc 5160 ctatcttata tggagatgag caaatttcct gcatctttaa tcaaatct caaagactg 5220 ccgagagaga aactgaaaaa attagcaag cttataatttg acttatcagc aggtgctgaa 5280 aatgactctt catatgcctt gcaagacagt gaagcacta atcaagtgca gtgagcatgg 5340 tcctgttttc attactatag aggtgatta catgatatgg actcatagg acttaaaaga 5400 agctttatct aatgggatag tgaagtctca tactacatt tacaatgtt atttagaaaa 5460 catagaaatt atatatgtca aggcttactt aagttagtaa aacacatca gagtgggata 5520 aatgacaatg atacattag atgtcattaa aagtgatggg tctcaaaaa catgtactca 5580 cctcaaaaaa ttaattaaag accactctgg taaagtgctt attgtactta agttaatatt 5640 agctttacta acatttctca cagtgacaat caccacaat tataaag window 5700 tctgcaaata tgtcagtca aaactgaatc agacaaaag gactcatcat caataccac 5760 5820 tcaaaggtat acaaactctg caataaacag agacacatgc tggaaaataa gcagaaatca 5880 atgcacaaac ataacaacat acaaattttt atgttttaaa tctgaagaaa caaaaaccaa 5940 caattgtgat aaactgacag atttatgcag aaacaaacca aaaccagctg ttgaagtgta 6000 tcacatagta gaatgccatt gtatatacac agttaaatgg aagtgctatc attacccaat 6060 agatgaaacc caatcctaaa taacactaga ttaggatcca tccaagtctg ttagttcaac 6120 aatttagtta tttaaaaata ttttgaaaac aagtaagttt ctatgatact tcataataat 6180 aagtaataat taattgctta atcatcatca caacattatt cgaaaccata actattcaat 6240 ttaagaagta aaaacaataa tatgggacaa gtagttatgg aggtgaaagt ggagaacatt 6300 cgaacaatag atatgctcaa agcaagagtg aaaaatcgtg tggcacgcag caaatgcttt 6360 aaaaatgcct ctttgatcct aataggaata actacattga gtatagccct caatatctat 6420 ctaatcataa actatacaat gcaagaaaac acatccgaat cagaacatca caccagctca 6480 tcacccatgg aatccagcag ggaaactcca acagcccta tggacaactc agacaccaat 6540 ccaggctcac agtatccac tcacagtcc acagaaggct ccacactcta ctttgcagcc 6600 tcagcaagct caccagagac agaccaca tcacaccag acacacaag ccgcccgccc 6660 ttcgtcgaca cacacacac actaccaagt gcaagcagaa caggacaag tccggcagtc 6720 cacacaaaaa acaatccaag ggcaagcccc agaacacatt ccccaccatg ggcaatgaca 6780 aggacggtcc gtggaaccac cactccgc acaagcagca caagaaaag accgtccaca 6840 gcatcagtcc aacctgacag cagcgcaca acccacaac acgagaagc aagcccagtg 6900 agcccgcaaa catctgcgag cacagcaca ccacaagg agggcatgga ggcagcaca 6960 tcaacat aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacaaca 7020 atagacacca acaattgaga agccaaagg aattcacaa tctctccaaa aaggcaacaa 7080 caccatatta gctccgctta aatctccctg gaaaaaacac tcgccatat accactata 7140 ccacaaccaaa aaagctgggt aaaaacacac ccacagaca aaacaatg 7200 gatcctctta atgaatccac tgttaatgtc tatctcccg actcatatct taaggagtg 7260 atttcttta gtgagactaa tgcaatttggt tcatgtctct taaaagacc tcacctaaaa 7320 aatgacaaca ctgcaaagt tgccatagag aatcctgtta tcgagcatgt tagactcaa 7380 aatgcaatca attctaagat gaaaatca gattacagga tagtagagcc agtaaacatg 7440 cacatgaaa ttatgagaa tgtacacagt tgtgagctca cattattaaa acagtttttta 7500 aaaggagta aaatattag cactcttaa ttaataatga tatgtgtg gctgcagtta 7560 aagtctacat cagatgatac ctcaatctta agttttag atgtagaatt tatacctagc 7620 tgggtaagca attggtttag taattggtac aatccaaca agttgattct ggaattcagg 7680 aagaagaag taatagaac tggttcaatc ttgtgtaggt cattgggtaa attagtttttt 7740 gttgtatcat catacggatg tatagtcag agcaaaaaa gcaaaggt gagcttctctc 7800 acatacaatc aactgttaac atggaagat gtgatgttaa gtagattca tgcaatttc 7860 tgtatatggg tagcacag tctgaatgaa atcagaag ggttaggtt gagaagtaat 7920 ctgcaaggca tattaactaa taagctatat gaaactgtag attatagct tagttttgtgt 7980 tgcaatgaag gtttctcact tgtgaagg ttgaggtt tattatgag tgaaatcctt 8040 aggattactg aacatgctca attcagtact agatttagaa atactttatt aaatggatta 8100 actgatcaat tgacaaatt aaaaaaay aacagactca gagttcatgg taccgtgtta 8160 gaaaataatg atttaccaat gtatgaagtt gtacttaaat tattagga tactttgaga 8220 tgtattaaat tattaatca taaaaactta gagaatgctg ctgaattata ctatatattt 8280 agaatattcg gtcaccaat ggtagatgaa agagatgcaa tggatgctgt caattaac 8340 aatgaaatca caaaatcct caggttggag agcttgacag aactaagagg ggcattcata 8400 ttaggatta tcaaggatt tgtagacaac aaaaaagat ggccgaaat taaaaactta 8460 aaagtgctta gtaaagatg gactatgtac ttcaagcaa aaagttaccc tagtcaactt 8520 gatttaagtg aaaagattt tttagagctt gctgcaatac agtttgaca agagttttct 8580 gttcctgaaa aaaccaacct tgagatgta ttaaatgata aagctatatc acctcctaaa 8640 agattaattt ggtctgtgta cccaaaaaat tacttacctg agacaataaa aaatcgatat ctagaagaga ctttcaatgc aagtgatagt ctcaaaacaa gaagagtact agagtactat ttgaagata ataaattcga ccaaaaaga cttaaaaagtt atgtggttaa acaagaat ttaatgata aggatcatat tgtctcgcta actggaaaag aaagagaatt aagtgtaggt agaatgtttg ctatgcaacc aggaaaacag cgacaatac aaatattggc tgaaaaattg ttagctgata atattgtacc ctttttccca gaaactttaa caaagtatgg tgatctagat cttcagagaa taatggaaat caaatcagaa ctttcttcta ttaaaaccag aagaaatgat agtataata attacattgc aagagcatcc atagtaacag atttaagtaa gttcaaccaa gcctttaggt atgaaactac agcgatctgt gcggatgtag cagatgaact acatggaaca caaagcctat tctgttggtt acatcttatc gttcctatga ctacaatgat atgtgcctat 9300. 9300. 9300. 9300. 9300. 9300. 9300. 9300. 9300. 9300. 9300 ggtttatata gatatcatat gggtggtatt gaggatggt gtcaaaaact ctggacaatg gaagctatat ctttattaga tgttgtatct gtaagacac gatgtcaat gatacttta 9420 ttaaacggtg acaaccaatc atagatgta agtaaaccag ttaagttatc tgaggtttta 9480 gatgagtga aagcagatta tagcttggct gtaaaaatgc taaaagaat aagagatgca 9540 tacagaata taggccaata acttaagaa ggggaaacat atatatcag agatcttcag 9600 tttataagta aggtgattca atctgaagga gtaatgcatc ctacccctat aaaaaagagatc 9660 ttaagagtgg gaccatggat aaacacaata ttagatgaca ttaaaaccag tgcagagtca 9720 attaggtc tatgtcagga attagaattt agggggaa gcatatagt tagtctgata 9780 taggaattt ttggctgta taatttac atgcatgaat caagcaca ccccctagca 9840 gggaagcagt tattcaaca actaaataa acatcacat cagtgcagag attttttgaa 9900 attaaaaagg aaatgaagt agtagatcta tggatgaca taccaatgca gtttggagga 9960 ggagatccag tagtctcta tagatctttc tatagaagga cccctgattt tttaactgaa 10020 gcaatcagtc atgtagatat tctgttaaaa atcagcca acatagaaa tgaagcgaaa 10080 ataagtttct tcaaagcctt actgtcaata gaaaaaaaatg aacgtgctac actgacaaca 10140 ctaatgagag accctcaagc tgtgggctca gagcgacaag caaaagtaac aagtgatatc 10200 aatagaacag cagttaccag catcttaagt ctttctccaa atcaactttt cagcgatagt 10260 gctatacact acagtagaaa tgaagaagag gtcggaatca ttgctgacaa cataacacct 10320 gtttatcctc atggactgag agttttgtat gaatcattac cttttcataa agctgaaaaa 10380 gttgtaaata tgatatcagg aacaaaatcc ataaccaact tattacagag aacatctgct 10440 attaatggtg aagatattga cagagctgta tccatgatgc tggagaacct aggattatta 10500 tctagaatat tgtcagtagt tgttgatagt atagaaattc caaccaaatc taatggtagg 10560 ctgatatgtt gtcagatatc tagaacccta agggagacat catggaataa tatggaaata 10620 gttggagtaa catcccctag catcactaca tgcatggatg tcatatatgc aactagctct 10680 catttgaaag ggataatcat tgaaaagttc agcactgaca gaactacaag aggtcaaaga 10740 ggtccaaaga gcccttgggt aggatcgagc acgcaagaga aaaaattagt tcctgtttat 10800 aaaaaaaa ttctttcaaaaaaaaaga gaacagctag aagcaattgg aaaaatgaga 10860 tgggtatata aagggacacc aggtttaaga cgattactca ataagatttg tcttggaagt 10920 ttaggcatta gttacaaatg tgtaaaacct ttattaccta ggtttatgag tgtaaatttc 10980 ctacacaggt tatctgtcag tagtagacct atgaattcc cagcagt tccagcttat 11040 agaacaacaattaccattt tgacactagt cctattaatc aagcactag tgaagattt 11100 gggaatgaag atattaattt ggtcttccaa atgcaatca gctgtggaat tagcataatg 11160 agtgtagtag aacaattaac tggtaggagt ccaaacagt tagttttaat acccaatta 11220 gagaaatag acattatgcc accaccagtg ttcaaggga attcatta tagctagta 11280 gataagataa cttctgacca acatactctc agtccagaca aaatagatat gttaacactg 11340 gggaaaatgc tcatgccac tataaaaggt cagaaaacag atcagttctt gaacaagaga 11400 gagaattatt tccatgggaa caatcttatt gagtctttgt cagcagcgtt agcatgtcat 11460 tggtgtggga tattaacaga gcaatgtata gaaaataata ttttcaagaa agactggggt 11520 gacgggttca tatcggatca tgcttttatg gacttcaaaa tattcctatg tgtctttaaa 11580 actaaacttt tatgtagttg gggatcccaa gggaaaaaca ttaaagatga agatatagta 11640 gatgaatcga tagataaact gttaaggatt gataatactt tttggagaat gttcagcaag 11700 gttatgtttg aatcaaaggt taagaaaagg ataatgttat atgatgtaaa atttctatca 11760 tttagggtt atatagggtt taagaattgg tttatagaac agttgagatc agctgagttg 11820 catgaggtac cttggattgt caatgccgaa ggtgatctgg ttgagatcaa gtcaattaaa 11880 atctatttgc aactgataga gcagagttta tttttaagaa taactgtttt gaactataca 11940 gatatggcac atgctctcac aagattaatc agaaagaagt tgatgtgtga taatgcacta 12000 ttaacttcaa ttccatcccc aatggttaac ttaactcaag ttattgatcc tacagaacaa 12060 ttagcttatt tccctaagat aacatttgaa aggctaaaaa attatgacac tagttcaaat 12120 tatgctaaag gaaagctaac aaggaattac atgatactgt tgccatggca acatgttaat 12180 agataact ttgtctttag ttctactga tgtaagtta gtctaaaac atgcattgga 12240 aaacttatga aagatctaaa ccctaaagtt ctgtacttta ttggagg ggcaggaat 12300 tggatggcca gaacagcatg tgaatatcct gatacaat ttgtatacag aagtttaaaa 12360 gatgaccttg atcatcatta tcctttggaa taccagagag tgataggaga attaagcagg 12420 aatagata gtggtgaagg gctttcaatg gaacaacag atgcaactca aaaaactcat 12480 tgggatttga tacacagagt aagcaagat gctttatta taacttg tgatgcagaa 12540 tttaggaca gagatgattt tttagatg gtaattctat ggaggaaca tgtattatca 12600 tgcagaattt gcactaccta tgggacagac cttattat tcgcaagta tcatgctaaa 12660 gactgcaata taaaattacc ttttgtg agatcagttg ccacctttat tatgcaaggt 12720 agtaaactgt caggctcgga atgctacata ctcttacac taggccacca cacaattta 12780 ccttgtcatg gagaaataca aattctaag atgaaatag cagcgtgtaa tgatttttat 12840 gctgcaaaaa aacttgacaa taatcaatt gagccaact gtaatcact tttatcagggg 12900 ctaagaatac cgaataata gaggaatta atagacaga gaggttatt aacactaca 12960 agcaaccatt cttctgtagc aacagttgga ggtagcaagg tcatagagtc taatggtta 13020 aaaaaagg siacaat attgattgg ttagaacata ttttaaattc tccaaaggt 13080 gattaatt atgattttt tgagcatta gaaatactt accctatat gattaacta 13140 atagataatc taggatgc agagattaa aactgatca aagtactgg atatatgctt 13200 gtaagtaaa atgaaaaat gataaaatg aataaatagg tgacaacttc attackatttcc 13260 aaagtaatca tttgattg cattatgta atagttaatt aaaactaa atcaaagt 13320 taaaaactaa taactgtcat taagttttatt aaaaataga attattaatt ggatgtatac 13380 ggttttttg ccgt 13394 <210> 2 <211> 12645 <212> DNA <213> wood chips <400> 2 gcgaaaaaaa cgcgtataaa ttagattaca aaaaaatatg ggacaagtga aaatgtctct 60 tcaagggatt cacctgagtg atctatcata caagcatgct atattaaag agtctcagta 120 cacataaaa agagatgtgg gtacaacac tgcagtgaca ccctcatcat tgcaacaa 180 aaacgctg ttgtgtggag aaattctgta tgctaaacat gctgattaca atatgctgc 240 agaaatagga atacaata tttaggacagc tttaggatca gagagagtgc agcagattct 300 gaggaactca ggcagtgaag tccaagtggt cttaaccaga acgtactctc tgggaagt 360 taaaaaacat aaggagaag atttacagat gttagacata cacggggtag agaagagctg 420 ggtagaagag atagacaaag aaagcaaggaa aacaatggca accttgctta aggaatcatc 480 aggtaatatc ccacaaatc agaggccctc agcaccagac accaccataa tcttattatg 540 tgtaggtgca ttaatatta ctaagctagc atcaccata gaagtgggac tagagaccac 600 agtcagaagg gctaaccgtg tactaagtga tgcactcaag agatacccta gatggacat 660 cccaaaaatt gccagatcct tctatgactt atttgacaa aaagtgtatc acagaagttt 720 gttcattgag tatggcaag cattaggctc atcatctaca ggcagcaag cagaaagtct 780 atttgttaat atattcatgc aagcttatgg agccggtcaa acaatgctaa ggtggggggt 840 cattgccagg tcatccaaca atataatgtt aggacatgta tctgtccaag ctgagttaaa 900 acaggtcaca gaagtctatg acttggtgcg agaaatgggc cctgaatctg gacttctaca 960 tttaaggcaa agcccaaaag ctggactgtt atcactagcc aactgtccca actttgcaag 1020 tgttgttctc ggaaatgcct caggcttagg cataatcggt atgtatcgtg ggagagtacc 1080 aaacacagaa ttattttcag cagcagaaag ttatgccaaa agtttgaaag agagcaataa 1140 aatcaatttc tcttcattag gacttacaga tgaagagaaa gaggctgcag aacatttctt 1200 aaatgtgagt gacgacagtc aaaatgatta tgagtaatta aaaaagtggg acaagtcaaa 1260 atgtctttcc ctgaaggaaa agatattcttt ttcatgggta atgaagcagc aaaattagca 1320 gaagctttcc agaaatcatt aaggaaacca agtcataaaa gatctcaatc tattatagga 1380 gaaaaagtga acactgtatc agaaacattg gaattaccta ctatcagtag acctgcaaaa 1440 ccaaccatac tgtcagaacc aaagttagca tggactgata aaggtggggc aatcaaaact 1500 gaaataagc aagcaatca agtcatcat cctattgagg aagaaggtc tactgagag 1560 aaggtgctgc cctccagtga tgggaaacc cctgcagaaa agaactgaa accatcact 1620 aacaccaaaa agaagtttc gtttacacca atgaaccag gaaaatac aaagttggaa 1680 aagatgctc tagattgct ctcagataat gagagaag atgcagaatc ttcaatctta 1740 acctttgaag aagagatac ttcatcgtta agcattgagg ccagattgga atcaatagag 1800 gagaaattaa gcatgatatt agggctatta agacactca acatgctac agcaggaccc 1860 accgagcaa gagatgggat acagagatgca atgattggcg tagagagga attatagca 1920s gaataata aggaagccaa aggaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa hasga havea 1980s cgatcaaaaa taggaaacgg tagtgtaaaaaaacagaaaagcaaaga gcttaaaaaa 2040 attgttgag atgaagcac aagtgagaa tctgagaag aaagaacc aaagacata 2100 cagacaata gtcacaga tgacattac cagttaatta tgtagtttta taaaataa 2160 caatgggaca agtaaaaatg gagtcctacc tagtagacac ttatcaggc attccttaca 2220 cagcagctgt tcaagttgat ctaataagaaa aggacctgtt acctgcaagc ctaacaatat 2280 ggttcccttt gtttcaggcc aacacaccac cagcagtgct gctcgatcag ttgaaaaccc 2340 taaaataac cactctgtat gctgcatcac aaaatggtcc aatactcaaa gtgaatgcat 2400 cagcccaagg tgcagcaatg tctgtacttc ccaaaaaatt tgaagtcaat gcgactgtag 2460 2520 2580 cagttggcaa aaaaaacacat gatctaatcg cactgtgtga tttatggat ctagaaaaaga 2640 acacacctgt tacaatacca gcattcatca aatcagtttc aatcaaagag agtgagtcag 2700 ctactgttga agctgctata agcagtgaag cagaccaagc tctaacacag gccaaaattg 2760 caccttatgc gggattgatt atgatcatga ctatgaacaa tcccaaaggc atattcaaaa 2820 agcttggagc tgggactcaa gttatagtag aactaggagc atatgtccag gctgaaagca 2880 tagtaaaat atgcaagact tggagccatc aagggacaag atatgtgttg aagtccagat 2940 aacagccaag caccttggcc aagagctact aactctatct catagattat aaagtcacca 3000 ttctagttat aaaaatca agttagaaca agaatttaat caatcaagaa tgggacaat 3060 aaaaatgtct tggaagtgg tgatcatttt ttcattgtta atacaccctc aacacggtct 3120 taaagagagc tattagag agtcatgtag cactactc gaaggatatc tcagtgttct 3180 gaggacaggt tggtatacca acgttttac actggaggta ggtgatgtag agaaccttac 3240 atgtgctgat ggacctagct tataaaaac agaattagac ctgaccaaaa gtgcactaag 3300 agaactcaga acagttctctg ctgatcaact ggcagagag gagcaattg agaatcccag 3360 acatctga ttgttctag gagcaatagc actcggtgtt gcaacagcag ctgcagttac 3420 agcaggtgtt gcaatgcca aaaccatccg gcttgaagt gaagtacag cattagaa 3480 tgccctcaa aagaccaatg aagcagtatc tacattgggg aatggagttc gagtgttggc 3540 aactgcagtg agggagctgg aagattttgt gagcaagaat ctacacgtg caatcaaca 3600 aaacaagtgc gatatgctg acctgaaaat ggccgttagc ttcagtcaat tcacagaag 3660 gtttctaat gttgtgcggc aatttcga caatgctgga ataacaccag caatatcctt ggacttaatg acagatgctg aactagccag agctgtttcc aacatgccaa catctgcagg acaaataaaa ctgatgttgg agaccgtgc aatggtaaga agaaaggggt tcggaatcct gataggagtt tacggagct ccgtaattta catggtgcaa ctgccaatct ttggagttat agacacgcct tgctggatag taaaagcggc cccttcttgc tcagaaaaaa agggaaacta tgcttgcctt ttaagagaag atcaaggatg gtattgtcag aatgcagggt caactgttta ctacccaaat gaaaaagact gcgaaacaag aggagaccat gtcttttgcg acacagcagc aggaatcaat gttgctgagc agtcaaagga gtgcaacatc aacatatcca ctactaatta cccatgcaaa gttagcacag gaagacaccc tatcagtatg gttgcactgt ctcctcttgg ggctttggtt gcttgctaca agggagtgag ctgttccatt ggcagcaaca gagtaggat 4260 catcaagcaa ctgaacaaag gctgctctta tataaccaac caagacgcag acacagtgac 4380. aatagacaac actgtatacc agctaagcaa agttgagggc gaacagcatg ttaaaagg aaggccagtg tcaagcagct ttgatccagt caatttcct gagatcaat tcaatgttgc 4440 acttgaccaa gtttcgaaa gcattgagaa cagtcaggcc tggtggatc atcaacag 4500 aatcctaagc agtgcagaga aaggaacac tggcttcatc attgtaataa ttctaattgc 4560 tgtccttggc tctaccatga tcctagtgag tgtttttac atataaaga