Therapeutic composition of camel milk

The Applicant hereby incorporates by reference the sequence listing contained in the ASCII text file titled 32490.44_Sequence_Listing_ST25, created on Jul. 3, 2017, and having 64 KB of data.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of South African Patent Application No. 2016/07084, filed on Oct. 14, 2016.

BACKGROUND OF THE INVENTION

1. Field of the Invention

A therapeutic composition of camel milk relates to a composition useful for medical treatment, and particularly, to a composition useful for the treatment of HIV/AIDS.

2. Description of the Related Art

Infection with Human Immunodeficiency Virus (HIV), a pathogenic retrovirus, can cause Acquired Immunodeficiency Syndrome (AIDS). AIDS is a major global health emergency, and is the sixth leading cause of early death in the world and the third in low-income countries. AIDS is considered an important cause of morbidity and mortality among young people. Moreover, it has reduced the life expectancy at birth in the African continent to 49 years from 53 years.

AIDS was first recognized in the United States in 1981 and has become one of the biggest problems facing the world today. A total of 36.9 million people were infected by HIV by the end of 2014. It has also been observed that the annual rate of new infection with HIV in the entire human population is not declining as 2 million people became newly infected with HIV in 2014 globally.

There are more than 21 million people who died from AIDS in the last decade. Although the rate of death due to AIDS has begun to drop in some nations, by an annualized rate of 2.8% between 2000 and 2013 as in the United States, primarily through the recent use of combination drug therapies against HIV infection and/or through the strict religious and moral codes. However, it is estimated that 16,000 people worldwide are being infected daily with HIV. Treatment success also has been limited by poor tolerance of the treatments by patients and the emergence of resistant strains of HIV. Thus, a need exists for an effective HIV treatment that is well tolerated, relatively inexpensive, and easily accessible.

Although great efforts have been dedicated to effective remedial and preventive methods for many years, there is no working vaccine or cure for HIV/AIDS yet. An ideal vaccine should be innocuous and capable of inducing neutralizing antibodies as well as persistent immune responses in the mucous membrane and blood. Many HIV vaccines currently developed in the world are still in the stages of animal trials. Although vaccines against HIV membrane proteins gp160 and gp120 have already moved into first, second, or third stages of clinical trials, the results of the trials are disappointing. Moreover, many vaccines that are effective to prevent HIV infection in small laboratory animals are not necessarily effective in humans. Although the subsequent or the second generation DNA vaccines seem to influence towards both humoral and cellular immune responses regardless of animal models used, researchers suggested that new modified DNA vaccines can be more efficient by broadly activate CD8+ cytotoxic T lymphocytes (CTLs) in larger animal models, compared with previous approved DNA methods. The fact that scientists are making little progress in HIV vaccine research could be attributed to the complexity and variability of HIV genetic materials. Although the use of combination drug therapies against HIV has proven to be effective in many patients, the current drug regimens are far from ideal due to several drawbacks and side effects documented in the literature.

Currently novel therapeutic approaches are being investigated at an encouraging rate and have the potential to improve the odds against the virus. Among them, immunotherapeutic approaches are one of the exciting areas and can be considered as adjunct to combined antiretroviral therapy for improving immune competence and can provide a sustained control of HIV replication.

Understanding the immune responses against HIV infection is essential for immunotherapy and the treatment of HIV/AIDS. Immunotherapy is meant to help the natural immune system in accomplishing control over viral infection. Different immunotherapy configurations have been assessed in either therapy-naive or therapy-experienced HIV-tainted patients throughout the last 20 years. These arrangements or configurations included non-antigen specific approaches, for example, cytokines that invigorate immunity or stifle the viral replication, and additionally antibodies that piece negative regulatory pathways. Various HIV-specific therapeutic vaccinations have additionally been proposed, utilizing inactivated virus in vivo, plasmid DNA encoding HIV antigens, or recombinant viral vectors containing HIV genes. These findings may give an objective and rational for immunotherapy in persons with progressive infection and have inspired an expanding effort to develop new strategies to enhance immunity.

Thus, a therapeutic composition solving the aforementioned problems is desired.

SUMMARY OF THE INVENTION

A therapeutic composition of camel milk can include an herbal composition having solid material or liquid extracts from the solid material of at least one of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. The solid material may include one or more of parts or the whole of the stem, the bark, the flowers and the roots of one or more, but preferably all of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. The therapeutic composition can include camel milk. The camel milk can include camel's milk having anti-HIV antibodies therein (HIV immunized camel milk). The therapeutic composition can include the herbal composition mixed with the camel milk to provide a treated camel milk. The HIV immunized camel milk can be obtained from a camel immunized against HIV. The camel can be immunized against HIV by administering to the camel the modified DNA plasmids of SEQ. ID No.: 3 and SEQ ID No.: 4.

These and other features of the present invention will become readily apparent upon further review of the following specification and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a flowchart showing the steps by which expression vectors were developed, tested, and demonstrated to produce seroconversion in camels.

FIG. 1B is a continued flowchart showing steps by which different combination HIV/AIDS therapies of formulated camel milk may be produced.

FIG. 2 is a graph showing pre-treatment and post-treatment weights for all seventeen patients treated with the treated milk.