aaacaaagaa 4620 acccacagga gcacctccag agctgagtgg tgtcacaaac aatggcttca taccacataa 4680 Tttagttatt aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 4740 aaaaaata aaatttggg aaatcata atgtctcgca aggctccatg caatatgaa 4800 gtgcggggca aatgcaatag aggaagtgag tgcaagttta accaatta ctggagttgg 4860 ccagatagat acttactaat aagatcaat tattatta atcaactttt aaggaacact 4920 gagagctg atggcttatc ataatatca ggchaggca gagagatag gachagat 4980 ttgtcctag gttccaccaa tgtggttcaa ggttatattg atgataacca aagcataaca 5040 aaagctgcag cctgttacag tctacataat atatcaac aactacaaga agttgaagtt 5100 aggcaggcta gagataacaa accatctgac agcaaacatg tggcacttca caacttagtc 5160 ctatcttata tggagatgag caaatttcct gcatctttaa tcaaatct caaagactg 5220 ccgagagaga aactgaaaaa attagcaag cttataatttg acttatcagc aggtgctgaa 5280 aatgactctt catatgcctt gcaagacagt gaagcacta atcaagtgca gtgagcatgg 5340 tcctgttttc attactatag aggtgatta catgatatgg actcatagg acttaaaaga 5400 agctttatct aatgggatag tgaagtctca tactacatt tacaatgtt atttagaaaa 5460 catagaaatt atatatgtca aggcttactt aagttagtaa aacacatca gagtgggaca 5520 agtagttatg gaggtgaag tggacaat tcgacaata gatatgctca aagcaagt 5580 gaaaaatcgt gtggcacgca gcaatgctt taaaaatgcc tctttgatcc tataggaat 5640 aactacattg agtatagccc tcaatcta tctaatcata acatacaca tgcaagaaaa 5700 cacatccgaa tcagaacatc acaccagctc atcaccatg gatccagca gggaaactcc 5760 aacagtccct atggacact cagacaccaa tccaggctca cagtatccaa ctcacagtc 5820 cacagaaggc tccacactct actttgcagc ctcagcaagc tcaccagaga cagaaccac 5880 atcaacca cacaacaa gccgcccgcc cttcgtcgac acacacacacacaccaag 5940 tgcaagcaga aagacaa gtccggcagt ccacacaaaaacaatccaa ggacaagccc 6000 cagaacacat tccccat gggcaatgac aaggacggtc cgtggaacca ccactccg 6060 cacaagcagc acaagaaaaa gaccgtccac agcatcagtc cacagcagc gcagcgcac 6120 aacccacaaa cacgagaag caagcccagt gagcccgcaa acatctgcga gcacagcaag 6180 accaaagg aagggcatgg aggccagcac atcaaca accaaccaa ctagttaaca 6240 aaaataca aaaactcta agataacca tatagacacc aaaaatgag aagccaaaag 6300 gcaattcaca atctctccaa aaggcaca acaccatatt agctccgctt aaatctccct 6360 ggaaaaaaca ctcgcccata taccactat accaacca tcccagaaa aaagctggg 6420 taaaacaca cccaagagac aaatacaat ggatccctctt atgaatcca ctgttaatgt 6480 ctatcttccc gactcatatc ttaaggagt gatttctttt agtgagacta atgcaatttgg 6540 ttcatgtctc ttaaaaagac cttacctaa aaatgacaac actgcaaag ttgccataga 6600 gatcctgtt atcgagcatg ttagactca aaatgcaatc attctaaga tgaaaatc 6660 attacagg agtagagc agattaaacat gcaacatgaa attagag attagag 6720 ttgtgagctc acattattaa aacagttttt aaaaggagt aaaaatatta gcacttta 6780 attaaatatg atatgtgatt ggctgcagtt aaagtctaca tcagatgata cctcaatctt 6840 aagttttata gatgtagaat ttatacctag ctgggtaagc aattggttta gtaattgta 6900 caatctcaac aagttgattc tggattcag gaagaaa gtaatagaa ctggttcaat 6960 cttgtgtagg tcattgggta aattagtttt tgttgtatca tcatacggat gtatagtca 7020 gagacaaa agxaaag tgagctctt cacatacaat gagacaaa catxaaag 7080 tgtgatgtta agtagattca atgcaattt ctgtatatgg gtaagcaaca gtctgaatga 7140 aaatcaagaa gggttaggggt tgagagtaa tctgcaaggc atacta atagctata 7200 tgaaactgta gattatatgc ttagttgtg ttgcaatgaa ggtttctcac ttgtgaaaga 7260 gtttgaggtt tttatga gtgaaatcct taggattact gaacatgctc aattcagtac 7320 tagattaga atactttat taaatggatt aactgatcaa ttgacaaat taaaaaata 7380 aaacagactc agagttcatg gtaccgtgtt agaaaataat gattatccaa tgtatgaagt 7440 tgtacttaaattattaggag atactttgag atgtattaaa ttattaatca aaaaactt 7500 agagaatgct gctgaattat actatatatt tagaatattc ggtcacccaa tggtagatga 7560 aagagatgca atggatgctg tcaattaa caatgaatc acaaaatcc tcaggttgga 7620 gagcttgaca gaactaagag gggcattcat atttaggatt atcaaggat ttgtagacaa 7680 siaaaaaga tggccgaaaa ttaaaaactt aaagtgctt agtaaagat ggactgta 7740 cttcaaagca aaagttacc ctagtcact tgattaagt gacaagatt ttttagagct 7800 tgctgcaata cagtttgaac aagagtttc tgttcctgaa aaaaccaacc ttgagatggt 7860 attaaatgat aaagctatat cacctcctaa aagattaatt tggtctgtgt acccaaaaaa 7920 ttacttacct gagacataa aaatcgata tctagagag acttcaatg caagtgatag 7980 tctcaaaaca agaagagtac tagagtacta tttgaaagat aataaattcg accaaaaaga 8040 acttaaaagt tatgtggtta aacaagaata tttaaatgat aaggatcata ttgtctcgct 8100 aactggaaaa gaaagagaat taagtgtagg tagaatgttt gctatgcaac caggaaaaca 8160 gcgacaaata caaatattgg ctgaaaaatt gttagctgat aatattgtac cctttttccc 8220 agaaacttta acaaagtatg gtgatctaga tcttcagaga ataatggaaa tcaaatcaga 8280 actttcttct attaaaacca gaagaaatga tagttataat aattacattg caagagcatc 8340 catagtaaca gatttaagta agttcaacca agcctttagg tatgaaacta cagcgatctg 8400 tgcggatgta gcagatgaac tacatggaac acaaagccta ttctgttggt tacatcttat 8460 cgttcctatg actacaatga tatgtgccta tagacatgca ccaccagaaa caaaaggtga 8520 atatgatata gataagatag aagagcaaag tggtttatat agatatcata tgggtggtat 8580 tgaaggatgg tgtcaaaaac tctggacaat ggaagctata tctttattag atgttgtatc 8640 tgtaaagaca cgatgtcaaa tgacatcttt attaaacggt gacaaccaat caatagatgt 8700 aagtaacca gttaagttat ctgaggtttt agatgaagtg aaagcagatt atagcttggc 8760 tgtaaaaatg ctaaaaagaa taagagatgc atacagaaat ataggccata aacttaaaga 8820 agggaaaca tatatatcaa gagatcttca gtttataagt aaggtgattc aatctgaagg 8880 agtaatgcat cctaccccta taaaaaagat cttaagagtg ggaccatgga taaacacaat 8940 attagatgac attaaaacca gtgcagagtc atagggagt ctatgtcagg attagaatt 9000 tagggggaa agcataatag ttagtctgat atggaggaat ttttggctgt ataatttata 9060 catgcatgaa tcaagcaac accccctagc agggagcag ttcaac aactaata 9120 aacattaaca tcagtgcaga gattttttga aattaaaag gaaatgaag tagtagatct 9180 atggatgaac ataccaatgc agtttggagg aggagatcca gtagtctct atagatcttt 9240 ctatagaagg acccctgatt tttactga agcaatcagt catgtagata ttctgttaaa 9300 atatcagcc aacataagaa atgagcgaa ataagtttc ttcaaagcct tactgtcaat 9360 agaaaaaaat gaacgtgcta cactgacaac actaatgaga gaccctcaag ctgtggggctc 9420 agagcgacaa gcaaagtaa caagtgaat chaatagaa gcagttacca gcatcttaag 9480 tctttctcca aatcacttt tcagcgaga tgctatacac tacagtaga atgagaag 9540 ggtcggaatc attgctgaca acatacacc tgtttatcct catggactga gagttttgta 9600 tgaatcatta cctttcata aagctgaaaa agttgtaaat atgatcag gaacaaatc 9660 cataaccac ttattacaga gaacatctgc tattaatggt gaagatattg acagagctgt 9720 atccatgatg ctggagaacc taggattat atctagaata tgtcagtag ttgttgatag 9780 tatagaaatt ccaaccaat ctaatggtag gctgatatgt tgtcagatat ctagaaccct 9840 aagggagaca tcatggaata atatggaat agttggagta acatccccta gcatcactac 9900 atgcatggat gtcatatatg caactagctc tcatttgaaa gggataatca ttgaaagtt 9960 cagcactgac agaactaca gaggtcaag agtccaaag agcccttggg taggatcgag 10020 cacgcaagag aaaaaattag ttcctgttta taacagacaa attctttca aaaaaaaaaaag 10080 agaacagcta gagcattg gaaaaatgag atgggtatt aagggacac caggttttag 10140 acgattactc aataagattt gtcttggaag tttaggcatt agttacaaat gtgtaaaacc 10200 tttattacct aggtttatga gtgtaaattt cctacacagg ttatctgtca gtagtagacc 10260 tatggaattc ccagcatcag ttccagctta tagaacaaca aattaccatt ttgacactag 10320 tcctattaat caagcactaa gtgaaagatt tgggaatgaa gatattaatt tggtcttcca 10380 aaatgcaatc agctgtggaa ttagcataat gagtgtagta gaacaattaa ctggtaggag 10440 tccaaaacag ttagttttaa taccccaatt agaagaaata gacattatgc caccaccagt 10500 gtttcaaggg aaattcaatt ataagctagt agataagata acttctgacc aacatatctt 10560 cagtccagac aaatagata tgttaacact ggggaaaatg ctcatgccaa ctataaaagg 10620 tcagaaaaca gatcagttct tgaacaagag agagaattat ttccatggga acaatcttat 10680 tgagtctttg tcagcagcgt tagcatgtca ttggtgtggg atattaacag agcaatgtat 10740 agaaaataat atttcaaga aagactgggg tgacgggttc atatcggatc atgcttttat 10800 ggacttcaaa atattcctat gtgtctttaa aactaaactt tttgtagtt ggggatccca 10860 agggaaaaac attaaagatg aagatatagt agatgaatcg atagataaac tgttaaggat 10920 tgataatact ttttggagaa tgttcagcaa ggttatgttt gaatcaaagg ttaagaaaag 10980 gataatgtta tatgatgtaa aatttctatc attagtaggt tatatagggt ttaagaattg 11040 gtttatagaa cagttgagat cagctgagtt gcatgaggta ccttggattg tcaatgccga 11100 aggtgatctg gttgagatca agtcaattaa aatctatttg caactgatag agcagagttt 11160 atttttaaga ataactgttt tgaactatac agatatggca catgctctca caagattaat 11220 cagaaagaag ttgatgtgtg ataatgcact attaacttca attccatccc caatggttaa 11280 cttaactcaa gttattgatc ctacagaaca attagcttat ttccctaaga taacatttga 11340 aaggctaaaa aattatgaca ctagttcaaa ttatgctaaa ggaaagctaa caaggaatta 11400 catgatactg ttgccatggc aacatgttaa tagatataac tttgtcttta gttctactgg 11460 atgtaaagtt agtctaaaaa catgcattgg aaaacttatg aaagatctaa accctaaagt 11520 tctgtacttt attggagaag gggcaggaaa ttggatggcc agaacagcat gtgaatatcc 11580 tgacatcaaa ttgtataca gaagtttaa agatgacctt gatcatcatt atccttttgga 11640 ataccagaga gtgataggag aattaagcag gatatagat agtggtgag ggctttcaat 11700 ggaaaaaaca gatgcaactc aaaaaactca ttgggatttg atacacagag taagcaaga 11760 tgctttatta atactttat gtgatgcaga atttaggac agagatgat ttttagat 11820 ggtaattcta tggaggaac atgtattatc atgcagaatt tgcactacct atgggacaga 11880 cctctattta ttcgcaagt atcatgctaa agactgcaat ataaattac ctttttttgt 11940 gagatcagtt gccaccttta ttatgcagg tagtaactg tcaggctcgg atgctacat 12000 actcttaaca ctaggccacc acaacaattt accttgtcat ggagaaatac aaaattctaa 12060 gatgaaaata gcagcgtgta atgattttta tgctgcaaa aaacttgaca aaatcaat 12120 tgaagccaac tgtaaatcac ttttatcagg gctaagaata ccgataaata agaaggaatt 12180 aaatagacag agaaggttat taacactaca aagcaccat tctctgtag siacagttgg 12240 aggtagcaag gtcatagagt ctaaatggtt aaaaacaag gcaacacaa tattgattg 12300 gttagaacat atttttaattt ctccaaaagg tgaatttaat tatgattttt ttgaagcatt 12360 agaaaattact taccctata tgattaact attagataat ctagggaatg cagagatta 12420 aaaactgatc aaagtaactg gatatatgct tgtaagtaa aaatgaaaa tgataaaaat 12480 gataaaatag gtgacaactt catactattc caaagtaatc atttgatt gcaattatgt 12540 atagttaat taaaaactaaaatcaaag ttaaaacta atactgtca ttagtttat 12600 taaaaatag aaattataat tggatgtata cggtttttt gccgt 12645 <210> 3 <211> 12473 <212> DNA <213> wood chips <400> 3 gcgaaaaaaa cgcgtataaa ttagattaca aaaaaatatg ggacaagtga aaatgtctct 60 tcaagggatt cacctgagtg atctatcata caagcatgct atattaaag agtctcagta 120 cacataaaa agagatgtgg gtacaacac tgcagtgaca ccctcatcat tgcaacaa 180 aaacgctg ttgtgtggag aaattctgta tgctaaacat gctgattaca atatgctgc 240 agaaatagga atacaata tttaggacagc tttaggatca gagagagtgc agcagattct 300 gaggaactca ggcagtgaag tccaagtggt cttaaccaga acgtactctc tgggaagt 360 taaaaaacat aaggagaag atttacagat gttagacata cacggggtag agaagagctg 420 ggtagaagag atagacaaag aaagcaaggaa aacaatggca accttgctta aggaatcatc 480 aggtaatatc ccacaaatc agaggccctc agcaccagac accaccataa tcttattatg 540 tgtaggtgca ttaatatta ctaagctagc atcaccata gaagtgggac tagagaccac 600 agtcagaagg gctaaccgtg tactaagtga tgcactcaag agatacccta gatggacat 660 cccaaaaatt gccagatcct tctatgactt atttgacaa aaagtgtatc acagaagttt 720 gttcattgag tatggcaag cattaggctc atcatctaca ggcagcaag cagaaagtct 780 atttgttaat atattcatgc aagcttatgg agccggtca acatgctaa ggtggggggt 840 cattgccagg tcatccaaca atataatgtt aggacatgta tctgtccaag ctgagttaaa 900 acaggtcaca gaagtctatg acttggtgcg agaaatgggc cctgaatctg gacttctaca 960 tttaaggcaa agcccaaag ctggactgtt atcactagcc aactgtccca actttgcaag 1020 tgttgttctc ggaaatgcct caggcttagg cataatcggt atgtatcgtg ggagagtacc 1080 aaaacacagaa ttattcag cagcagaaag ttatgccaaa agtttgaaag agagcataa 1140 aatcaatttc tctcattag gacttacaga tgagagaa gaggctgcag aacattctt 1200 aaatgtgagt gacgacagtc aaatgatta tgagtatta aaaagtggg acagtcaaa 1260 atgtctttcc ctgaaggaaa agatattcttt ttcatgggta atgaagcagc aaaattagca 1320 gaagctttcc agaatcatt aaggaacca agtcataaa gatctcaatc tattagga 1380 gaaaaagtga acactgtatc agaaacattg gattaccta ctatcagtag acctgcaaa 1440 ccaaccatac tgtcagaacc aaagttagca tggactgata aaggtggggc aatcaaact 1500 gaaataagc aagcaatca agtcatcat cctattgagg aagaaggtc tactgagag 1560 aaggtgctgc cctccagtga tgggaaacc cctgcagaaa agaactgaa accatcact 1620 aacaccaaaa agaagtttc gtttacacca atgaaccag gaaaatac aaagttggaa 1680 aagatgctc tagattgct ctcagataat gagagaag atgcagaatc ttcaatctta 1740 acctttgaag aagagatac ttcatcgtta agcattgagg ccagattgga atcaatagag 1800 gagaaattaa gcatgatatt agggctatta agacactca acatgctac agcaggaccc 1860 accgagcaa gagatgggat acagagatgca atgattggcg tagagagga attatagca 1920s gaataata aggaagccaa aggaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa hasga havea 1980s cgatcaaaaa taggaaacgg tagtgtaaaaaaacagaaaagcaaaga gcttaaaaaa 2040 attgttgag atgaagcac aagtgagaa tctgagaag aaagaacc aaagacata 2100 cagacaata gtcacaga tgacattac cagttaatta tgtagtttta taaaataa 2160 caatgggaca agtaaaaatg gagtcctacc tagtagacac ttatcaggc attccttaca 2220 cagcagctgt tcaagttgat ctaatagaaa aggacctgtt acctgcaagc ctaacaat 2280 gttccctt gttcaggcc aacacaccac cagcaggct gctcgatcag ttgaaaaccc 2340 taaaataac cactctgtat gctgcatcac aaaatggtcc aatactcaaa gtgaatgcat 2400 cagcccaagg tgcagcaatg tctgtacttc ccaaaaaatt tgaagtcaat gcgactgtag 2460 2520 2580 cagttggcaa aaaaaacacat gatctaatcg cactgtgtga tttatggat ctagaaaaaga 2640 acacacctgt tacaatacca gcattcatca aatcagtttc aatcaaagag agtgagtcag 2700 ctactgttga agctgctata agcagtgaag cagaccaagc tctaacacag gccaaaattg 2760 caccttatgc gggattgatt atgatcatga ctatgaacaa tcccaaaggc atattcaaaa 2820 agcttggagc tgggactcaa gttatagtag aactaggagc atatgtccag gctgaaagca 2880 tagtaaaat atgcaagact tggagccatc aagggacaag atatgtgttg aagtccagat 2940 aacagccaag caccttggcc aagagctact aactctatct catagattat aaagtcacca 3000 ttctagttat ataaaaatca agttagaaca agaattaaat caatcaagaa tgggacaaat 3060 aaaaatgtct tggaagtgg tgatcatttt ttcattgtta atacaccctc aacacggtct 3120 taaagagagc tattagag agtcatgtag cactactc gaaggatatc tcagtgttct 3180 gaggacaggt tggtatacca acgttttac actggaggta ggtgatgtag agaaccttac 3240 atgtgctgat ggacctagct tataaaaac agaattagac ctgaccaaaa gtgcactaag 3300 agaactcaga acagttctctg ctgatcaact ggcagagag gagcaattg agaatcccag 3360 acatctga ttgttctag gagcaatagc actcggtgtt gcaacagcag ctgcagttac 3420 agcaggtgtt gcaatgcca aaaccatccg gcttgaagt gaagtacag cattagaa 3480 tgccctcaa aagaccaatg aagcagtatc tacattgggg aatggagttc gagtgttggc 3540 aactgcagtg agggagctgg aagattttgt gagcaagaat ctacacgtg caatcaaca 3600 aaacaagtgc gatatgctg acctgaaaat ggccgttagc ttcagtcaat tcacagaag 3660 gtttctaaat gttgtgcggc aattttcaga caatgctgga atacaccag caatcctt 3720 ggacttaatg acgatgctg aactagccag agctgttttcc aacatgccaa catctgcagg 3780 acaaataaaa ctgatgttgg agaccgtgc aatggtaaga agaaaggggt tcggaatcct gataggagtt tacggagct ccgtaattta catggtgcaa ctgccaatct ttggagttat agacacgcct tgctggatag taaaagcggc cccttcttgc tcagaaaaaa agggaaacta tgcttgcctt ttaagagaag atcaaggatg gtattgtcag aatgcagggt caactgttta ctacccaaat gaaaaagact gcgaaacaag aggagaccat gtcttttgcg acacagcagc aggaatcaat gttgctgagc agtcaaagga gtgcaacatc aacatatcca ctactaatta cccatgcaaa gttagcacag gaagacaccc tatcagtatg gttgcactgt ctcctcttgg ggctttggtt gcttgctaca agggagtgag ctgttccatt ggcagcaaca gagtaggat 4260 catcaagcaa ctgaacaaag gctgctctta tataaccaac caagacgcag acacagtgac 4380. aatagacaac actgtatacc agctaagcaa agttgagggc gaacagcatg ttaaaagg aaggccagtg tcaagcagct ttgatccagt caaatttcct gaagatcaat tcaatgttgc acttgacca gttttcgaa gcattgaga cagtcaggcc ttggtggatc aatcaaacag aatcctaagc agtgcagaga aaggaacac tggcttcatc attgtaataa ttctaattgc 4560 tgtccttggc tctaccatga tcctagtgag tgtttttac atataaaga aaacaaagaa 4620 acccacagga gcacctccag agctgagtgg tgtcacaaac aatggcttca taccacataa 4680 Tttagttatt aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 4740 aaaaaata aaatttggg aaatcata atgtctcgca aggctccatg caatatgaa 4800 gtgcggggca aatgcaatag aggaagtgag tgcaagttta accaatta ctggagttgg 4860 ccagatagat acttactaat aagatcaat tattatta atcaactttt aaggaacact 4920 gagagctg atggcttatc ataatatca ggchaggca gagagatag gachagat 4980 ttgtcctag gttccaccaa tgtggttcaa ggttatattg atgataacca aagcataaca 5040 aaagctgcag cctgttacag tctacataat atatcaac aactacaaga agttgaagtt 5100 aggcaggcta gagataacaa accatctgac agcaaacatg tggcacttca caacttagtc 5160 ctatcttata tggagatgag caaatttcct gcatctttaa tcaaatct caaagactg 5220 ccgagagaga aactgaaaaa attagcaaag cttataattg acttatcagc aggtgctgaa 5280 aatgactctt catatgcctt gcaagacagt gaaagcacta atcaagtgca gtgagcatgg 5340 tcctgttttc attactatag aggttgatta catgatatgg actcataagg acttaaaaga 5400 agctttatct aatgggatag tgaagtctca tactaacatt tacaattgtt atttagaaaa 5460 catagaaatt atatatgtca aggcttactt aagttagtaa aaacacatca gagtgggata 5520 aatgacaatg ataacattag atgtcattaa aagtgatggg tcttcaaaaa catgtactca 5580 cctcaaaaaa ttaattaaag accactctgg taaagtgctt attgtactta agttaatatt 5640 agctttacta acatttctca cagtgacaat caccatcaat tatataaaaag tagaaaacaa 5700 tctgcaaata tgtcagtcaa aaactgaatc agacaaaaag gactcatcat caaataccac 5760 atcagtcaca accaagacta ctctaaatca tgatataaca fòttttta aaagtttgat 5820 tcaaaggtat acaaactctg caataaacag agacacatgc tggaaaataa gcagaaatca 5880 atgcacaaac ataacaacat acaaattttt atgttttaaa tctgaagaaa caaaaaccaa 5940 caattgtgat aaactgacag atttatgcag aaacaaacca aaaccagctg ttgaagtgta 6000 tcacatagta gaatgccatt gtatatacac agttaaatgg aagtgctatc attacccaat 6060 agatgaaacc caatcctaaa taacactaga ttaggatcca tccaagtctg ttagttcaac 6120 aatttagtta tttaaaaata ttttgaaaac aagtaagttt ctatgatact tcataataat 6180 aagtaataat taattgctta atcatcatca caacattatt cgaaaccata actattcaat 6240 ttaagaagta aaaacaataa tatgagacaa ataacaatgg atcctcttaa tgaatccact 6300 gttaatgtct atcttcccga ctcatatctt aaaggagtga tttcttttag tgagactaat 6360 gcaattggtt catgtctctt aaaaagacct tacctaaaaa atgacaacac tgcaaaagtt 6420 gccatagaga atcctgttat cgagcatgtt agactcaaaa atgcaatcaa ttctaagatg 6480 aaaatatcag attacaggat agtagagcca gtaaacatgc aacatgaaat tatgaagaat 6540 gtacacagtt gtgagctcac attattaaaa cagtttttaa caaggagtaa aaatattagc 6600 actcttaaat taaatatgat atgtgattgg ctgcagttaa agtctacatc agatgatacc 6660 tcaatcttaa gttttataga tgtagaattt attackactagct gggtaagcaa tggtttagt 6720 aattggtaca atctcaaca gttgattctg gattcagga aagaagt aagaact 6780 ggttcaatct tgtgtaggtc attgggtaaa ttagttttg ttgtatcatc atacggatgt 6840 atagtcaag gcaaaag haaagagtg agcttcttca catacaatca actgttaca 6900 tggaagatg tgatgttaag tagattcaat gcaatttct gtatatggt aagcaacagt 6960 ctgaatgaaa atcagaagg gttagggttg agaagtaatc tgcaaggcat attack 7020 aagctatatg aaactgtaga ttatatgctt agtttgtgtt gcaatgaagg tttctcactt 7080 gtgaaaggt ttgaggtttt tattatgagt gaatcctta ggattactga acatgctcaa 7140 ttcagtacta gatttagaaa tactttatta aatgattaa ctgatcatt ghaaaaatta 7200 aaaaaayaa acagactcag agttcatggt accgtgttag aaaataatga ttatccaatg 7260 tatgagttg tacttaatt attaggagat actttgagat gtattaatt attaatcaat 7320 aaaaacttag agatgctgc tgaattatac tatattatta gatattcgg tcaccaatg 7380 gtagatgaaa gagatgcaat ggatgctgtc aaattaaaca atgaaatcac aaaaatcctc 7440 aggttggaga gcttgacaga actaagaggg gcattcatat taaggattat caaaggattt 7500 gtagacaaca acaaaagatg gccgaaaatt aaaaacttaa aagtgcttag taaaagatgg 7560 actatgtact tcaaagcaaa aagttaccct agtcaacttg aattaagtga acaagatttt 7620 ttagagcttg ctgcaataca gtttgaacaa gagttttctg ttcctgaaaa aaccaacctt 7680 gagatggtat taaatgataa agctatatca cctcctaaaa gattaatttg gtctgtgtac 7740 ccaaaaaatt acttacctga gacaataaa aatcgatatc tagaagac tttcaatgca 7800 agtgatagtc tcaaaacaag aagagtacta gagtactatt tgaaagataa taaattcgac 7860 caaaaagaac ttaaaagtta tgtggttaaa caagaatatt taaatgataa ggatcatatt 7920 gtctcgctaa ctggaaaaga aagagaatta agtgtaggta gaatgtttgc tatgcaacca 7980 ggaaaacagc gacaaataca aatattggct gaaaaattgt tagctgataa tattgtaccc 8040 ttttcccag aaactttaac aaagtatggt gatctagatc ttcagagaat aatggaaatc 8100 aaatcagaac ttctcttat taaaaccaga agaatgata gttatata ttacattgca 8160 agagcatcca tagtacaga tttaagtag ttcaccaag cctttaggta tgaaactaca 8220 gcgatctgtg cggatgtagc agatgaacta catggacac aaagcctatt ctgttggtta 8280 catcttatcg ttcctatgac tacaatgata tgtgcctata gatagcacc accagaaaca 8340 aaggtgaat atgatataga window gagcaagtg gtttatatag atatcatatg 8400 ggtgtattg aaggatgtg tcaaaaactc tggacaatg aagctatatc tttagat 8460 gttgtatctg aagacacg atgtcaatg acatctttat aacggtga spacecaatca 8520 atgatgtaa gtaaaccagt tagttatct gaggttttag atgagtga agcagatt 8580 agcttggctg taaaaatgct aaagaaata agagatgcat acagaatat agccataaa 8640 cttaaagag gggaacata ttcaga gatcttcagt tttaagtaa ggtgattcaa 8700 tctgaaggag taatgcatcc taccctata aaaagatct tagagtgg accatgata 8760 aacacaat tagatgacat taaaccagt gcagagtca taggtct atgtcagga 8820 ttagaattta gggggaag catatagtt agtctgatat taggaattt tggctt 8880 aatttataca tgcatgaatc aaagcacac cccctagcag ggagcagtt attcaacaa 8940 ctaaaaaa cattacacatc agtgcagaga tttttgaaa ttaaaagga aaatgaagta 9000 gtagatctat ggatgaacat accaatgcag tttggaggag gagatccagt agtcttctat 9060 agatctttct atagaaggac ccctgatttt ttactgaag caatcagtca tgtagatatt 9120 ctgttaaaaa tatcagccaa cataagaat gaagcgaaaa taagttttctt caaagcctta 9180 ctgtcaatag aaaaaaatga acgtgctaca ctgacacac tatgagaga ccctcaagct 9240 gtgggctcag agcgacaagc aaagtaca agtgatatca atagacagc agttaccagc 9300 atcttaagtc tttctccaaa tcaactttc agcgatagtg ctatacacta cagtagaaat 9360 gagaagagg tcggaatcat tgctgacaac atacacctg ttatcctca tggactgaga 9420 gttttgtag atcattacc tttcataaa gctgaaaag ttgtaataat gatatcagga 9480 aaaaatcca taaccactt attacagaga acatctgcta ttaatggtga agatattgac 9540 agagctgtat ccatgatgct ggagaaccta ggattat ctagaatatt gtcagtagtt 9600 gttgatagta tagaaattcc aaccaaatct aatggtaggc tgatatgttg tcagatatct 9660 agaaccctaa gggagacatc atggaataat atggaatag tggagtaac atcccctagc 9720 atcactacat gcatggatgt catatatgca actagctctc atttgaaagg gataatcatt 9780 gaaaagttca gcactgacag aactacaaga ggtcaaag gtccaaag cccttgggta 9840 ggatcgagca cgcaagagaa aaaattagtt cctgttata acagacaaat tcttcaaa 9900 aaaaagag aacagctga agcaattga aaatgagat gggtataata agggacacca 9960 ggtttaagac gattactca windowttgt cttggaagtt taggcattag ttacaatgt 10020 gtaaaacctt tattacctag gtttatgagt gtaaatttcc tacacaggtt atctgtcagt 10080 agtagaccta tggaatttccc agcatcagtt ccagcttata gacacaaa ttaccatttt 10140 gaactagtc ctattaatca agcactagt gaagatttg gaatgaga tattattg 10200 gtcttccaaa atgcaatcag ctgtggaattt agcataatga gtgtagtaga acaattaact 10260 ggtaggagtc caaacagtt agttttata cccaattag aagaataga cattatgcca 10320 ccaccagtgt ttcaagggaa attcattat aagctagtag atagataac ttctgaccaa 10380 catatcttca gtccagacaa atagatatg ttaacactgg ggaaaatgct catgccaact 10440 aaaaggtc agaaaaacaga tcagttcttg aaaaagagag agaattattt ccatgggaac 10500 aatcttattg agtctttgtc agcagcgtta gcatgtcatt ggtgtgggat atttacagag 10560 caatgtatag aaaatatat ttcaagaa gactggggtg acggggttcat atcggatcat 10620 gcttttatgg acttcaaaat attcctatgt gtctttaaaa ctaaactttt atgtagttgg 10680 ggatcccaag ggaaaaacat taagatgaa gatatagtag atgaatcgat agataactg 10740 tattaggattg ataatacttt tggagaatg ttcagcagg tattgttg atcaaggtt 10800 aagaaaagga tatgttata tgatgtaaaa tttctcat tagtaggtta taggtttt 10860 aagaatttggt ttatagaaca gttgagatca gctgagttgc atgaggtacc ttggattgtc 10920 aatgccgaag gtgatctggt tgagatcaag tcaattaaaa tctatttgca actgatagag 10980 cagagtttat ttttagaat aactgttttg aactatacag atatggcaca tgctctcaca 11040 agattaatca gaagaagtt gatgtgtgat aatgcactat taacttcaat tccatcccca 11100 atggttaact taactcaagt tattgatcct acagaacaat tagcttattt ccctaagata 11160 acatttgaaa gctaaaaaa tttgacact agttcaaatt atgctaagg aaagctaaca 11220 aggaattaca tgatactgtt gccatggcaa catgttaata gatataactt tgtctttagt 11280 tctactggat gtaagttag tctaaaaaca tgcattggaa aacttatgaa agatctaaac 11340 cctaaagttc tgtactttat tggaaggg gcaggaaatt ggatggccag aacagcatgt 11400 gatatcctg acatcaatt tgtatacaga agtttaaag atgaccttga tcatcattat 11460 cctttggaat accagagt gatgagaga taatagatag tggtgaaggg 11520 ctttcaatgg aaaacaga tgcaactca aaaactcatt gggatttgat accagagta 11580 agcaagatg ctttaat aactttatgt gatgcagaat ttaggacag agatgattt 11640 tttaagatgg taattctatg gaggaacat gtattatcat gcagaatttg cacatacctat 11700 gggacagacc tctatttatt cgcaagtat catgctaaag actgcaat aaaattacct 11760 ttttttgtga gatcagttgc caccttttt atgcaggta gtaaactgtc aggctcggaa 11820 tgctacatac tcttacact aggccaccac aacaatttac cttgtcatgg agaatacaa 11880 aattctaaga tgaaaatagc agcgtgtaat gatttttatg ctgcaaaaaa acttgacaat 11940 aaatcaatg aagccaactg taaatcactt ttcagggc tagaatacc gataaatag 12000 aaggaattta atagacagag aaggttatta acactacaa gcaaccattc ttctgtagca 12060 acagttggag gtagcaggt catagagtct aaatggttaa haaaaaggc aaacacaata 12120 attgattggt tagacatat tttaaatttct ccaaaggtg aattaatta tgattttttt 12180 gagcattag aaatactta ccctatatg attackata gatataatct agggaatgca 12240 gagattaaaactgatcaa agtaactgga tatatgcttg taagtaaaa atgaaaatg 12300 aaaaatga taaaataggt gandacttca tactattcca aagtaatcat ttgattgc 12360 aattatgtaa tagttaatta aaaactaaaa atcaaaagtt aaaaactaat aactgtcatt 12420 aagtttatta aaaataagaa attataattg gatgtatacg gtttttttgc cgt 12473 <210> 4 <211> 14141 <212> DNA <213> Artificial Sequence <220> <223> GFP gene inserted between positions 40 and 784 <400> 4 gcgaaaaaaa cgcgtataaa ttagattaca aaaaaatatg ggacaagtga aaatggtgag 60 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt 120 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct 180 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac 240 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga 300 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga 360 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg 420 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga 480 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa 540 ggtgaacttc aagatccgcc acacatcga ggacggcagc gtgcagctcg ccgaccacta 600 ccagcagaac accccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag 660 cacccagtcc gccctgagca aagacccca cgagaagcgc gatcacatgg tcctgctgga 720 gttcgtgacc gccgccggga tcactcgg catggacgag ctgtacaagt aagttaatta 780 aaagtggga caagtgaaaa tgtctcttca agggattcac ctgagtgatc tatcatacaa 840 gcatgctata ttaaaaggt ctcagtacac aaaaaga gatgtgggta ciacactgc 900 agtgacaccc tcatcattgc aacaagaaat aacgctgttg tgtggagaaa ttctgtagc 960 taaacatgct gattacaat atgctgcaga ataggata caatatta gcacagcttt 1020 aggatcag agagtgcagc agatctgag gaactcaggc agtgagtcc aagtggtctt 1080 aaccagaacg tactctctgg ggaagttaaaaacataaa ggagagatt tacagatgtt 1140 agacatacac ggggtagaga agagctgggt agagagata gaagaag aagaaaac 1200 aatggcaacc ttgcttaagg aatcatcagg tatatccca caaaatcaga ggccctcagc 1260 accagacaca cccataatct tattatgtgt aggtgcatta atattacta agctagcatc 1320 aaccatagaa gtgggactag agaccacagt cagaagggct aaccgtgtac taagtgatgc 1380 actcaagaga taccctagaa tggacatccc aaaaattgcc agatccttct atgacttatt 1440 tgaacaaaaa gtgtatcaca gaagttttgtt cattgagtat ggcaaagcat taggctcatc 1500 atctacaggc agcaaagcag aaagtctatt tgttaatata ttcatgcaag cttatggagc 1560 cggtcaaaca atgctaaggt ggggggtcat tgccaggtca tccaacaata taatgttagg 1620 acatgtatct gtccaagctg agttaaaaca ggtcacagaa gtctatgact tggtgcgaga 1680 aatgggccct gaatctggac ttctacattt aaggcaaagc ccaaaagctg gactgttatc 1740 actagccaac tgtcccaact ttgcaagtgt tgttctcgga aatgcctcag gcttaggcat 1800 aatcggtatg tatcgtggga gagtaccaaa cacagaatta ttttcagcag cagaaagtta 1860 tgccaaaagt ttgaaagaga gcaataaaat caatttctct tcattaggac ttacagatga 1920 agaaagag gctgcagaac atttcttaaa tgtgagtgac gacagtcaaa atgattatga 1980 gtaattaaaa aagtgggaca agtcaaaatg tctttccctg aagaaaaga tattctttc 2040 atgggtaatg aagcagcaaa attagcagaa gctttccaga aatcattaag gaaccaagt 2100 cataaaagat ctcaatctat tataggaa aaagtgaaca ctgtatcaga aacattggaa 2160 ttacctacta tcagtagacc tgcaaaacca accatactgt cagaaccaaa gttagcatgg 2220 actgataaag gtggggcaat caaaactgaa ataaagcaag caatcaaagt catggatcct 2280 attgagaag aagagtctac tgagaagaag gtgctgccct ccagtgatgg gaaaacccct 2340 gcaagaaaga aactgaaacc atcaactaac accaaaaaga aagtttcgtt tacaccaaat 2400 gaaccaggaa aatatacaaa gttggaaaaa gatgctctag atttgctctc agataatgaa 2460 gaagagatg cagaatcttc aatcttaacc tttgaagaaa gagatacttc atcgttaagc 2520 attgaggcca gattggaatc aatagaggag aaattaagca tgatattagg gctattaaga 2580 acactcaaca ttgctacagc aggacccaca gcagcaagag atgggatcag agatgcaatg 2640 attggcgtaa gagaggaatt aatagcagac aataaagg aagccaaagg gaaagcagca 2700 gaaatgatgg aaggaaat gagtcaacga tcaaaaatag gaacggtag tgtaaaacta 2760 acagagaaag caaaagagct taacaaaatt gttgaagatg aagcacaag tggagaatct 2820 gaagaaag aagaaccaaa agacatacaa gacaatagtc aagaagatga catttaccag 2880 ttaattatgt agtttaataa aataacaa tgggacaagt aaaaatggag tcctacctag 2940 tagacactta tcaaggcatt ccttacacag cagctgttca agttgatcta atagaaaagg 3000 acctgttacc tgcaagccta aaatatggt tccctttgtt tcaggccaac acaccaccag 3060 cagtgctgct cgatcagttg aaaaccctaa caataaccac tctgtatgct gcatcacaaa 3120 atggtccaat actcaaagtg aatgcatcag cccaaggtgc agcaatgtct gtacttccca 3180 3240 aactcacagt ctgtgaagta aaaacagttt acttaaaac catgaaacca tacgggatgg 3300 tatcaaaatt tgtgagctca gccaaatcag ttggcaaaaa aacacatgat ctaatcgcac 3360 tgtgtgattt tatggatcta gaaagaaca cacctgttac ataccagca ttcatcaat 3420 cagtttcaat aaagagt gagtcagcta ctgttgaagc tgctataagc agtgaagcag 3480 accaagctct aacacaggcc aaaattgcac cttatgcggg attgattg atcatgacta 3540 tgaacaatcc caaggcata ttcaaaagc tggagctgg gactcaagtt atagtagaac 3600 taggagcata tgtccaggct gaagcata gtaaaatg taggacttgg agccatcaag 3660 ggacaagata tgtgttgaag tccagataac agccaagcac cttggccaag agctactaac 3720 tctatctcat agattataaa gtcaccattc tagttatata aaatcaagt tagacaaga 3780 attaaatcaa tcaagaatgg gataataa atgtcttgg aaagtggtga tcatttttc 3840 attgttaata acacctcaac acggtcttta agagagctat ttagagagt catgtagcac 3900 tataactgaa ggatatctca gtgttctgag gataggttgg tataccac tttttacact 3960 ggaggtaggt gatgtagaga accttacatg tgctgatgga cctagcttaa taaaaacaga 4020 attagacctg accaaagtg cactaagaga actcagaaca gttctgctg atcaactggc 4080 aagagaggag caaattgaga atcccagaca atctagattt gttctaggag caatagcact 4140 cggtgttgca acagcagctg cagttacagc aggtgttgca attgccaaaa ccatccggct 4200 tgaaagtgaa gtaacagcaa ttaagaatgc cctcaaaaag accaatgaag cagatacttac 4260 attgggggaat ggagttcgag tgttggcaac tgcagtgagg gagctgggaag attttgtgag 4320 caagaatcta acacgtgcaa tcaacaaaaa caagtgcgac attgctgacc tgaaaatggc 4380 cgttagcttc agtcaattca acagaaggtt tctaaatgtt gtgcggcaat tttcagacaa 4440 tgctgggaata acaccagcaa tatccttgga cttaatgaca gatgctgaac tagccagagc 4500 tgtttccaac atgccaaacat ctgcaggaca ataaaaactg atgttggaga accgtgcaat 4560 ggtaagaa aaggggttcg gaatcctgat aggagtttac ggaagctccg taatttacat 4620 ggtgcaactg ccaatctttg gagttataga cacgccttgc tggatagtaa aagcggcccc 4680 ttcttgctca gaaaaaaaaagg gaaactatgc ttgcctttta agagaagatc aaggatggta 4740 ttgtcagaat gcagggtcaa ctgtttacta cccaaatgaa aaagactgcg aaacaagagg 4800 agaccatgtc ttttgcgaca cagcagcagg aatcaatgtt gctgagcagt caaaggagtg 4860 caacatcaac atatccacta ctaattaccc atgcaaagtt agcacaggaa gacaccctat 4920 cagtatggtt gcactgtctc ctcttggggc tttggttgct tgctacaagg gagtgagctg 4980 ttccattggc agcaacagag tagggatcat caagcaactg aaaaggct gctcttatat 5040 aaccaaccaa gacgcagaca cagtgacaat agacaacact gtataccagc taagcaaagt 5100 tgagggcgaa cagcatgtta taaaaggaag gccagtgtca agcagctttg atccagtcaa 5160 atttcctgaa gatcaattca atgttgcact tgaccaagtt ttcgaaagca ttgagaacag 5220 tcaggccttg gtggatcaat caaacagaat cctaagcagt gcagagaaag gaaacactgg 5280 cttcatcatt gtaataattc taattgctgt cttggctct accatgatcc tagtgagtgt 5340 tttatcata ataaagaaaa caaagaaacc cacaggagca cctccagagc tgagtggtgt 5400 cacaaacaat ggcttcatac cacataatta gttaattaaa aataaagtaa attaaattaa 5460 aataaataa attaaaatt aaaataaaat aaaaataaaa atttgggaca aatcataatg 5520 tctcgcaagg ctccatgcaa attgaagtg cggggcaaat gcaatagagg aagtgagtgc 5580 aagtttaacc acaattactg gagttggcca gatagatact tactaatag atcaattat 5640 ttattaatc aacttttag gacactgat agagctgatg gcttatcaat atatcagga 5700 gcaggcagag agataggac acagatttt gtcctaggtt ccaccaatgt ggttcaggt 5760 tatattgatg ataccaaag cataacaaaa gctgcagcct gttacagctct acatatata 5820 atcaacaac tachaagagt tgaagttagg caggctgag atacaacc atctgacagc 5880 aaacatgtgg cacttcacaa cttagtccta tcttatatgg agatgagcaa aattcctgca 5940 tctttaatca acatctca aagactgccg agagagaac tgaaaaaatt agcaagctt 6000 ataattgact tatcagcagg tgctgaaat gactctcat atgccttgca agacagtgaa 6060 agcactaatc aagtgcagtg agcatggtcc tgtttcatt actatagagg ttgattacat 6120 gatatggact caataggact taaaagaagc tttatctaat gggatagtga agtctcatac 6180 tacaatttac attgttatt tagaaacat agaaattata tatgtcagg cttacttaag 6240 ttagtaaaa cacatcagag tgggataat cacaatgata acattagatg tcattaaaag 6300 tgatgggtct tcaaaacat gtactcacct CAaaaatta attaagacc actctggtaa 6360 agtgcttatt gtacttaagt taatattagc tttactaca tttctcacag tgacaatcac 6420 help aaaaacaatct gcaatgt cagtcaaaa ctgaatcaga 6480 caaaaaggac tcatcatca attackcatc agtcacaacc aagactactc taaatcatga 6540 tataacacag tattttaaaa gtttgattca aaggtataca aactctgca taaacagaga 6600 cacatgctgg aaataagca gaatcaatg cacaacata acataca aatttttatg 6660 ttttaaatct gagaaaaaaaccaaaa ttgtgaaaa ctgacagatt tatgcagaaa 6720 caaaccaaaa ccagctgttg aagtgtatca catagtagaa tgccattgta tatacacagt 6780 taaatggaag tgctatcatt acccaataga tgaaacccaa tcctaaataa cactagatta 6840 ggatccatcc aagtctgtta gttcaacaat ttagttattt aaaaatattt tgaaaacaag 6900 taagttttcta tgatacttca taataatag taatattaa ttgctatc atcatcacaa 6960 cattattcga aaccatact attcaattta agagtaaaa acatatat gggacaagta 7020 gttatggagg tgaagtgga gaacattcga acatagata tgctcaaagc agagtgaaa 7080 aatcgtgtgg cacgcagcaa atgctttaaa atgccctt tgatcctaat aggaataact 7140 acattgagta tagccctca tatctatcta atcataact atacaatgca agaaaacaca 7200 tccgaatcag aaccacac cagctcatca cccatggaat ccagcaggga aactccaaca 7260 gtccctatgg acactcaga caccaatcca ggctcacagt atccactca acagtccaca 7320 gaaggctcca cacttactt tgcagcctca gcaagctcac cagagacaga accacatca 7380 acaccagaca accaagccg cccgccctc gtcgacacac accaacact accaagtgca 7440 agcagaaaa ggacaagtcc ggcagtccac aaaaaaaaaaaaaaaaaccaaggac aagccccaga 7500 acacattccc caccatgggc aatgacagg acggtccgtg gaaccac tctccgcaca 7560 agcagcacaa gaaaaagacc gtccacagca tcagtccac ctgacagcag cgcacaacc 7620 cacaacacg agaagcaag cccagtgagc ccgcaaacat ctgcgagcac agcaagacca 7680 7740. snow gcatggagg snow snow snow snow snow snow snow snow attack actctaagat aaaccatata gacaccaaca attgagaagc caaaaggcaa ttcacaatct ctccaaaaag gcacaacac catattagct ccgcttaaat ctccctgga 7920. aaaacactcg cccatatacc aactatacca caccatccc aagaaaaaaa gctgggtaaa acaacaccca agagacaaat aacaatggat cctcttaatg aatccactgt taatgtctat cttcccgact catatcttaa aggagtgatt tcttttagtg agactaatgc aattggttca tgtctcttaa aaagacctta cctaaaaaat gacaacactg caaaagttgc catagagaat cctgttatcg agcatgttag actcaaaaat gcaatcaatt ctaagatgaa aatatcagat 8220. 8220. 8220. 8220. 8220. 8220. 8220. 8220. 8220 gagctcacat tattaaaca gtttttaaca aggagtaaa atattagcac tcttaaatta gtgattggct gcagttaag tctacatcag atgatacctc aatcttaagt ttttatagtg tagtttt acctagctgg gtagcaatt ggtttagtaa ttggtacaat ctcacaagt tgattctgga attcaggaa gagaagtaa tagactgg ttcaatcttg 8460 tgtaggtcat tgggtaatt agttttgtt gtatcatcat acggatgtat agtcaagagc 8520 aaaaaagca aagagtgag cttcttcaca tacaatcaac tgttaacatg gaagatgtg 8580 atgttaagta gattcaatgc aaatttctgt atatgggtaa gcacagtct gatgaaat 8640 caagggtt taggttgag aagtaatctg caggcaat windows gctatatgaa 8700 actgtagatt atatgcttag ttgtgttgc aatgaaggtt tctcacttgt gaaagagttt 8760 gaggggtttta ttatgagtga aatccttagg attactgaac atgctcatt cagtactga 8820 tttagaaata cttattaaa tggattaact gatcattga CAAATTAAAAAAAAAC 8880 agactcagag ttcatggtac cgtgttagaa ataatgatt atccaatgta tgaagttgta 8940 cttaaattat taggagatac tttgagatgt atttaattat taatcataa aaacttagag 9000 aatgctgctg aattatacta tatattaga atattcggtc acccaatggt agatgaaaga 9060 gatgcaatgg atgctgtcaa attackaacaat gaatcaca aaatcctcag gttggagagc 9120 ttgacagaac taagaggggc attcatatta aggattatca aaggatttgt agacaacaac 9180 aaaagatggc cgaaaattaa aaacttaaaa gtgcttagta aaagatggac tatgtacttc 9240 aaagcaaaaa gttaccctag tcaacttgaa ttaagtgaac aagatttttt agagcttgct 9300 gcaatacagt ttgaacaaga gttttctgtt cctgaaaaaa ccaaccttga gatggtatta 9360 aatgataaag ctatatcacc tcctaaaaga ttaatttggt ctgtgtaccc aaaaaattac 9420 ttacctgaga caataaaaaa tcgatatcta gaagagactt tcaatgcaag tgatagtctc 9480 aaaacaagaa gagtactaga gtactatttg aaagataata aattcgacca aaaagaactt 9540 aaaagttatg tggttaaaca agaatattta aatgataagg atcatattgt ctcgctaact 9600 ggaaaagaaa gagaattaag tgtaggtaga atgtttgcta tgcaaccagg aaaacagcga 9660 caaatacaaa tattggctga aaaattgtta gctgataata ttgtaccctt tttcccagaa 9720 actttaacaa agtatggtga tctagatctt cagagaataa tggaaatcaa atcagaactt 9780 tcttctatta aaaccagaag aaatgatagt tataataatt acattgcaag agcatccata 9840 gtaacagatt taagtaagtt caccaagcc tttaggtag aaactacagc gatctgtgcg 9900 gatgtagcag atgaactaca tggacacaa agcctattct gttggttaca tcttatcgtt 9960 cctatgacta caatgatatg tgcctataga catgcaccac cagaaacaa aggtgaat 10020 gatatagata agatagaga gcaagtggt ttatatagat atcatatggg tggtattgaa 10080 ggatggtgtc aaaaactctg ɣaatggaa gctatatctt tattagatt tgtatctgta 10140 aagacacgat gtcaatgac atctttatta aacggtgaca accaatcaat agatgtaagt 10200 aaaccagtta agttatctga gggtttagat gaagtgaag cagattatag cttggctgta 10260 aaatgctaa aagaataag agatgcatac agaataag gccataact taagaaggg 10320 gaaacatata tatcagaga tctcagtttt ataagtagg tgatcaatc tgaggagta 10380 atgcatccta cccctataa aagatctta agagtgggac catggataa cacaatatta 10440 gatgacatta aaaccagtgc aggatcata gggagtctat gtcaggaatt agaatttagg 10500 ggggaaagca tatagttag tctgatatta aggaatttt ggctgtataa ttatacatg 10560 catgaatcaa agcacacccc cctagcagggg aagcagttat tcaaaact aaaaaaca 10620 ttacatcag tgcagagatt tttgaatt aaaaggaaa atgagtagt agatctatgg 10680 atgaacatac caatgcagtt tggagga gatccagtag tcttctatag atctttctat 10740 agaaggaccc ctgattttt aactgaagca atcagtcatg tagatattct gttaaaaata 10800 tcagccaaca tagaaatga agcgaaaata agttttctca aagccttact gtcaatagaa 10860 aaaaatgaac gtgctacact vakaacacta atgagacc ctcaagctgt gggctcagag 10920 cgacaagcaa agtacaag tgatatcaat agaacagcag ttaccagcat cttaagctctt 10980 tctccaaatc aacttttcag cgatagtgct atacactaca gtagaatga agaagaggtc 11040 ggaatcattg ctgacacat aacacctgtt tatcctcatg gactgagagt ttgtatgaa 11100 tcattacctt ttcataagc tgaaaagtt gtaaatatga tatcaggaac aaatccata 11160 accaacttat tacagagaac atctgctatt aatggtgaag atttgacag agctgtatcc 11220 atgatgctgg agaacctagg attattatct agaatattgt cagtagttgt tgatagtata 11280 gaaattccaa ccaaatctaa tggtaggctg atatgttgtc agatatctag aaccctaagg 11340 gagacatcat ggaataatat ggaatatagtt ggagtaacat cccctagcat cactacatgc 11400 atggatgtca tatatgcaac tagctctcat ttgaaaggga taatcattga aaagttcagc 11460 actgacagaa ctacaagagg tcaaagaggt ccaaagagcc cttgggtagg atcgagcacg 11520 caagagaaaa aattagttcc tgtttataac agacaaattc tttcaaaaca acaaagagaa 11580 cagctagaag caattggaaa aatgagatgg gtatataaag ggacaccagg tttaagacga 11640 ttactcaata agatttgtct tggaagttta ggcattagtt acaaatgtgt aaaaccttta 11700 ttacctaggt ttatgagtgt aaatttccta cacaggttat ctgtcagtag tagacctatg 11760 gaattcccag catcagttcc agcttataga acaacaaatt accattttga cactagtcct 11820 attaatcaag cactaagtga aagatttggg aatgaagata ttaatttggt cttccaaaat 11880 gcaatcagct gtggaattag cataatgagt gtagtagaac aattaactgg taggagtcca 11940 aaacagttag ttttaatacc ccaattagaa gaatagaca ttatgccacc accagtgttt 12000 caagggaaat tcaattataa gctagtagat aagataactt ctgaccaaca tatcttcagt 12060 ccagacaaaa tagatatgtt aacactgggg aaaatgctca tgccaactat aaaaggtcag 12120 aaaacagatc agttcttgaa caagagagag aattatttcc atgggaacaa tcttattgag 12180 tctttgtcag cagcgttagc atgtcattgg tgtgggatat taacagagca atgtatagaa 12240 aataatattt tcaagaaaga ctggggtgac gggttcatat cggatcatgc tttatggac 12300 ttcaaaatat tcctatgtgt cttaaaact aaacttttat gtagttgggg atcccaaggg 12360 aaaaacatta aagatgaaga tatagtagat gaatcgatag ataaactgtt aaggattgat 12420 aatacttttt ggagaatgtt cagcaaggtt atgtttgaat caaaggttaa gaaaaggata 12480 atgttatatg atgtaaaatt tctatcatta gtaggttata tagggtttaa gaattggttt 12540 atagaacagt tgagatcagc tgagttgcat gaggtacctt ggattgtcaa tgccgaaggt 12600 gatctggttg agatcaagtc aattaaaatc tatttgcaac tgatagagca gagtttattt 12660 ttagataa ctgttttgaa ctatacagat atggcacatg ctctcacag attaatcaga 12720 aagaagttga tgtgtgataa tgcactatta acttcattc catccccaat ggttaactta 12780 actcaagtta ttgatcctac agacaatta gcttatttcc ctaagataac atttgaagg 12840 ctaaaaaatt atgacactag ttcaattat gctaaaggaa agctaacaag gattacactg 12900 atactgttgc catggcaca tgttaataga tataacttg tctttagttc tactgtgt 12960 aaagttagtc taaaaacatg cattggaaa cttatgaaag atctaaccc taaagttctg 13020 tactttattg gagaaggggc aggaatttgg atggccagaa cagcatgtga atatcctgac 13080 atcaatttg tatacagaag tttaaagat gaccttgatc atcattatcc tttggaatac 13140 cagagagtga taggatt aagcaggata atagatagtg gtgaagggct ttcaatggaa 13200 aaacagatg acaccaaa aactcattgg gatttgaac acagagtaag aagatgct 13260 ttattaataa ctttatgtg tgcagaattt aaggacagag atgattttt tagatggta 13320 attctatgga ggaaacatgt attcatgc agaatttgca ctacctatgg gacagacctc 13440. tatttattcg caaagtatca tgctaaagac tgcaatataa aattaccttt ttttgtgaga tcagttgcca cctttattat gcaaggtagt aaactgtcag gctcggaatg ctacatactc ttacactag gccaccacaa caatttacct tgtcatggag aaatacaaaa ttctaagatg aaaatagcag cgtgtaatga tttttatgct gcaaaaaaac ttgacaata atcaattgaa gccaactgta aatcactttt atcagggcta agaataccga taataaga ggaattaat 13740. ggttattaac actacaaagc aaccattctt ctgtagcaac agttggaggt agcaaggtca taggtctaa atggttaaca aacaaggcaa acacaata tgattggtta 13860. 13860. 13860. 13860. 13860. 13860. 13860. 13860. 13860. 13860 aatacttacc ctaatatgat taaactatactag ggaatgcaga gattaaaaaa ctgatcaaag taactggata tatgcttgta agtaaaaaat gaaaaatgat aaaaatgata aaataggtga caacttcata ctattccaaa gtaatcattt gattatgcaa ttatgtaata 14040 gttaattaaa aactaaaaat caaaagttaa aaactaataa ctgtcattaa gtttattaaa 14100 aataagaaat tataattgga tgtatacggt ttttttgccg t 14141 <210> 5 <211> 13392 <212> DNA <213> Artificial Sequence <220> <223> GFP gene inserted between positions 40 and 784 <400> 5 gcgaaaaaaa cgcgtataaa ttagattaca aaaaaatatg ggacaagtga aaatggtgag 60 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt 120 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct 180 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac 240 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga 300 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga 360 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg 420 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga 480 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa 540 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta 600 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag 660 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga 720 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aagttaatta 780 aaaagtggga caagtgaaaa tgtctcttca agggattcac ctgagtgatc tatcatacaa 840 gcatgctata ttaaaagagt ctcagtacac aataaaaaga gatgtgggta caacaactgc 900 agtgacaccc tcatcattgc aacaagaaat aacgctgttg tgtggagaaa ttctgtatgc 960 taaacatgct gattacaaat atgctgcaga aataggaata caatatatta gcacagcttt 1020 aggatcagag agagtgcagc agattctgag gaactcaggc agtgaagtcc aagtggtctt 1080 aaccagaacg tactctctgg ggaaagttaa aaacaataaa ggagaagatt tacagatgtt 1140 agacatacac ggggtagaga agagctgggt agaagagata gacaaagaag caaggaaaac 1200 aatggcaacc ttgcttaagg aatcatcagg tatatccca caaaatcaga ggccctcagc 1260 accagacaca cccataatct tattatgtgt aggtgcatta atattacta agctagcatc 1320 aaccatagaa gtgggactag agaccacagt cagaagggct aaccgtgtac taagtgatgc 1380 actcaagaga taccctagaa tggacatccc aaaaattgcc agatccttct atgacttatt 1440 tgaacaaaaa gtgtatcaca gaagttttgtt cattgagtat ggcaaagcat taggctcatc 1500 atctacaggc agcaaagcag aaagtctatt tgttaatata ttcatgcaag cttatggagc 1560 cggtcaaaca atgctaaggt ggggggtcat tgccaggtca tccaacaata taatgttagg 1620 acatgtatct gtccaagctg agttaaaaca ggtcacagaa gtctatgact tggtgcgaga 1680 aatgggccct gaatctggac ttctacattt aaggcaaagc ccaaaagctg gactgttatc 1740 actagccaac tgtcccaact ttgcaagtgt tgttctcgga aatgcctcag gcttaggcat 1800 aatcggtatg tatcgtggga gagtaccaaa cacagaatta ttttcagcag cagaaagtta 1860 tgccaaaagt ttgaaagaga gcaataaat caatttctct tcattaggac ttacagatga 1920 agaaagag gctgcagaac atttcttaaa tgtgagtgac gacagtcaaa atgattatga 1980 gtaattaaaa aagtgggaca agtcaaaatg tctttccctg aagaaaaga tattctttc 2040 atgggtaatg aagcagcaaa attagcagaa gctttccaga aatcattaag gaaccaagt 2100 cataaaagat ctcaatctat tataggaa aaagtgaaca ctgtatcaga aacattggaa 2160 ttacctacta tcagtagacc tgcaaaacca accatactgt cagaaccaaa gttagcatgg 2220 actgataaag gtggggcaat caaaactgaa ataaagcaag caatcaaagt catggatcct 2280 attgagaag aagagtctac tgagaagaag gtgctgccct ccagtgatgg gaaaacccct 2340 gcaagaaaga aactgaaacc atcaactaac accaaaaaga aagtttcgtt tacaccaaat 2400 gaaccaggaa aatatacaaa gttggaaaaa gatgctctag atttgctctc agataatgaa 2460 gaagagatg cagaatcttc aatcttaacc tttgaagaaa gagatacttc atcgttaagc 2520 attgaggcca gattggaatc aatagaggag aaattaagca tgatattagg gctattaaga 2580 acactcaaca ttgctacagc aggacccaca gcagcaagag atgggatcag agatgcaatg 2640 attggcgtaa gagaggaatt aatagcagac aataaagg aagccaaagg gaaagcagca 2700 gaaatgatgg aaggaaat gagtcaacga tcaaaaatag gaacggtag tgtaaaacta 2760 acagagaaag caaaagagct taacaaaatt gttgaagatg aagcacaag tggagaatct 2820 gaagaaag aagaaccaaa agacatacaa gacaatagtc aagaagatga catttaccag 2880 ttaattatgt agtttaataa aataacaa tgggacaagt aaaaatggag tcctacctag 2940 tagacactta tcaaggcatt ccttacacag cagctgttca agttgatcta atagaaaagg 3000 acctgttacc tgcaagccta aaatatggt tccctttgtt tcaggccaac acaccaccag 3060 cagtgctgct cgatcagttg aaaaccctaa caataaccac tctgtatgct gcatcacaaa 3120 atggtccaat actcaaagtg aatgcatcag cccaaggtgc agcaatgtct gtacttccca 3180 3240 aactcacagt ctgtgaagta aaaacagttt acttaaaac catgaaacca tacgggatgg 3300 tatcaaatt tgtgagctca gccaaatcag ttggcaaaa aacacatgat ctaatcgcac 3360 tgtgtgattt tatggatcta gaaagaaca cacctgttac ataccagca ttcatcaat 3420 cagtttcaat aaagagt gagtcagcta ctgttgaagc tgctataagc agtgaagcag 3480 accaagctct aacacaggcc aaaattgcac cttatgcggg attgattg atcatgacta 3540 tgaacaatcc caaggcata ttcaaaagc tggagctgg gactcaagtt atagtagaac 3600 taggagcata tgtccaggct gaagcata gtaaaatg taggacttgg agccatcaag 3660 ggacaagata tgtgttgaag tccagataac agccaagcac cttggccaag agctactaac 3720 tctatctcat agattataaa gtcaccattc tcttata aaatcaagt tagacaaga 3780 attaaatcaa tcaagaatgg gataataa atgtcttgg aaagtggtga tcatttttc 3840 attgttaata acacctcaac acggtcttta agagagctat ttagagagt catgtagcac 3900 tataactgaa ggatatctca gtgttctgag gataggttgg tataccac tttttacact 3960 ggaggtaggt gatgtagaga accttacatg tgctgatgga cctagcttaa taaaaacaga 4020 attagacctg accaaaagtg cactagaaga actcagaaca gtttctgctg atcaactggc 4080 aagagaggag caaattgaga atcccagaca atctagattt gttctaggag caatagcact 4140 cggtgttgca acagcagctg cagttacagc aggtgttgca attgccaaaa ccatccggct 4200 tgaaagtgaa gtaacagcaa ttaagaatgc cctcaaaaag accaatgaag cagatacttac 4260 attgggggaat ggagttcgag tgttggcaac tgcagtgagg gagctgggaag attttgtgag 4320 caagaatcta acacgtgcaa tcaacaaaaa caagtgcgac attgctgacc tgaaaatggc 4380 cgttagcttc agtcaattca acagaaggtt tctaaatgtt gtgcggcaat tttcagacaa 4440 tgctgggaata acaccagcaa tatccttgga cttaatgaca gatgctgaac tagccagagc 4500 tgtttccaac atgccaaacat ctgcaggaca ataaaaactg atgttggaga accgtgcaat 4560 ggtaagaa aaggggttcg gaatcctgat aggagtttac ggaagctccg taatttacat 4620 ggtgcaactg ccaatctttg gagttataga cacgccttgc tggatagtaa aagcggcccc 4680 ttcttgctca gaaaaaaaaagg gaaactatgc ttgcctttta agagaagatc aaggatggta 4740 ttgtcagaat gcagggtcaa ctgtttacta cccaaatgaa aaagactgcg aaacaagagg agccatgtc ttttgcgaca cagcagcagg aatcaatgtt gctgagcagt caaaggagtg 4920. snowflake snowflake snowflake 4920. snowflake cagtatggtt gcactgtctc ctcttggggc tttggttgct tgctacaagg gagtgagctg 4980 ttccattggc agcaacagag tagggatcat caagcaactg aacaaaggct gctcttatat aaccaacca gacgcagaca cagtgacaat agacaacact gtataccagc tagcaaagt tgagggcgaa cagcatgtta taaaaggaag gccagtgtca agcagctttg atccagtcaa atttcctgaa gatcaattca atgttgcact tgaccaagtt ttcgaaagca ttgagaacag tcaggccttg gtggatcaat caaacagaat cctaagcagt gcagagaaag gaaacactgg cttcatcatt gtaataattc taattgctgt ccttggctct accatgatcc roofgagtgt ttttatcata ataaagaaa caaagaacc cacaggagca cctccagagc tgagtggtgt cacaaacaat ggcttcatac cacaatatta gttaatta aataagtaa attaatta aaaaaaaaaattt aaaaaaaaattgggaca aatcaatg 5520 tctcgcaagg ctccatgcaa attgaagtg cggggcaaat gcaatagagg aagtgagtgc 5580 aagtttaacc acaattactg gagttggcca gatagatact tactaatag atcaattat 5640 ttattaatc aacttttag gacactgat agagctgatg gcttatcaat atatcagga 5700 gcaggcagag agataggac acagatttt gtcctaggtt ccaccaatgt ggttcaggt 5760 tatattgatg ataccaaag cataacaaaa gctgcagcct gttacagctct acatatata 5820 atcaacaac tachaagagt tgaagttagg caggctgag atacaacc atctgacagc 5880 aaacatgtgg cacttcacaa cttagtccta tcttatatgg agatgagcaa aattcctgca 5940 tctttaatca acatctca aagactgccg agagagaac tgaaaaaatt agcaagctt 6000 ataattgact tatcagcagg tgctgaaat gactctcat atgccttgca agacagtgaa 6060 agcactaatc aagtgcagtg agcatggtcc tgtttcatt actatagagg ttgattacat 6120 gatatggact caataggact taaaagaagc tttatctaat gggatagtga agtctcatac 6180 taacatttac aattgttat tagaaaacat agaaattata tatgtcaagg cttacttaag 6240 ttagtaaaaa cacatcagag tgggacaagt agttatggag gtgaaagtgg agaacattcg 6300 aacaatagat atgctcaaag caagagtgaa aaatcgtgtg gcacgcagca aatgctttaa 6360 aaatgcctct ttgatcctaa taggaataac tacattgagt atagccctca atatctatct 6420 aatcataaac tatacaatgc aagaaaacac atccgaatca gaacatcaca ccagctcatc 6480 acccatggaa tccagcagg aaactccaac agtccctatg gacaactcag acaccaatcc 6540 aggctcacag tatccaactc aacagtccac agaaggctcc acactctact ttgcagcctc 6600 agcaagctca ccagagacag aaccaacatc aacaccagac acaacaagcc gcccgccctt 6660 cgtcgacaca cacacaacac taccaagtgc aagcagaaca agcaagtc cggcagtcca 6720 cacaaaaaac aatccaagga caacccccag aacacattcc ccaccatggg caatgacaag 6780 gacggtccgt ggaaccacca ctctccgcac aagcagcaca agaaaaagac cgtccacagc 6840 atcagtccaa cctgacagca gcgcaacaac ccacaaacac gaagaagcaa gcccagtgag 6900 cccgcaaaca tctgcgagca cagcagacc achaaggaag ggcatggagg cagcacacatc 6960 aaaaacatac aaccaaaaaaaaaaaaaacaaaat aactctaga aaaccatat 7020 agacaccaac attgagaag ccaaaggca attcacaatc tctccaaaaa ggcacaaca 7080 ccatattagc tccgttaaa tctccctgga aaaaacactc gcccatatac caactatacc 7140 acaaccacaaaaaaaaa agctgggtaa aaaacaccc agagacaaa taacaatgga 7200 tcctcttaat gatccactg ttaatgtcta tcttcccgac tcatatctta aaggtgat 7260 ttctttagt gagactaatg caatggttc atgtctctta aaagacctt acctaaaaaa 7320 tgacaacact gcaaagttg ccatagagaa tcctgttatc gagcatgtta gactcaaaa 7380 tgcaatcaat tctaagatga aaatatcaga ttacaggata gtagagccag taaacatgca 7440 acatgaaatt atgaagaatg tacacagttg tgagctcaca ttattaaac agttttaac 7500 aaggagtaaaatattagca ctcttaattt aaatatgata tgtgattggc tgcagttaaa 7560 gtctacatca gatgatacct caatcttaag ttttagat gtagaattta tacctagctg 7620 ggtaagcaat tggtttagta attggtacaa tctcaacaag ttgattctgg aattcaggaa 7680 agaagaagta ataagaactg gttcaatctt gtgtaggtca ttgggtaaat tagttttgt 7740 tgtatcatca tacggatgta tagtcaagag caaaaagc aaaagagtga gcttcttcac 7800 atacaatcaa ctgttaacat ggaaagatgt gatgttaagt agattcaatg caaatttctg 7860 tatatgggta agcaacagtc tgaatgaaaa tcaagaaggg ttagggttga gaagtaatct 7920 gcaaggcata ttaactaata agctatatga aactgtagat tatatgctta gtttgtgttg 7980 caatgaaggt ttctcacttg tgaaagagtt tgagggtttt attatgagtg aaatccttag 8040 gattactgaa catgctcaat tcagtactag atttagaaat actttattaa atggattaac 8100 tgatcaattg acaaaattaa aaaataaaaa cagactcaga gttcatggta ccgtgttaga 8160 aaatatgat tatccaatgt atgaagttgt acttaaatta ttaggagata ctttgagatg 8220 tattaaatta ttaatcaata aaaacttaga gaatgctgct gaattatact atatatttag 8280 aatattcggt cacccaatgg tagatgaaag agatgcaatg gatgctgtca aattaaacaa 8340 tgaaatcaca aaaatcctca ggttggagag cttgacagaa ctaagagggg cattcatatt 8400 aagattatc aaaggatttg tagacaacaa caaaagatgg ccgaaaatta aaaacttaaa 8460 agtgcttagt aaaagatgga ctatgtactt caaagcaaaa agttacccta gtcaacttga 8520 attaagtgaa caagattttt tagagcttgc tgcaatacag tttgaacaag agttttctgt 8580 tcctgaaaaa accaaccttg agatggtatt aaatgataaa gctatatcac ctcctaaaag 8640 attaatttgg tctgtgtacc caaaaaatta cttacctgag acaataaaaa atcgatatct 8700 agaagagact ttcaatgcaa gtgatagtct caaaacaaga agagtactag agtactattt 8760 gaaagataat aaattcgacc aaaaagaact taaaagttat gtggttaaac aagaatattt 8820 aaatgataag gatcatattg tctcgctaac tggaaagaa agaagaattaa gtgtaggtag 8880 aatgtttgct atgcaaccag gaaaacagcg acaaatacaa atattggctg aaaaattgtt 8940 agctgataat attgtaccct ttttcccaga aactttaaca aagtatggtg atctagatct 9000 tcagagaata atggaaatca aatcagaact ttcttctatt aaaaccagaa gaaatgatag 9060 ttatataat tacattgca gagcatccat agtaacagat ttaagtaagt tcaccaagc 9120 ctttaggtat gaaactacag cgatctgtgc ggatgtagca gatgaactac atggaacaca 9180 aagcctattc tgttggttac atcttatcgt tcctatgact acatgatat gtgcctatag 9240 acatgcacca ccagaaaaaaggtgaata tgatatagat aagatagaag agcaaagtgg 9300 tttatataga tatcatatgg gtgtattga aggatggtgt caaaactct ggacaatgga 9360 agctatatct ttattagatg ttgtatctgt aaagacacga tgtcaaatga catctttatt 9420 aaacggtgac aaccaatca tagagtag taaaccagtt aagttatctg agggtttaga 9480 tgaagtgaaa gcagattata gcttggctgt aaaaatgcta aaagaata gagatgcata 9540 cagaaatata ggccataac ttaagaagg ggaacatat atcaagg atcttcagtt 9600 tataagtaag gtgattcaat ctgaggagt atgcatcct acccctataa aaagatctt 9660 aagagtggga ccatggataa acacaatatt agatgacattt aaaaccagtg cagagtcaat 9720 agggagtcta tgtcaggaat tagaatttag gggggaaagc atatagtta gtctgatatt 9780 aaggaatttt tggctgtata attacat gcatgaatca aagcaacacc ccctagcagg 9840 gaagcagtta ttcaacaac taaataaac attackaca gtgcagagat ttttgaat 9900 taaaaaggaaaatgaagtag tagatctg gatgacata ccaatgcagt tggaggagg 9960 agatccagta gtctctata gatctttcta taactgaagc cctgattttt taactgaagc 10020 aatcagtcat gtagatattc tgttaaaaat atcagccaac ataagaatg aagcgaaaat 10080 aagtttctc aaagccttac tgtcaataga aaaaatgaa cgtgctacac tgacacact 10140 aatgagagac cctcaagctg tgggctcaga gcgacaagca aaagtaacaa gtgatatcaa 10200 tagacagca gttaccagca tcttaagtct ttctccaat caactttca gcgatagtgc 10260 tatacactac agtagaatg agagaggt cggaatcatt gctgacaca taacacctgt 10320 ttatcctcat ggactgagag tttgtatga atcattacct ttcataaag ctgaaaaagt 10380 tgtaaatatg atatcaggaa caaatccat aaccaactta ttacagagaa catctgctat 10440 taatggtgaa gatttgaca gagctgtatc catgatgctg gagaacctag gatttattac 10500 tagaatattg tcagtagttg ttgatgat agaaattcca accaaatcta atggtaggct 10560 gatatgttgt cagatatcta gaaccctaag gagacatca tggaatata tggaatgt 10620 tggagtaca tcccctagca tcactacatg catggatgtc atatatgcaa ctagctctca 10680 tttgaaaggg ataatcattg aaagttcag cactgacaga actacaagag gtcaaagagg 10740 tccaaagagc ccttgggtag gatcgagcac gcaagagaaa aattagttc ctgtttataa 10800 cagacaatt ctttcaaac aaaagaga acagctagaa gcaattgaa aaatgagatg 10860 ggtatataa gggacaccag gtttaagacg attackcaat aagatttgtc ttggaagttt 10920 aggcattagt tacaaatgtg taaaaccttt attacctagg tttagtg taaatttcct 10980 acacaggtta tctgtcagta gtagacctat ggaattcca gcatcagttc cagcttatag 11040 aaaacaaat taccattttg acactagtcc tattaatcaa gcactagtg aagatttgg 11100 gatgagat attaatttgg tcttccaaaa tgcaatcagc tgtggatta gcataatgag 11160 tgtagtagaa cattaactg gtaggagtcc aaaacagtta gttttatac cccaattaga 11220 agaaatagac attatgccac caccagtgtt tcaagggaaa ttcaattata agctagtaga 11280 window tctgaccac atatctcag tccagacaa atagatgt windowsggg 11340 gaaaatgctc atgccaacta taaaaggtca gaaaacagat cagttcttga acaagagaga 11400 gattatttc catgggaaca atcttattga gtctttgtca gcagcgttag catgtcattg 11460 gtgtgggata ttcacagagc atgtataga aaataatatt ttcaagaag actggggtga 11520 cgggttcata tcggatcatg cttttatgga cttcaaaata ttcctatgtg tctttaaaac 11580 taaactttta tgtagttggg gatcccaagg gaaaaacatt aagatgaag atagtaga 11640 tgaatcgata gataactgt taggattga tatactttt tggaatgt tcagcaggt 11700 tatgtttgaa tcaaggtta agaaaaggat aatgttatat gatgtaaaat ttctatcatt 11760 agtaggttat ataggtttta agaatttggtt tatagacag ttgagatcag ctgagttgca 11820 tgaggtacct tggattgtca atgccgagg tgatctggtt gagatcaagt cattaaaat 11880 ctatttgcaa ctgatagagc agagtttatt tttaagaata actgttttga actatacaga 11940 tatggcacat gctctcacaa gattaatcag aaagaagttg atgtgtgata atgcactatt 12000 aacttcaatt ccatccccaa tggttaactt aactcaagtt attgatccta cagaacaatt 12060 agcttatttc cctaagataa catttgaaag gctaaaaaat tatgacacta gttcaaatta 12120 tgctaaagga aagctaacaa ggaattacat gatactgttg ccatggcaac atgttaatag 12180 atataacttt gtctttagtt ctactggatg taaagttagt ctaaaaacat gcattggaaa 12240 acttatgaaa gatctaaacc ctaaagttct gtactttatt ggagaagggg caggaaattg 12300 gatggccaga acagcatgtg aatatcctga catcaaattt gtatacagaa gtttaaaaga 12360 tgaccttgat catcattatc ctttggaata ccagagagtg ataggagaat taagcaggat 12420 aatagatagt ggtgaagggc tttcaatgga aacaacagat gcaactcaaa aaactcattg 12480 ggatttgata cacagagtaa gcaaagatgc tttattaata actttatgtg atgcagaatt 12540 taaggacaga gatgattttt ttaagatggt aattctatgg aggaaacatg tattatcatg 12600 cagaatttgc actacctatg ggacagacct ctatttattc gcaaagtatc atgctaaaga 12660 ctgcaatata aaattacctt tttttgtgag atcagttgcc acctttatta tgcaaggtag 12720 taaactgtca ggctcggaat gctacatact cttaacacta ggccaccaca acaatttacc 12780 ttgtcatgga gaaatacaaa attctaagat gaaaatagca gcgtgtaatg attttatgc 12840 tgcaaaaaaa cttgacaata aatcaattga agccaactgt aaatcacttt tatcagggct 12900 aagaataccg ataaataaga aggaattaaa tagacagaga aggttattaa cactacaaag 12960 caaccattct tctgtagcaa cagttggagg tagcaaggtc atagagtcta aatggttaac 13020 aaacaaggca aacacaataa ttgattggtt agaacatatt ttaaattctc caaaaggtga 13080 attaaattat gatttttttg aagcattaga aaatacttac cctaatatga ttaaactaat 13140 agataatcta gggaatgcag agattaaaaa actgatcaaa gtaactggat atatgcttgt 13200 aagtaaaaaaa tgaaaaatga taaaaatgat aaaataggtg acaacttcat actattccaa 13260 agtaatcatt tgattatgca attatgtaat agttaattaa aaactaaaaa tcaaaagtta 13320 aaaactaata actgtcatta agtttattaa aaataagaaa ttataattgg atgtatacgg 13380 tttttttgcc gt 13392 <210> 6 <211> 13220 <212> DNA <213> Artificial Sequence <220> <223> GFP gene inserted between positions 40 and 784 <400> 6 gcgaaaaaaa cgcgtataaa ttagattaca aaaaaatatg ggacaagtga aaatggtgag 60 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt 120 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct 180 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac 240 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga 300 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga 360 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg 420 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga 480 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa 540 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta 600 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag 660 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga 720 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aagttaatta 780 aaaagtggga caagtgaaaa tgtctcttca agggattcac ctgagtgatc tatcatacaa 840 gcatgctata ttaaaagagt ctcagtacac aataaaaaga gatgtgggta caacaactgc 900 agtgacaccc tcatcattgc aacaagaaat aacgctgttg tgtggagaaa ttctgtatgc 960 taaacatgct gattacaaat atgctgcaga aataggaata caatatatta gcacagcttt 1020 aggatcagag agagtgcagc agattctgag gaactcaggc agtgaagtcc aagtggtctt 1080 aaccagaacg tactctctgg ggaaagttaa aaacaataaa ggagaagatt tacagatgtt 1140 agacatacac ggggtagaga agagctgggt agaagagata gacaaagaag caaggaaaac 1200 aatggcaacc ttgcttaagg aatcatcagg tatatccca caaaatcaga ggccctcagc 1260 accagacaca cccataatct tattatgtgt aggtgcatta atattacta agctagcatc 1320 aaccatagaa gtgggactag agaccacagt cagaagggct aaccgtgtac taagtgatgc 1380 actcaagaga taccctagaa tggacatccc aaaaattgcc agatccttct atgacttatt 1440 tgaacaaaaa gtgtatcaca gaagttttgtt cattgagtat ggcaaagcat taggctcatc 1500 atctacaggc agcaaagcag aaagtctatt tgttaatata ttcatgcaag cttatggagc 1560 cggtcaaaca atgctaaggt ggggggtcat tgccaggtca tccaacaata taatgttagg 1620 acatgtatct gtccaagctg agttaaaaca ggtcacagaa gtctatgact tggtgcgaga 1680 aatgggccct gaatctggac ttctacattt aaggcaaagc ccaaaagctg gactgttatc 1740 actagccaac tgtcccaact ttgcaagtgt tgttctcgga aatgcctcag gcttaggcat 1800 aatcggtatg tatcgtggga gagtaccaaa cacagaatta ttttcagcag cagaaagtta 1860 tgccaaaagt ttgaaagaga gcaataaat caatttctct tcattaggac ttacagatga 1920 agaaagag gctgcagaac atttcttaaa tgtgagtgac gacagtcaaa atgattatga 1980 gtaattaaaa aagtgggaca agtcaaaatg tctttccctg aagaaaaga tattctttc 2040 atgggtaatg aagcagcaaa attagcagaa gctttccaga aatcattaag gaaccaagt 2100 cataaaagat ctcaatctat tataggaa aaagtgaaca ctgtatcaga aacattggaa 2160 ttacctacta tcagtagacc tgcaaaacca accatactgt cagaaccaaa gttagcatgg 2220 actgataaag gtggggcaat caaaactgaa ataaagcaag caatcaaagt catggatcct 2280 attgagaag aagagtctac tgagaagaag gtgctgccct ccagtgatgg gaaaacccct 2340 gcaagaaaga aactgaaacc atcaactaac accaaaaaga aagtttcgtt tacaccaaat 2400 gaaccaggaa aatatacaaa gttggaaaaa gatgctctag atttgctctc agataatgaa 2460 gaagagatg cagaatcttc aatcttaacc tttgaagaaa gagatacttc atcgttaagc 2520 attgaggcca gattggaatc aatagaggag aaattaagca tgatattagg gctattaaga 2580 acactcaaca ttgctacagc aggacccaca gcagcaagag atgggatcag agatgcaatg 2640 attggcgtaa gagaggaatt aatagcagac aataaagg aagccaaagg gaaagcagca 2700 gaaatgatgg aaggaaat gagtcaacga tcaaaaatag gaacggtag tgtaaaacta 2760 acagagaaag caaaagagct taacaaaatt gttgaagatg aagcacaag tggagaatct 2820 gaagaaag aagaaccaaa agacatacaa gacaatagtc aagaagatga catttaccag 2880 ttaattatgt agtttaataa aataacaa tgggacaagt aaaaatggag tcctacctag 2940 tagacactta tcaaggcatt ccttacacag cagctgttca agttgatcta atagaaaagg 3000 acctgttacc tgcaagccta aaatatggt tccctttgtt tcaggccaac acaccaccag 3060 cagtgctgct cgatcagttg aaaaccctaa caataaccac tctgtatgct gcatcacaaa 3120 atggtccaat actcaaagtg aatgcatcag cccaaggtgc agcaatgtct gtacttccca 3180 aaaaatttga agtcaatgcg actgtagcac tcgatgaata tagcaaattg gaatttgaca aactcacagt ctgtgaagta aaaacagttt acttaaaac catgaaacca tacgggatgg 3300 tatcaaatt tgtgagctca gccaaatcag ttggcaaaa aacacatgat ctaatcgcac 3360 tgtgtgattt tatggatcta gaaagaaca cacctgttac ataccagca ttcatcaat 3420 cagtttcaat aaagagt gagtcagcta ctgttgaagc tgctataagc agtgaagcag 3480 accaagctct aacacaggcc aaaattgcac cttatgcggg attgattg atcatgacta 3540 tgaacaatcc caaggcata ttcaaaagc tggagctgg gactcaagtt atagtagaac 3600 taggagcata tgtccaggct gaagcata gtaaaatg taggacttgg agccatcaag 3660 ggacaagata tgtgttgaag tccagataac agccaagcac cttggccaag agctactaac 3720 tctatctcat agattataaa gtcaccattc tagttatata aaatcaagt tagacaaga 3780 attaaatcaa tcaagaatgg gataataa atgtcttgg aaagtggtga tcatttttc 3840 attgttaata acacctcaac acggtcttta agagagctat ttagagagt catgtagcac 3900 tataactgaa ggatatctca gtgttctgag gataggttgg tataccac tttttacact 3960 ggaggtaggt gatgtagaga accttacatg tgctgatgga cctagcttaa taaaaacaga 4020 attagacctg accaaaagtg cactagaaga actcagaaca gtttctgctg atcaactggc 4080 aagagaggag caaattgaga atcccagaca atctagattt gttctaggag caatagcact 4140 cggtgttgca acagcagctg cagttacagc aggtgttgca attgccaaaa ccatccggct 4200 tgaaagtgaa gtaacagcaa ttaagaatgc cctcaaaaag accaatgaag cagatacttac 4260 attgggggaat ggagttcgag tgttggcaac tgcagtgagg gagctgggaag attttgtgag 4320 caagaatcta acacgtgcaa tcaacaaaaa caagtgcgac attgctgacc tgaaaatggc 4380 cgttagcttc agtcaattca acagaaggtt tctaaatgtt gtgcggcaat tttcagacaa 4440 tgctgggaata acaccagcaa tatccttgga cttaatgaca gatgctgaac tagccagagc 4500 tgtttccaac atgccaaacat ctgcaggaca ataaaaactg atgttggaga accgtgcaat 4560 ggtaagaa aaggggttcg gaatcctgat aggagtttac ggaagctccg taatttacat 4620 ggtgcaactg ccaatctttg gagttataga cacgccttgc tggatagtaa aagcggcccc 4680 ttcttgctca gaaaaaaaaagg gaaactatgc ttgcctttta agagaagatc aaggatggta 4740 ttgtcagaat gcagggtcaa ctgtttacta cccaaatgaa aaagactgcg aaacaagagg agccatgtc ttttgcgaca cagcagcagg aatcaatgtt gctgagcagt caaaggagtg 4920. snowflake snowflake snowflake 4920. snowflake cagtatggtt gcactgtctc ctcttggggc tttggttgct tgctacaagg gagtgagctg 4980 ttccattggc agcaacagag tagggatcat caagcaactg aacaaaggct gctcttatat aaccaacca gacgcagaca cagtgacaat agacaacact gtataccagc tagcaaagt tgagggcgaa cagcatgtta taaaaggaag gccagtgtca agcagctttg atccagtcaa atttcctgaa gatcaattca atgttgcact tgaccaagtt ttcgaaagca ttgagaacag tcaggccttg gtggatcaat caaacagaat cctaagcagt gcagagaaag gaaacactgg cttcatcatt gtaataattc taattgctgt ccttggctct accatgatcc roofgagtgt ttttatcata ataaagaaa caaagaacc cacaggagca cctccagagc tgagtggtgt cacaaacaat ggcttcatac cacaatatta gttaatta aataagtaa attaatta aaaaaaaaaattt aaaaaaaaattgggaca aatcaatg 5520 tctcgcaagg ctccatgcaa attgaagtg cggggcaaat gcaatagagg aagtgagtgc 5580 aagtttaacc acaattactg gagttggcca gatagatact tactaatag atcaattat 5640 ttattaatc aacttttag gacactgat agagctgatg gcttatcaat atatcagga 5700 gcaggcagag agataggac acagatttt gtcctaggtt ccaccaatgt ggttcaggt 5760 tatattgatg ataccaaag cataacaaaa gctgcagcct gttacagctct acatatata 5820 atcaacaac tachaagagt tgaagttagg caggctgag atacaacc atctgacagc 5880 aaacatgtgg cacttcacaa cttagtccta tcttatatgg agatgagcaa aattcctgca 5940 tctttaatca acatctca aagactgccg agagagaac tgaaaaaatt agcaagctt 6000 ataattgact tatcagcagg tgctgaaat gactctcat atgccttgca agacagtgaa 6060 agcactaatc aagtgcagtg agcatggtcc tgtttcatt actatagagg ttgattacat 6120 gatatggact caataggact taaaagaagc tttatctaat gggatagtga agtctcatac 6180 tacaatttac attgttatt tagaaacat agaaattata tatgtcagg cttacttaag 6240 ttagtaaaa cacatcagag tgggataat cacaatgata acattagatg tcattaaaag 6300 tgatgggtct tcaaaacat gtactcacct CAaaaatta attaagacc actctggtaa 6360 agtgcttatt gtacttaagt taatattagc tttactaca tttctcacag tgacaatcac 6420 help aaaaacaatct gcaatgt cagtcaaaa ctgaatcaga 6480 caaaaaggac tcatcatca attackcatc agtcacaacc aagactactc taaatcatga 6540 tataacacag tattttaaaa gtttgattca aaggtataca aactctgca taaacagaga 6600 cacatgctgg aaataagca gaatcaatg cacaacata acataca aatttttatg 6660 ttttaaatct gagaaaaaaaccaaaa ttgtgaaaa ctgacagatt tatgcagaaa 6720 caaaccaaaa ccagctgttg aagtgtatca catagtagaa tgccattgta tatacacagt 6780 taaatggaag tgctatcatt acccaataga tgaaacccaa tcctaaataa cactagatta 6840 ggatccatcc aagtctgtta gttcaacaat ttagttattt aaaaatattt tgaaaacaag 6900 taagtttcta tgatacttca tataataag tataattaa ttgcttaatc atcatcacaa 6960 cattattcga aaccataact attcaattta agaagtaaaa acaataatat gagacaaata 7020 acaatggatc ctcttaatga atccactgtt aatgtctatc ttcccgactc atatcttaaa 7080 ggagtgattt cttttagtga gactaatgca attggttcat gtctcttaaa aagaccttac 7140 ctaaaaaatg acaacactgc aaaagttgcc atagagaatc ctgttatcga gcatgttaga 7200 ctcaaaaatg caatcaattc taagatgaaa atatcagatt acaggatagt agagccagta 7260 aacatgcaac atgaaattat gaagaatgta cacagttgtg agctcacatt attaaaacag 7320 ttttaacaa ggagtaaaaa tattagcact cttaaattaa atatgatatg tgattggctg 7380 cagttaaagt ctacatcaga tgatacctca atcttaagtt ttatagatgt agaatttata 7440 cctagctggg taagcaattg gtttagtaat tggtacaatc tcaacaagtt gattctggaa 7500 ttcaggaaag aagaagtaat aagaactggt tcaatcttgt gtaggtcatt gggtaaatta 7560 gttttgttg tatcatcata cggatgtata gtcaagagca acaaaagcaa aagagtgagc 7620 ttctcacat acatcaat gttaacatgg aagatgtga tgttaagtag attcaatgca 7680 aatttctgta tattgggtag cacagtctg aatgaaatc aagaagggtt agggttgaga 7740 agtaatctgc aaggcatatt aactaataag ctatatgaaa ctgtagatta tatgcttagt 7800 ttgtgttgca atgaaggttt ctcacttg aaagagtttg agggttttat tatgagtgaa 7860 atccttagga ttactgaaca tgctcaatc agtactagat ttagaatac tttaaat 7920 ggattaactg atcattgac aaattaaaa aaaaaaca gactcagagt tcatggtacc 7980 gtgttagaaa atatgatta tccaatgtat gaagttgtac ttaattatt aggagatact 8040 ttgagatgta ttaaattatt aatcaataaa acttagaga atgctgctga attackatactat 8100 atatttagaa tattcggtca cccaatggta gatgaagag atgcaatgga tgctgtcaa 8160 ttaaacaatg aaatcacaa aatcctcagg ttggagct tgacagact aagaggggca 8220 ttcatattaa ggattatca aggatttgta gacaaca aagatggcc gaaaattaaa 8280 aacttaaaag tgcttagtaa aagatggact atgtactca aagcaaaag tccctagt 8340 caacttgaat taagtgaaca agatttttta gagcttgctg caatacagtt tgaacaagag 8400 ttttctgttc ctgaaaaaac caaccttgag atggtattaa atgataaagc tatatcacct 8460 cctaaaagat taatttggtc tgtgtaccca aaaaattact tacctgagac aataaaaaat 8520 cgatatctag aagagacttt caatgcaagt gatagtctca aaacaagaag agtactagag 8580 tactatttga aagataataa attcgaccaa aaagaactta aaagttatgt ggttaaacaa 8640 gaatatttaa atgataagga tcatattgtc tcgctaactg gaaaagaaag agaattaagt 8700 gtaggtagaa tgtttgctat gcaaccagga aaacagcgac aaatacaaat attggctgaa 8760 aaattgttag ctgataatat tgtacccttt ttcccagaaa ctttaacaaa gtatggtgat 8820 ctagatcttc agagaataat ggaaatcaaa tcagaacttt cttctattaa aaccagaaga 8880 aatgatagtt ataataatta cattgcaaga gcatccatag taacagattt aagtaagttc 8940 aaccaagcct ttaggtatga aactacagcg atctgtgcgg atgtagcaga tgaactacat 9000 ggaacacaaa gcctattctg ttggttacat cttatcgttc ctatgactac aatgatatgt 9060 gcctatagac atgcaccacc agaaacaaa ggtgaatg atatagataa gatagagag 9120 caaagtggtt tatatagata tcatatgggt ggtattgaag gatggtgtca aaactctgg 9180 acatggaag ctatatcttt attagatgtt gtatctgtaa agacacgatg tcaatgaca 9240 tctttattaa acggtgacaa ccaatcaata gatgtagta aaccagttaa gttatctgag 9300 ggtttagatg aagtgaagc agattatagc ttggctgtaa aaatgctaaa agaataaga 9360 gatgcataca gaaatagg ccataactt aaagaagggg aacatatat atcaagagat 9420 cttcagttta tagtaggt gattcaatct gagagtaa tgcatcctac ccctataaaa 9480 aagatcttaa gagtgggacc atggataac acatattag atgacatta aaccagtgca 9540 gagtcaatag ggagtctatg tcaggaatta gatttaggg gggaagcat atagttagt 9600 ctgatattaa ggaattttg gctgtataat ttatacatgc atgaatcaa gcaacccc 9660 ctagcaggga agcagttatt storm aaaaacat ctagcagt gcagagattt 9720 tttgaaatta aaaaggaaaa tgaagtagta gattatgga tgaacatacc atgcagtttt 9780 ggaggaggag atccagtagt cttctataga tctttctata gaaggacccc tgatttttta 9840 actgaagcaa tcagtcatgt agatattctg ttaaaaatat cagccacat agaaatgaa 9900 gcgaaaataa gtttcttcaa agccttactg tcaatagaaa aaatgaacg tgctacactg 9960 aaacactaa tgagaccc tcaagctgtg ggctcagagc gawagcaa agtacaagt 10020 gatatcaata gaacagcagt taccagcatc ttaagtcttt ctccaaatca acttttcagc 10080 gatatgcta tacactacag tagaatgaa gagaggtcg gatcattgc tgacacata 10140 acacctgttt atcctcatgg actgagagtt tgtatgaat cattacctttt tcataaagct 10200 gaaaaagttg t'ataatgat acagaca aaatccaata ccaacttatt acagaca 10260 tctgctatta atggtgaaga tattgacaga gctgtatcca tgatgctgga gaacctagga 10320 ttattatcta gatattgtc agtagttgtt gatagtatag aattccaac caaatctaat 10380 ggtaggctga tatgttgtca gatatctga accctaaggg agacatcatg gaataatatg 10440 gaatagttg gagtacatc ccctagcatc actacatgca tggatgtcat atatgcaact 10500 agctctcatt tgaagggat aatcattgaa aagttcagca ctgacagac tacagaggt 10560 aaagaggtc haaagagcccc ttggggtagga tcgagcacgc aagaaaaa attagttcct 10620 gtttataaca ghaaatttct ttcaaaaaaaaagaac agctagaagc aattggaaaa 10680 atgagatggg tataaagg wakaccaggt ttagacgat tactcataa gatttgtctt 10740 ggaagtttag gcattagtta caatgtgta aaacctttat tacctaggtt tatgagtgta 10800 aatttcctac acaggttatc tgtcagtagt agacctatgg aattcccagc atcagttcca 10860 gcttatagaa caaaatta ccatttgac actagtccta ttaatcaagc actaagtgaa 10920 agatttggga atgagatat taatttggtc ttccaaatg caatcagctg tggaatttagc 10980 ataatgagtg tagtagaca attackactggt aggagtcca aacagttagt tttaataccc 11040 cattagaag aaatagacat tatgccacca ccagtgtttc aagggaatt cattataag 11100 ctagtagata agataacttc tgaccacat atcttcagtc cagacaaat agatagtta 11160 acactgggga aaatgctcat gccactata aaaggtcaga aacagatca gttcttgaac 11220 aagagagaga attatttcca tgggaacaat cttattgagt ctttgtcagc agcgttagca 11280 tgtcattggt gtgggatatt aacagagcaa tgtatagaaa atatattt caaagac 11340 tggggtgacg gttcatatc ggatcatgct tttatggact tcaaatatt cctatgtgtc 11400 tttaaaacta aacttttag tagttgggga tcccaaggga aaaacattaa agatgagat 11460 atagtagatg atcgataga taaactgtta aggattgata atactttg gagaatgttc 11520 agcaggtta tgtttgaatc aaagttaag aaaggataa tgttatatga tgtaaaattt 11580 ctatcattag taggttatat agggtttaag aattggtttta tagacagtt gagatcagct 11640 gagttgcatg aggtaccttg gattgtcaat gccgaaggtg atctggttga gatcaagtca 11700 attaaaatct atttgcaact gatagagcag agtttattt windowataac tgttttgaac 11760 tatacagata tggcacatgc tctcacaga ttaatcagaa agaagttgat gtgtgataat 11820 gcactattaa cttcaatcc atcccaatg gttaacttaa ctcaagttat tgatcctaca 11880 gaacaattag cttattccc windowaca ttgaaaggc taaaaaatta tgacactagt 11940 tcaattatg ctaaaggaaa gctaacaagg attackacatga tactgttgcc atggcaacat 12000 gttaatagat atactttgt ctttagttct actgatgta aagttagtct aaaaacatgc 12060 attggaaac tttagaaga tctaaccct aaagttctgt actttattgg agaaggggca 12120 ggaaattgga tggccagaac agcatgtgaa tatcctgaca tcaatttgt atacagaagt 12180 ttaaaagatg accttgatca tcattatcct ttggaatacc agagagtgat aggagatta 12240 agcaggataa tagatagtgg tgaagggctt tcaatggaa aacagatgc aactcaaaa 12300 actcattggg atttgataca cagagtaagc aagatgctt tattaac tttatgtgat 12360 gcagaattta aggacagaga tgattttt aagatggtaa ttctatggag gaaacatgta 12420 ttatcatgca gatttgcac tacctatggg acagacctct atttattcgc aaagtatcat 12480 gctaaagact gcaatataaa attacctttt ttgtgagat cagttgccac ctttattatg 12540 caagtagta aactgtcagg ctcggaatgc tacatactct tacactagg caccacac 12600 aatttacctt gtcatggaga atacaaat tctagatga aaatagcagc gtgtaatgat 12660 ttttatgctg caaaaaaact tgacataaa tcaattgaag ccaactgtaa atcacttta 12720 tcagggctaa gataccgat aaatagaag gatttaatta gagagagag gttattaca 12780 ctacaaagca accattctc tgtagcaaca gttggaggta gcaaggtcat agagtctaaa 12840 tggttaacaa aaggcaa cacaataatt gattggttag aacatatttt aaattctcca 12900 aaggtgaat taaattatga ttttttgaa gcattagaaa attackaccc taatatgatt 12960 aaactaatag atatctagg gatgcagag atttaaaaac tgatcaagt aactgatat 13020 atgcttgtaa gtaaaaatg aaaatgata aaatgataa ataggtgac aacttcatac 13080 tattccaaag taatcattg attatgcaat tattgtaatag tattaaaa actaaaaatc 13140 aaagttaaaactaataac tgtcattaag tttattaaaaatagaaatt atattggat 13200 gtatacggtt ttttgccgt 13220 <210> 7 <211> 13280 <212> DNA <213> wood chips <400> 7 acgcgaaaaa acgcgtata aattaattc caacaaac gggacaata aaatgtctc 60 ttcagggat tcacctaagt gatctgtcat aaacatgc tatattaaaa gagtctcaat 120 acacataaa aagagatgta ggcaccacaa ctgcagtgac accttcatca ttgcagcaag 180 agatacact ttgtgtgga gagattcttt acactaaca tactgattac aaatatgctg 240 spagagatagg gatataat atttgcacgg ctctaggatc agaagagta spacaattt 300 taagaaattc aggcagtgaa gttcaggtgg ttctaccaa gatactct ttagggaag 360 gtaaaaatag taaagggaa gagttgcaaa tgttagatat acatggagtg gaaagagtt 420 gggtagaga atagacaaa gaggcagaa aaacaatggt gactttgcta aaggaatcat 480 caggtaacat cccacaaac cagaggcctt cagcaccaca cacaccaata attttat 540 gtgtaggtgc tttaatattc actaaactag catcacaat agaagttgga ctagagacta 600 cagttagaag agctaacaga gtgctaagtg atgcgctcaa aagataccct agggtagata 660 taccgaagat tgctagatct ttctatgaac tatttgagca gaagtgtat tacaggagtc 720 tattcattga gtatgggaaa gctttaggct catctcaac aggaagcaaa gcagaaagtt 780 tgtttgtaaa tatatttatg caagcttatg gagccggtca aacaatgcta aggtggggtg 840 tcattgccag atcatctaac aacataatgc taggacatgt gtctgtgcaa gctgaattga 900 agcaagttac agaggtttat gatttggtga gagaaatggg tcctgaatct gggctttac 960 atctaagaca aagtccaag gcaggactgt tatcgttggc caatgcccc gattttgcta 1020 gtgttgttct tggtaatgct tcaggtctag gtataatcgg aatgtacaga ggaagagtgc 1080 ciaacacaga gctattttct gcagcagaaa gttatgccag aagcttaaa gaagcaaca 1140 aaatcaactt ctcctcatta gggctcacag acgaaaa agaagctgca gaacacttct 1200 taaacatgag tgatgacaat aaaaactgg xgagtca 1260 aatgtcattc cctgaaggaa aagatatcct gttcatgggt aatgaagcag caaaatagc 1320 agaagctttc cagaaatcac taaaagatc agtcacaa agaacccagt ctattgtagg 1380 ggaaaaaagta aacactatat cagaaactct agagctacct accatcagca aacctgcacg 1440 atcatctaca ctgctagagc caaaattggc atgggcagac agcagcagag ccaccaaaac 1500 cacagaaaaa caaacaacca aaacaacaga tcctgttgaa gaaggaac tcaatgaaaa 1560 gaagatatca ccttccagtg atgggaagac tcccgcagag aaaaaatcaa aatctccaac 1620 caatgtaaaa aagaaagttt ccttcacatc aaatgaacca gggaaatata ccaaactaga 1680 aaaagatgcc ctagatttgc tctcagacaa tgaggaagaa gacgcagagt cctcaatctt 1740 1800 agagaagcta agcatgatat taggactgct tcgtacactt aacattgcaa cagcaggacc 1860 1920 agaataata aaagaagcaa agggaaaagc agctgaaatg atggagaagg aaatgaatca 1980 aaggtcaaaa ataggtaatg gcagtgtaaa actaaccgag aaggcaaaag aacttaataa 2040 aattgttgaa gacgagagca caaccggtga atcagaagaa gaagagaac caaaagaaac 2100 tcaggataac atcaggag agatattta ccagttaatc atgtagtttta aaaaata 2160 acatgggac aagtcagat ggagtcctat ctagtggaca cttatcagg cattccctac 2220 acagctgctg ttcaagttga tctggtagaa aaagacttac taccagcaag tttgacaata 2280 tggttttcctc tattccaagc CACACACA ccagcggttt tgctcgatca gctaaagacc 2340 ttgacataa caacctgta tgctgcatca cagaatggtc caatactcaa ggtaaatgca 2400 tcagctcagg gtgctgctat gtctgtactt cccaaaaaat tcagtaaa tgcaactgtg 2460 gcacttgatg atacagcaa acttgacttt gatacagttaa cggttttgcga tgttaaaaca 2520 gtttatttga caaccatgaa accatatggg atggtgtcaa atttgtgag ttcagccaaa 2580 tcagttggca acagacaca tgatctaatt gcactgtgtg acttcatgga cctagagaaa 2640 atatacctg tgacaatacc agcattcata aagtcagtttt caatcaaga gagtgagtca 2700 gccactgttg aagctgcaat aagcagtgag gccgaccaag cattacaca agccaaaatt 2760 gcacctatg caggactaat catgatcatg accatgaaca atccaaaagg tatattcaag 2820 aaactaggag ctggaacaca agtgagta gagctagggg catatgttca agccgagagc 2880 atcagcagga tctgcagag ctggagtcac siaggaaca gatatgtact aaaatccaga 2940 taaaaataac tgtcctaatc ataatttgct tatataatct taagatca tgagcttatt 3000 attaagtta tadaaaaaaaaaaaaaaaaaaaaaaaaaaggtaaggtatt attagaaagc gggaaaaaaaaggta 3060 aaaatgtctt ggaagtgat gattatcatt tcgttactca taacacctca gcacggacta 3120 aaggaaagtt atttagaga atcatgtagt actataacctg aaggatatct cagtgtttta 3180 agaacaggtt ggtacaccaa tgtctttaca ttagaagttg gtgatgttga aaatcttaca 3240 tgtactgatg gacctagctt atcaaaca gaacttgacc taaccaaag tgctctaaga 3300 gaactcaaa cagttctctgc tgatcagtta gcgagagaag aacaattga aaatcccaga 3360 caatcaggt tgtcctagg tgcaatagct cttgtgttg ccacagcagc agcagtcaca 3420 gcaggcattg cgatagccaa aaccataagg cttgagagtg aagtgaatgc atcaaggt 3480 gctctcaaaa siaccaatga ggcagtatcc acactaggaa atggagtgcg agtcctagcc 3540 accgcagtaa gagagctgaa agaatttgtg agcaaaaacc tgactagtgc aattaacaag 3600 aacaaatgtg acattgctga tctgaagatg gctgtcagct tcagtcaatt caacagaaga 3660 ttcctaaatg ttgtgcggca gttttcagac aatgcaggga taacaccagc aatatcattg 3720 gacctaatga ctgatgctga gctggccaga gctgtatcat acatgccaac atctgcagga 3780 cagataaaac taatgttaga gaaccgtgca atggtgagga gaaaaggatt tggaatcttg 3840 ataggggtct acggaagctc cgtgatttac atggtccagc tgccgatctt tggtgtcata 3900 gatacacctt gttggataat caaggcagct ccctcttgtt cagaaaaaga tggaaactat 3960 gcttgcctcc taagagagga tcaagggtgg tattgtaaaa atgcaggatc cactgtttac 4020 tacccaaata aaaaagactg cgaaacaaga ggtgatcatg ttttttgtga cacagctgca 4080 gggatcaatg ttgctgagca atcaagagaa tgcaacatca acatatctac aaccaactac 4140 ccatgcaaag tcagcacagg aagacaccct atcagcatgg ttgcactatc acctctcggt 4200 gctttggtgg cttgctacaa aggggttagc tgttcaattg gcagtaatcg ggttggaata 4260 atcaaacaac tacctaaagg ctgctcatac ataactaacc aggacgcaga cactgtaaca 4320 attgacaaca ctgtgtatca actaagcaaa gttgagggtg aacagcatgt aataaaaggg 4380 agaccagttt caagcagttt cgatccaatc aagtttcctg aggatcagtt caatgttgcg 4440 cttgatcaag tctttgaaag cattgaaaac agtcaagcac tagtggacca gtcaaacaaa 4500 attctgaaca gtgcagaaaa aggaaacact ggcttcatta ttgtaataat tttgattgct 4560 gttcttgggt taaccatgat ttcagtgagc atcatcatca taatcaaaaa aacaaggaaa 4620 cccacagggg cacctccaga gctgaatggt gttaccaacg gcggttttat accgcatagt 4680 tagttaatta aaaaatggga caaatcatca tgtctcgtaa agctccatgc aaatatgaag 4740 tacggggcaa gtgcaacagg ggaagtgagt gcaaattcaa ccacaattac tggagttggc 4800 ctgataggta tttattgtta agatcaaatt atctcttgaa tcagctttta agaaacactg 4860 ataaggctga tggtttgtca ataatatcag gagcaggtag agaagacagg actcaagact 4920 ttgttcttgg ttctactaat gtggttcaag ggtacattga tgacaatcaa ggaataacaa 4980 aggctgcagc ttgctatagt ctacataaca taataaaaca gctacaagaa atagaagtaa 5040 gacaggccag agataataag ctttctgaca gcaaacatgt ggcacttcac aacttgatat 5100 tatcctatat ggagatgagc aaaactcctg catccctgat taataaacta aagaaactac 5160 caagaaaa actgaaaaaa ttagcgaaat taataattga tttatcagca ggaactgata 5220 5280 cccaaattca tcaccataga ggcagatgat atgatatgga cacacaaaga attaaaagag 5340 acactgtctg atgggatagc aaaatcacac accaatattt acagttgtta tttagaaaat 5400 atagaaataa tatatgttaa agcttactta agttagtaaa aataaatag aatgggataa 5460 5520 ctcaaaaaaa taataaaaaa acactcaggt aaattgctta ttgcatcaaa accgacattg 5580 gccttattga cgtccttcac agtaacaatt actgtcaact atacaaaagt agaaataat 5640 ttgcaggcat gtcaattaaa aaatgaatca gacaaaaagg acacaaagct aaataccaca 5700 tcaacaa tcagacccat tcctgatcta aatgcagtac agtacctgaa aaggctgatt 5760 cagaacaca cacaacctgt cacaaagac agagatacct gttggagaat accacacgaat 5820 caatgcacaa atataaaaat ataagttc ttagttttg ggtctatgaa ttcacaaat 5880 acagactgtg aagaaccaac agttctatgc vakaaaaagt aaaaaccat gagaaaaa 5940 cataggaaag cagagtgtca ccgtccacat acaccgagt ggtggtgcca ttatctttaa 6000 gagaaaactc agttttcaac atttaaatca gacaaatca tatctagatc tattatata 6060 atagtctagt tatttaaaa ctctaaatat tgtctagact tcacacacc ctgcggtcat 6120 atgcaataat caatggtcaa accactgttg caaacccacc tataatacaa tcactgagta 6180 atacaaaca agaaatggg acagtggcc atggaagcaa gagtgagaa cattcggggca 6240 atagacatgt tcaagcaa gatgaaaaac cgtataagaa gtagcaagtg ccatagaat 6300 gctacactga tccttattgg atcacagca ccaagtatgg cactcacac ccttttaatc 6360 attgatcatg cacatcaa aaacatgacc aaagtggac actgtgtca catgccgccg 6420 gtagaaccaa gcaagagac cccaatgacc tctgcagcag acccaacac caacccaat 6480 ccacagcagg spacacagct gaccacag gattcacat cttagcagc aaccctagag 6540 gaccatctac acacagggac aactccaca ccagatgcaa cagtctccca gcaaccaca 6600 gagatchacc stormgct gagatchacc AACCAG 6660 aaaaagccaaaaaaaaaaaaaaaccaaaaaaaccaaa ctcgaaccac aagcacagct 6720 gcaaccaaa cactcacac caccaccaa actagcaatg gagagaggc aaccacaaa 6780 tctgccagat ccagaaaaaaaaagcagcg atcaacaaaaaaaaaaaacaaaaaaaaacaacaaca 6840 gaccaagct cccaacaca gaccacacag aaagcacaa gaccacaca 6900 acatctctctc caagtagtta aaaaaaac taaaaata ccatgaaaac aaaaaaacta 6960 gaaagttaa ttgaactca gaaagaacaacacat atgaattgtt tgagcgtata 7020 tactaatgaa atagcatctg tttgtgcatc ataatacca tcattattta agaaataaga 7080 agaagctaaa attcaaggga CAAACAA tggatccgtt ttgtgaatcc actgtcaatg 7140 tctatcttcc tgattcatat ctcaaaggag tatatcttt cagtgaaacc aatgcaattg 7200 gctcatgcct tttgaaaaga ccctatctta aaaaagataa cactgctaaa gttgctgtag 7260 aaaccctgt tgttgaacat gtcagactta gaaatgcagt catgaccaaa atgaagatat 7320 cagattataa agtggttgaa ccaattaata tgcagcatga aataatgaaa aatatacaca 7380 gttgtgagct cacattatta aaacaattct taacaagaag taaaacatt agctccctaa 7440 aattaagtat gatatgtgat tggttacagt taaaatccac ctcagataac acatcaattc 7500 ttaattttat agatgtggag tttatacccg tttgggtgag caattggttt agtaactggt 7560 ataatctcaa taaattaatc ttagagttta gaagagagga agtaataaga actggttcaa 7620 tttatgcag atcactaggc aagttagttt tcattgtatc atcttatggg tgtgtagtaa 7680 aaagcaacaa aagtaaaaga gtaagttttt tcacatataa ccaactgtta acatggaaag 7740 atgtgatgtt aagtaggttc aatgcaaact tttgtatatg ggtaagtaac aacctgaaca 7800 aaaatcaaga aggactagga tttagaagta atctacaagg tatgttaact aataaattat 7860 atgaaactgt tgattatatg ttaagtctat gtagcaatga agggttctca ctagtgaaag 7920 agttcgaagg ctttattatg agtgaaatttc ttaaattac tgagcatgct cattcagta 7980 ctaggtttag gatactta ttaaatgggt tgactgaaca atttacaatg ttgaagcta 8040 aaaaacagatc tagttctt ggcactatat taggaaaaaaaaaacaccc atgtgag 8100 tagtactta attattagggg gandactttga aaagtataa atttaattt aacaagaatt 8160 tagaaaatgc tgcagaatta tattatat tcagaatttt tggacaccct atggtagatg 8220 agagggagc aatggatgct gttaaattta atgaatgagat tacaaaattt cttaaactgg 8280 agagcttaac agaactaga ggagcattta tactagaat tataaaaggg ttgtagata 8340 ataataaag atggcctaaa attaagaatt taaaagtgct cagtaaaga tgggttatgt 8400 atttcaaagc taaaagttac cctagccaac ttgagctaag tgtacagat ttttagaac 8460 ttgctgcagt acaattcgaa caggaatttt ctgtccctga aaaaaccaat cttgagatgg 8520 tattaaatga taaagcaata tctccaccaa aaagttaat atggtcggta tatccaaaaa 8580 attatctacc tgaaattata aaaaatcaat atttagaaga ggtcttcaat gcaagtgaca 8640 gtcaaagaac gaggagagtc ttagaattttt acttaaaaga ttgcaaattt gatcaaaaag 8700 acctcaaacg ttatgtaact aaacaagagt atctaaatga caaagaccac attgtctcat 8760 taactgggaa agaaagagaa ttaagtgtag gcaggatgtt tgcaatgcaa cctggcaaac 8820 aaagacaaat acagatacta gccgagaaac ttttagctga tatattgta cccttttttcc 8880 cagaaacttt aaaagtat ggtgacttgg atctccaaag aattatggaa atgaaatcag 8940 aactttcttc cattaaaact aggaagaatg atagttacaa caattatatt gcaagagcct 9000 ccatagtaac agacctaagt aaattcaatc aagcctttag atatgaaacc acagctatct 9060 gcgcagacgt agcagatgag ttacatggca cgcaaagctt atttgttgg ttacatctta 9120 ttgttcccat gaccacaatg atatgtgcat acagacatgc accaccagaa acaaaggggg 9180 agtatgatat agacaaaata gaagagcaaa gtgggctata cagataccat atgggaggga 9240 ttgaagggtg gtgtcagaag tttggacaa tggaggcgat atccttgtta gatgtagtat 9300 ctgttaagac tcgttgtcag atgaccctc tattaacgg agacaatca tcaatagatg 9360 tcagtaaacc agtaaaattg tctgaggta taggagt aaagcagat tagcttag 9420 cattaaaat gcttaaag atagagatg cctataaaaa cattggccat aaactcaaag 9480 aaggtgaaac atatatca agagatctc aatttataag taagtgatt caatctgagg 9540 gggtcatgca tcctacccc ataaaaaga tattaagggt aggtccctgg ataatacaa 9600 tactagatga cattaaaact agtgcagaat tactagggag tctgtgtcaa gaactagagt 9660 tcagaggaga aagtatacta gttagcttga tattaggaa tttctggctg tataacttat 9720 acatgcatga gtcaaacag catccgttag ctggaaaaca actgtttaaa cattgaaca 9780 aaacactaac atctgtgcaa agattttg agctgaagaa agaaatgat gtggttgacc 9840 tatggatgaa tataccaatg cagttggag ggggagaccc agtagttttt tacagatctt 9900 tttacagaag gactcctgat ttctgactg aagcaatcag ccatgtggat ttactgttaa 9960 aagtttcaaa caatattaaa atgagacta agatacgatt ctttaaagcc ttattatcta 10020 tagaaagaa tgaacgtgct acattaacaa cactaatgag agacccccag gcggtaggat 10080 cggaagaca agctaggta acaagtgata windowagtact agcagtact agcatactga 10140 gtctatctcc gatcagcta tttgtgata gtgctataca ctatagcaga atgaagaag 10200 aagtagggat cattgcagac aacatacac ctgtttatcc tcacggattg agagtgctct 10260 atgaatcact accttttcat aaggctgaaa aggttgtca tatgatca ggtacaaagt 10320 ctataactaa cctattgcag agaacatctg ctatcaatgg tgagatatt gatagagcag 10380 tgtctatgat gttagagaac ttaggttgt tatctaggat attgtcagta attattaata 10440 gtatagaaat accaattaag tccaatggca gattgatatg ctgtcaaatt tctaagactt 10500 tgagagaaaa atcatggaac atatggaaa tagtaggt gatactcca agtattgtaa 10560 catgtatgga tgttgtgtat gcgactagtt ctcatttaaa aggaataatt attgaaaaat 10620 tcagtactga caaccaca agaggtcaga ggggaccaaa aagcccttgg gtaggatcaa 10680 gcactcaaga gaaaaatta gttcctgttt atacagaca aattctttca aaaaacaaa 10740 aagagcaact ggaagcaata ggaaaaatga ggtgggtgta taaaggaact ccagggctaa 10800 gaagattgct caataagatt tgcataggaa gtttaggtat tagctataaa tgtgtaaaac 10860 ctctattacc aagatttatg agtgtaaact tcttacatag gttatctgtt agtagcagac 10920 ccatggaatt cccagcttct gttccagctt ataggacaac aaattaccac tttgacacta 10980 gtccaatcaa ccaagcatta agtgagaggt tcgggaacga agacattaat ctagtgttcc 11040 aaaatgcaat cagctgcgga attagtataa tgagtgttgt agaacagtta actggtagaa 11100 gcccaaaaca attagtctta atcccccaat tagaagagat agatattatg ccccctcctg 11160 tatttcaagg aaaattcaat tataaactag ttgataaaat aacctccgat caacacatct 11220 tcagtcctga caaatagac atattaacac tagggaagat gcttatgcct actataaaag 11280 gtcaaaaaac tgatcagttc ttaaataaga gagaaaacta tttccatgga aataatttaa 11340 ttgaatcttt atctgcagca cttgcatgcc attggtgtgg aatattaaca gaacagtgtg 11400 11460. tagaaaacaa tatctttagg aaagactggg gtgatgggtt catatcagat catgccttca tggatttcaa gatatttcta tgtgtattta aaaccaaact tttatgtagt tggggatccc aagggaaaaa tgtaaaagat gaagatata tagagatc cattgacaaa ttattaaga ttgacaacac tttttggaga atgttcagca aagtcatgtt tgaatcaaag gtcaaaaaaa 11700. 11700. 11700. 11700. 11700. 11700. 11700. 11700. 11700 ggtttataga gcagttaaga gtagtagaat tgcatgaagt accctggatt gtcaatgctg aaggggagct agttgaaatt aaaccaatca aaatttattt gcagttaata gaacaaagtc fathers fathers fathers fathers cagacatggc acatgctctt acacgataa ttaggaga attgatgtgt gataatgcac tctttaatcc aagttcatca ccaatgttta gtctaactca cctacaacc ttttcctaag gtaattttg aaaggttaaa aagttatgac accagttcag actacaacaa agggaagtta acaagaaatt acatgacatt attaccatgg cagcacgtaa acaggtata ttttgtcttt agttcaacag gatgtaaaat cagcttgaag acatgcatcg ggaaattgat aaaggactta aaccctaagg ttctttactt tattggagaa ggagcaggta actggatggc aagaacagca tgtgagtatc ctgacataaa atttgtatat aggagtttaa aggatgatct tgatcaccat tacccattag aatatcaaag ggtaataggt gatttaaata gagtaatag tggtggtgaa ggattatcaa 12420. tggagaccac agatgcaact caaaagactc attgggactt gatacacaga ataagtaaag atgctttatt gataacattg tgtgatgcag aattcaaaaa cagagatgat ttctttaaaa tggtaattct ttggagaaaa catgtattat catgtagaat ctgtacagct tatggaacag atctttactt atttgcaaag tatcatgcga cggactgcaa tataaagtta ccattttttg taaggtctgt agctactttt attatgcaag gaagcaaatt gtcaggatca gaatgttaca tacttttac attaggtcat cacaatatc tgccatgtca cggagaata caaattcca aaatgagaat agcagtgtgt aatgatttcc atgcctcaaa aaaactagac aacaaatcaa ttgaagcaaa ctgcaaatct cttctatcag gattaagaat accaataaac aaaaaagagt 12840 taaatagaca aaagaaactg ttaacactac aaagcaatca ttcttccata gcaacagttg 12900 gcggaagtaa gattatagaa tccaaatggt taaagaataa agcaagtaca ataattgatt 12960 ggttagagca tatcttgaat tctccaagag gtgaattaaa ctatgatttc tttgaagcat 13020 tagagaacac atatcccaat atgatcaagc ttatagataa cctgggaaat gcagagataa 13080 aaaaactaat caaagttacc gggtatatgc ttgtgagtga gaagtaataa taataataat 13140 aatcaaccat aatctcacac aactgagaaa atgatcatct aacagtttaa ttgaccatta 13200 gttaattaaa aattataaat tagtaactaa ttgataaaaa ataagaaatt gaaattgaat 13260 gtatacggtt tttttgccgt 13280 <210> 8 <211> 14028 <212> DNA <213> Artificial Sequence <220> <223> GFP gene inserted between positions 41 and 785 <400> 8 acgcgaaaaa aacgcgtata aattaaattc caaacaaaac gggacaaata aaaatggtga 60 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg 120 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc 180 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga 240 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg 300 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg 360 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc 420 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg 480 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca 540 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact 600 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga 660 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg 720 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag taagttaatt 780 aaaaaactgg gataaaa atgtctctt cagggattc acctaagtga tctgtcatat 840 aaacatgcta tattaaaga gtctcaatac acataaaa gagatgtagg caccacaact 900 gcagtgacac cttcatcatt gcagcaagag atacactt tgtgtggaga gattctttac 960 actaaacata ctgattacaa atatgctgca gagataggga acataacata ttgcacggct 1020 ctaggatcag aagagtaca acagatttta agaattcag gcagtgaagt tcaggtggtt 1080 ctaaccaaga catactcttt agggaaggt aaaaatagta aagggaa gttgcaatg 1140 ttagatatac atggagtgga aaagagttgg gtagaagaaa tagacaaaa ggcaaaaa 1200 acatggtga ctttgctaaa ggaatcatca ggtaacatcc cacaaacca gaggccttca 1260 gcaccagaca caccaataat tttatgt gtaggtgctt taatattcac taaactagca 1320 tcaacatag aagttggact agagactaca gttagagag ctacagagt gctaagtgat 1380 gcgctcaaaa gataccctag ggtagatata ccgagattg ctagatcttt ctgaacta 1440 ttgagcaga aagtgtatta caggagtcta tcattgag atgggaagc tttaggctca 1500 1560 gccggtcaaa caatgctaag gtggggtgtc attgccagat catctaacaa cataatgcta 1620 1680 gaaatgggtc ctgaatctgg gcttttacat ctaagacaaa gtccaaaggc aggactgtta 1740 tcgttggcca attgccccaa ttttgctagt gttgttcttg gtaatgcttc aggtctaggt 1800 ataatcggaa tgtacagagg aagagtgcca aacacagagc tattttctgc agcagaaagt 1860 tatgccagaa gcttaaaaga aagcaacaaa atcaacttct cctcattagg gctcacagac 1920 gaaaaag aagctgcaga acacttctta aacatgagtg atgacaatca agatgattat 1980 2040 tcatgggtaa tgaagcagca aaaatagcag aagctttcca gaaatcacta aaaagatcag 2100 gtcacaaaag aacccagtct attgtagggg aaaaagtaaa cactatatca gaaactctag 2160 agctacctac catcagcaaa cctgcacgat catctacact gctagagcca aaattggcat 2220 gggcagacag cagcagagcc accaaaacca cagaaaaaaaaaaaaaaaacagatc 2280 ctgttgaaga agaggaacctc aatgaaaga agatacacc ttccagtgat gggaagactc 2340 ccgcagagaa aaatcaaaa tctccaacca atgtaaaaa gaagtttcc ttcacatcaa 2400 atgaaccagg gaaatacc aaactagaaa aagatgccct agatttgctc tcagacaatg 2460 aggagaga cgcagagtcc tcaatcttaa cctttgaga gagacaca tcatcacta 2520 gcattgaggc tagactagaa tcatagaag agagctaag catgatatta ggactgcttc 2580 gtacacttaa cattgcaaca gcaggaccaa cggctgcaag agatggaatc agagatgcaa 2640 tgattggtat agagagaa ctaatagcag aaataataa aggaagcaag ggaaagcag 2700 ctgaatgat ggagaggaa atgaatcaa ggtcaaaat aggtaatggc agtgtaaaac 2760 taaccgagaa ggcaaagaa cttataaaa ttgttgaga cgagagcaca agcggtgaat 2820 agaaaaaaaaaaaaccc agaaaaaaaaaaaaaaaaaaaackacc 2880 agttaatcat gtagtttaat aaaaataac aatgggacaa gtcaagatgg agtcctatct 2940 agtggacact tatcaggca ttccctac agctgctgtt caagttgatc tggtagaaaa 3000 agacttacta ccagcaagtt tgacaatag gtttcctcta ttccaagcca acacaccacc 3060 agcggttttg ctcgatcagc taagacctt gataaca actctgtatg ctgcatcaca 3120 gatggtcca atactcagg taaatgcatc agctcagggt gctgctatgt ctgtacttcc 3180 caaaaaatttc gaagtaatg caactgtggc acttgatgaa tacagcaac tgactttga 3240 caagttaacg gtttgcgatg ttaaaacagt tattttgaca accatgaaac catatgggat 3300 ggtgtcaaaa ttgtgagtt cagccaaatc agttggcaac agacacatg atctaattgc 3360 actgtgtgac ttcatggacc tagagaaaa atacctgtg acataccag cattcataaa 3420 gtcagtttca atcaagaga gtgagtcagc cactgttgaa gctgcaataa gcagtgaggc 3480 cgaccaagca tcacacaag ccaaaattgc accctatgca ggactaatca tgatcatgac 3540 catgaacaat ccaaaggta tattcagaa actaggagct ggaacacag tgatagtaga 3600 gctaggggca tatgttcaag ccgagagcat cagcaggatc tgcagagct ggagtcacca 3660 aggaaaga tatgtacta atccagata aaaatactg tcctaatca taattgctta 3720 tataatctta aagatcaatg agcttatt tatagtta taaaaaattt tagatagg 3780 aaggtatta tagaagcgg ghaagtaaa atgtcttgg aaagtgatga ttatcattc 3840 gttactcata acacctcagc acggactaa ggaagttat ttagagaat catgtagtac 3900 tataactgaa ggatatctca gtgttttaag aacaggttgg tacaccaatg tctttacatt 3960 agaagttggt gatgttgaaa atcttacatg tactgatgga cctagcttta tcaaacaga 4020 acttgaccta accaaagtg ctctaagaga acccaaaca gtttctgctg atcagttagc 4080 gagagagaa caattgaaa atcccagaca atcaggtttt gtcctaggtg caatagctct 4140 tggtgttgcc acagcagcag cagtcacagc aggcattgcg atagccaaaa ccataggct 4200 tgagagtgaa gtgaatgcaa tcaaggtgc tctcaaaca accaatgagg cagtatccac 4260 actaggaaat ggagtgcgag tcctagccac cgcagtaaga gagctgaaag aatttgtgag 4320 caaaaacctg actagtgcaa tcaacaa aaatgtgac attgctgatc tgaagatggc 4380 tgtcagcttc agtcaattca acagaagatt cctaaatgtt gtgcggcagt tttcagacaa 4440 tgcagggata acaccagcaa tatcattgga cctaatgact gatgctgagc tggccagagc 4500 tgtatcatac atgccaacat ctgcaggaca gataaaacta atgttagaga accgtgcaat 4560 ggtgaggaga aaaggatttg gaatcttgat aggggtctac ggaagctccg tgatttacat 4620 ggtccagctg ccgatctttg gtgtcataga tacaccttgt tggataatca aggcagctcc 4680 ctcttgttca gaaaaagatg gaaactatgc ttgcctccta agagaggatc aagggtggta 4740 ttgtaaaaat gcaggatcca ctgtttacta cccaaatgaa aaagactgcg aaacaagagg 4800 tgatcatgtt ttttgtgaca cagctgcagg gatcaatgtt gctgagcaat caagagaatg 4860 caacatcaac atatctacaa caactaccc atgcaaagtc agcacaggaa gacaccctat 4920 cagcatggtt gcactatcac ctctcggtgc tttggtggct tgctacaaag gggttagctg 4980 ttcaattggc agtaatcggg ttggaataat caaacaacta cctaaaggct gctcatacat 5040 aactaaccag gacgcagaca ctgtaacaat tgacaacact gtgtatcaac taagcaaagt 5100 tgagggtgaa cagcatgtaa taaaagggag accagtttca agcagtttcg atccaatcaa 5160 gtttcctgag gatcagttca atgttgcgct tgatcaagtc tttgaaagca ttgaaaacag 5220 tcaagcacta gtggaccagt caaacaaaat tctgaacagt gcagaaaaag gaacactgg 5280 cttcattatt gtaataattt tgattgctgt tcttgggtta accatgattt cagtgagcat 5340 catcatcata atcaaaaaaa caaggaaacc cacaggggca cctccagagc tgaatggtgt 5400 taccaacggc ggttttatac cgcatagtta gttaattaaa aaatgggaca aatcatcatg 5460 tctcgtaaag ctccatgcaa atagtaagta cggggcaagt gcaacagggg aagtgagtgc 5520 agattcaacc aaattactg gagttggcct gataggtatt tattgttaag atcaaattat 5580 ctcttgaatc agcttttaag aaacactgat aaggctgatg gtttgtcaat aatatcagga 5640 gcaggtagag aagacaggac tcaagacttt gttcttggtt ctactaatgt ggttcaaggg 5700 5760 ataaaacagc tacaagaaat agaagtaaga caggccagag ataaataagct ttctgacagc 5820 aaacatgtgg cactcacaa cttgatatta tcctatatgg agatgagcaa aactcctgca 5880 tccctgatta atacctaaa gaactacca agagaaaaac tgaaaaaatt agcgaaatta 5940 atattgatt tatcagcagg aactgatat gactctcat atgccttgca agacagtgaa 6000 agcactaatc aagtgcagta agcatggtcc caaattcatt accatagagg cagatgatat 6060 gatgagaat t'aagagac actgtctgat gggatagta atcacac 6120 caatattac agttgttatt tagaaatat agaataata tatgttaag cttacttaag 6180 tttaaaaa aataatagaa tgggaataat ghaatgaaa acattagatg tcataaaag 6240 tgacggatcc tcagaaacat gtaatcaact CAaaaaata ataaaaaac actcaggtaa 6300 attgcttatt gcattaaaac tgatattggc cttattgacg ttttcacag taacaattac 6360 tgttaactat aaaagtag aaataattt gcaggcatgt cattaaaaa atgaatcaga 6420 haaaaaggac aaagctaa ataccacatc aaaaacaatc agacccattc ctgatctaaa 6480 tgcagtacag tacttgaaaa ggctgattca gaacacacc aacttgtca taaagacag 6540 agatacctgt tggaatac acacgaatca atgcacaat aaaaataat ataagttctt 6600 atgttttggg tctatgaatt cacaatac agactgtgaa gaactacag ttttatgcga 6660 caaaaagtca aaaaaccatga cagaaaaaca taggaagca gagtgtcact gtctacatac 6720 aaccgagtgg tggtgttatt atctttaga gaaaactcag ttttcacat taaaatcaga 6780 aaatcata tctagatcta tattatatat agtttagtta tttaaaact ctaaatattg 6840 tcgacttc acacactt gcggtcatat gcaataatca atggtcaac cactgttgca 6900 aactcaccta taatataatc actgagtaat aaaaacaag aaatgggac aagtggccat 6960 ggaagtaaga gtggagaaca ttcggcaat agacatgttc aaagcaaga tgaaaaaccg 7020 tataagagt agcaagtgct atagaatgc tacactgatc cttattggat spacaccact 7080 aagtatggca ctcaattt ttttaatcat tgattgca acatcaaaa acatgaccaa 7140 agtggaacac tgtgtcaata tgccgccggt agaaccaagc aagagaccc caatgaccctc 7200 tgcagtagac ccaaacacca aaccaatcc acagcaggca accagctga ccacagagga 7260 ttcacatct ctagcagcaa ccctagagga ccatctacac acagggacaa ctccaacacc 7320 agatgcaca gtctctcagc aaaccacaga cgagcacaca acactgctga gatcaccaa 7380 cagacagacc accaaaaaaaagccaacc aggaaaaaaaaaga 7440 aaccacaact cgaaccacaa gcacagctgc aaccaaaca ctcaacca ccaaccaac 7500 tagcaatgga agaggcaa ccacacatc tgccagatcc agaacaatg ccacacatca 7560 aagcagcgat siacaaccc aggcagcaga cccaagctcc caatcacaac atacacagaa 7620 aagcacaaca acacaca acacacac atcttctcca agtagttaac aaaaaaacta 7680 taaaatacc atgaaaacca aaaaactaga aaagttaatt tgaactcaga aagaacaca 7740 aacactatat gattgtttg agcgtatata ctaatgaaat agcatctgtt tgtgcatcaa 7800 taataccatc attattag aaataagaag agctaaat tcaagggaca ataacaatg 7860 gatccgtttt gtgaatccac tgtcaatgtc tatctcctg attcatatct caaggagta 7920 atatctttca gtgaaaccaa tgcaattggc tcatgccttt tgaaagacc ctactttaaa 7980 aaagataaca ctgctaaagt tgctgtagaa aaccctgttg ttgaacatgt cagacttaga 8040 aatgcagtca tgaccaaaat gaagatatca gattataaag tggttgaacc aattaatatg 8100 cagcatgaaa taatgaaaaa tatacacagt tgtgagctca cattattaaa acaattctta 8160 acaagaagta aaaacattag ctccctaaaa ttaagtatga tatgtgattg gttacagtta 8220 aaatccacct cagataacac atcaattctt aattttatag atgtggagtt tataccccgtt 8280 tgggtgagca attggtttag taactggtat aatctcaata aattaatctt agagtttaga 8340 agagaggaag tataagaac tggttcaatt tttgcagat cactaggcaa gttagttttc 8400 attgtatcat cttatgggtg tgtagtaaaa agcaacaaaa gtaaaagagt aagttttttc 8460 acatataacc aactgttaac atggaaagat gtgatgttaa gtaggttcaa tgcaaacttt 8520 tgtatatggg taagtaacaa cctgaacaaa aatcaagaag gactaggatt tagaagtaat 8580 ctacaaggta tgttaactaa taaattatat gaaactgttg attatatgtt aagtctatgt 8640 agcaatgaag ggttctcact agtgaaagag ttcgaaggct ttatatgag tgaaattctt 8700 aaaattactg agcatgctca attcagtact aggtttagga atactttt aaatggttg 8760 actgaacat tatcaatgtt gaagctaa aacagatcta gagttctttgg cactatatta 8820 gaaaacaatg attacccat gtatgaagta gtacttaat tattaggga cactttgaa 8880 agtataaaat tattatta cagaattta gaaatgctg summer tattattc 8940 agaattttg vakaccctat ggtagatgag agggaagcaa tggatgctgt taattaat 9000 aatgagatta caaaatttct taactggag agcttacag aactaagagg agcatttata 9060 ctaagatta t'aaagggtt tgtagattat aaaaagat ggcctaaat t'agaattta 9120 aaagtgctca gtaaagatg ggttatgtat ttcaaagcta aaagttaccc tagccaactt 9180 gagctaagtg tacagattt tttagaactt gctgcagtac aattcgaaca ggaatttct 9240 gtccctgaaa aaaccaatct tgagatgta ttaatgata aagcaatc tccaccaaaa 9300 aagttaatat gtcggtata tccaaaaat tatctactg aaattataaa aaatcaat 9360 ttagagagg tcttcaatgc aagtgacagt ttagaachga ggagagtctt agaattttac 9420 ttaaaagatt gcaatttga tcaaaagac ctcaacgtt atgtaacta acagagta 9480 ctaaatgaca aagaccacat tgtctcatta actgggaag aagagaatt aagtgtaggc 9540 aggatgtttg caatgcaacc tggcaacaa agacaacaac agatactagc cgagaaactt 9600 ttagctgata atattgtacc ctttttccca gaactttaa caaagtatgg tgacttggat 9660 ctccaaagaa tttaggaat gaatcagaa ctttcttcca ttaaaactag gaagaatgat 9720 agttacaaca attatattgc aagagccctc atagtacag acctaagtaa attcaatca 9780 gcctttagat atgaaccac agctatctgc gcagacgtag cagatgagtt acatggcacg 9840 caaagcttat ttgttggtt acatcttatt gttcccatga ccacaatgat atgtgcatac 9900 agacatgcac caccagaaac aaaggggggg tatgattag aaaaataga agaghaagt 9960 gggctataca gataccatat gggaggtt gaagggtggt gtcagaagtt atggacaatg 10020 gaggcgatat ccttgttaga tgtagtatct gttaagactc gttgtcagat gaccctcta 10080 ttaaacggag acaatcaatc atagatgtc agtaaaccag taaaattgtc tgaaggtata 10140 gatgagtaaagcagatta tagcttagca atttaaatgc ttaagagat aagagatgcc 10200 tataaaaaca ttggccataa actcaagaa ggtgaaacat atatatcaag agatcttcaa 10260 tttataagta aggtgattca atctgagggg gtcatgcatc ctaccccat aaaaaagata 10320 ttaagggtag gtccctggat aaatacaata ctagatgaca ttaaaactag tgcagaatca 10380 ataggtc tgtgtcaaga actagagttc agaggagaaa gtatacttagt tagcttgata 10440 ttaggaatt tctgctgta taacttatac atgcatgt CAAACAGCA tccgttagct 10500 ggaaaacaac tgtttaaca attgaacaa acactacat ctgtgcaaag atttttgag 10560 ctgaagaag aaatgatgt gttgaccta tggatgata taccaatgca gtttggaggg 10620 ggagacccag tagtttttta cagatcttt tacagaagga ctcctgattt cttgactgaa 10680 gcaatcagcc atgtggatt actgttaaaa gtttcaaca atattaaaaa tgagactaag 10740 atacgattct ttaaagcctt attatctata gaaaagaatg aacgtgctac attackaca 10800 ctaatgagag acccccaggc ggtaggatcg gaagacaag ctaagtaac aagtgatata 10860 atagaacag cagttactag catactgagt ctatctccga atcagctatt ttgtgatagt 10920 gctatacact atagcagaaa tgagagaa gtagggatca ttgcagacaa catacacct 10980 gtttatccctc acggattgag agtgcttat gatcactac cttttcataa ggctgaaaag 11040 gttgtcaata tgatatcagg tachaagctct attackactacc tattgcag aacatctgct 11100 atcaatggtg aagatattga tagagcagtg tctatgatgt tagagaactt agggttgtta 11160 tctaggatat tgtcagtaat attatagt atagaatac cattaagtc caatggcaga 11220 ttgatatgct gtcaatttc tagactttg agagaaaaat catggacaaa tatggaaata 11280 gtaggagtga catctccaag tattgtaca tgtatggatg tgtgtatgc gactagttct 11340 cattaaaag gaatattat tgaaaaatttc agtactgaca agaccacaag aggtcagagg 11400 ggaccaaaaa gcccttgggt aggatcaagc actcaagaga aaaaattagt tcctgtttat 11460 aaaaaaaa ttcttcaaaaaaaaaaa gagcaactgg aagcaatagg aaaaatgagg 11520 tggtgtata aaggaactcc agggctaaga agattgctca aagatttg cataggaagt 11580 ttaggtatta gcttaaatg tgtaaaacct ctattaccaa gatttatgag tgtaaacttc 11640 ttacataggt tatctgttag tagcagaccc atggaattcc cagctctgt tccagcttat 11700 aggacaacaa attaccactt tgacactagt ccaatcaacc aagcattaag tgagaggttc 11760 gggaacgaag acattaatct agtgttccaa atgcaatca gctgcggaat tagtataatg 11820 agtgttgtag aacagttaac tggtagaagc ccaaacaat tagtcttaat ccccaatta 11880 gagagatag atattatgcc ccctcctgta ttcaggaa attcatta taaactagtt 11940 gataaaaay cctccgatca acacatctc agtcctgaca aaatagacat attackacacta 12000 gggaagatgc tatgcctac tataaaaggt caaaaactg atcagttctt aataagaga 12060 gaaaactatt tccatggaaa taatttaatt gatctttat ctgcagcact tgcatgccat 12120 tggtgtggaa tattacaga acagtgtgta gaaaacaata tctttaggaa agactggggt 12180 gatggttca tatcagatca tgccttcatg gatttcaaga tattctatg tgtatttaaa 12240 accaaacttt tatgtagttg gggatcccaa gggaaaaatg tAAagatga agatataata 12300 gatgaatcca ttgacaattt attagaatt vakaacactt ttggagaat gttcagcaaa 12360 gtcatgtttg atcaaggt CAaaaaaga atatgttat atgatgtaaa attcctatca 12420 tagtaggtt attaggatt taaaaactgg attagagc attagagt agtagattg 12480 catgaagtac cctggattgt caatgctgaa ggggagctag tgaattaa accaatcaa 12540 atttattgc agttaataga aaagtcta tctttaagaa taactgtttt gattataca 12600 gataggcac atgctcttac acgattaatt aggaagaat tgatgtgtga taatgcactc 12660 tttaatccaa gttcatcacc atgtttagt ctaactcag ttattgatcc tacacacag 12720 ctagactatt ttcctaggt atatttgaa aggttaaaaa gttatgacac cagttcagac 12780 tachaaaag ggaattac aagaaattac atgacattat taccatggca gcacgtaac 12840 aggtataattt ttgtctttag ttcacagga tgtaaaatca gcttgaagac atgcatcggg 12900 aaattgataa aggacttaaa ccctaaggtt ctttacttta ttggagaagg agcaggtaac 12960 tggatggcaa gaacagcatg tgagtatcct gacataaaaat ttgtatatag gagtttaaag 13020 gatgatcttg atcaccatta cccattagaa tatcaaaggg taataggtga tttaaataga 13080 gtaatagatg gtggtgaagg attatcaatg gagaccacag atgcaactca aaagactcat 13140 tgggacttga tacacagaat aagtaaagat gctttattga taacattgtg tgatgcagaa 13200 ttcaaaaaca gagatgattt ctttaaaatg gtaattcttt ggagaaaaca tgtattatca 13260 tgtagaatct gtacagctta tggaacagat ctttacttat ttgcaaagta tcatgcgacg 13320 gactgcaata taaagttacc attttttgta aggtctgtag ctacttttat tatgcaagga 13380 agcaaattgt caggatcaga atgttacata cttttaacat taggtcatca caataatctg 13440 ccatgtcacg gagaaataca aaattccaaa atgagaatag cagtgtgtaa tgatttccat 13500 gcctcaaaaa aactagacaa caaatcaatt gaagcaaact gcaaatctct tctatcagga 13560 ttaagaatac caataaacaa aaaagagtta aatagacaaa agaaactgtt aacactacaa 13620 agcaatcatt cttccatagc aacagttggc ggaagtaaga ttatagaatc caaatggtta 13680 aagaataaag caagtacaat aattgattgg ttagagcata tcttgaattc tccaaaaggt 13740 gaattaaact atgatttctt tgaagcatta gagaacacat atcccaatat gatcaagctt 13800 atagataacc tgggaaatgc agagataaaa aaactaatca aagttaccgg gtatatgctt 13860 13920 gatcatctaa cagtttaatt gaccattagt taattaaaaa ttataaatta gtaactaatt 13980 gataaaaaat aagaaattga aattgaatgt atacggtttt tttgccgt 14028

Claims

1. An attenuated virus strain derived from human metapneumovirus strain rC-85473, which has a genome sequence shown in SEQ ID NO: 2 or SEQ ID NO:

3.

2. The attenuated virus strain according to claim 1, further comprising at least one exogenous gene.

3. The attenuated virus strain according to claim 2, wherein the exogenous gene encodes a viral antigen.

4. Use of the attenuated virus strain according to claim 2 as an in vitro expression vector of at least one exogenous gene in a target cell of a human metapneumovirus.

5. Use of the attenuated virus strain according to claim 1 in the preparation of a medicament for preventing infection caused by human metapneumovirus.

6. A vaccine composition comprising at least one attenuated virus strain according to claim 1, and optionally an adjuvant, in a pharmaceutically acceptable carrier.

7. The vaccine composition according to claim 6, characterized in that The vaccine composition is in a pharmaceutical form intended for administration by inhalation.

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