Similar reference characters denote corresponding features consistently throughout the attached drawings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A therapeutic composition of camel milk can include an herbal composition having solid material or liquid extracts from the solid material of at least one of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. The solid material may include one or more of parts or the whole of the stem, the bark, the flowers and the roots of one or more, but preferably all of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. For example, the solid material may include parts or the whole of the stem and the bark of Saussurea acrophila Diels and Saussurea ceratocarpa, and parts or the whole of the roots of Aucklandia lappa Decne. The solid material may be in finely divided particulate form and may be dry or wet, e.g. in suspension. The composition may include a mixture of solid material of two or more of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne.

The therapeutic composition can include camel milk. The camel milk can include camel's milk having anti-HIV antibodies therein (HIV-immunized camel milk). The therapeutic composition can include treated camel's milk. The treated camel's milk can include a mixture of camel milk and the herbal composition. The treated camel's milk can include a concentration of about 800 g of the herbal composition per 5-6 liters of the camel milk.

The liquid extract may include liquid solvent extracts from the solid material of one or more, but preferably all of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. The extracts may be obtained by treatment of solid material of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne with a solvent. The solvent may be ethanol. The ethanol may be about 96% ethanol, more preferably 99.9% ethanol. In the case of the composition comprising liquid extracts, the composition may comprise a mixture of liquid solvent extracts from solid material of two or more of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne.

The solid material may comprise one or more of parts or the whole of the stem, the bark, the flowers and the roots of one or more but preferably all of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. More preferably, the solid material may comprise parts or the whole of the stem and the bark of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne, and parts or the whole of the roots of Aucklandia lappa Decne. The solid material can be previously harvested. The harvested material can be formed into finely divided particulate, e.g., powdered, form, e.g., by grinding.

On a dry basis, the herbal composition may comprise from about 10-20% by weight, preferably 14% by weight, of the solid material of Saussurea acrophila Diels or liquid extract thereof, from about 10-20% by weight, preferably 14% by weight, of the solid material of Saussurea ceratocarpa or liquid extract thereof and about 45-75% by weight, preferably 72% by weight, of the solid material of Aucklandia lappa Decne or liquid extract thereof.

The herbal composition may be provided in an aqueous form for use, particularly for use as a medicament. The aqueous form may, for example, comprise from about 0.5 g to about 1 g, preferably about 0.8 g of extract per ml water, i.e. preferably 800 g of extract per liter of water. Of the 800 g of extract, about 112 g is extract of Saussurea acrophila Diels, about 112 g is extract of Saussurea ceratocarpa, and about 576 g is extract of Aucklandia lappa Decne. The water is preferably distilled water. The quantity can be scaled-up according to the requirements of a subject to which it is to be administered, or to the need of the manufacturer.

An effective amount of the therapeutic composition can be administered to a patient in need thereof for medical treatment. The medical treatment can include treatment of human immunodeficiency virus (HIV), HIV/AIDS or prevention of HIV infection. The medical treatment can include treatment for or prevention of any one or more of viral infections, including, but not limited to Epstein Barr virus, Herpes simplex, Herpes zoster, influenza, and cancer. The medical treatment can include administering to a patient in need thereof an effective amount of treated camel milk. The treated camel milk can include about 6.8 g of extract of Saussurea acrophila Diels, about 6.8 g of extract of Saussurea ceratocarpa, about 34.6 g of extract of Aucklandia lappa Decne, and about 300 ml of camel milk.

The composition need not strictly comprise the liquid extracts. It may, in one embodiment, comprise an emulsion or a mixture of solid material of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne.

For commercial and industrialized purposes the composition can be further processed by spray-drying, or other similar drying techniques, and subsequent granulation thereof into particles which can be further encapsulated, tableted or filled into sachets or blister packets as required.

The method of preparing the composition may include combining the solid material of at least one but preferably all of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. The method may include forming a mixture of solid material of at least one but preferably all of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. The method may include a prior step of harvesting the solid material. The method may include another prior step of forming the harvested solid material into finely divided particulate, powdered, form, e.g. by grinding. Thus, the composition of solid material may be one or a mixture of the harvested solid material in finely divided particulate form.

A composition comprising liquid extracts from solid material of at least one of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne can be prepared by forming a mixture of liquid extracts from solid material of at least one of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. This method may include preparing the liquid extracts by treating solid material of at least one of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne with a solvent. The solvent may be ethanol, typically about 96% ethanol, or about 99.9% ethanol.

The solid material used to produce the liquid extracts may comprise one or more of parts or the whole of the stem, the bark, the flowers and the roots of one or more but preferably all of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. The solid material may comprise parts or the whole of the stem and the bark of Saussurea acrophila Diels and Saussurea ceratocarpa, and parts or the whole of the roots of Aucklandia lappa Decne.

Treatment of the solid material with the solvent may be in a mass ratio of 2:1 solvent:material, particularly when the solvent is ethanol.

The liquid extracts may be individually prepared by treating the solid material of one or more of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne separately. The composition may then comprise one of the extracts, or may be prepared by mixing two or more of the extracts.

The liquid extracts may be prepared as a combination of extracts, by preparing a mixture of solid material of two or more of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne and treating the mixture of solid material with the solvent. On a dry basis, the mixture may comprise from about 10-20% by weight, or about 14% by weight, of the solid material of Saussurea acrophila Diels, from about 10-20% by weight, or about 14% by weight, of the solid material of Saussurea ceratocarpa and about 45-75% by weight, or about 72% by weight, of the solid material of Aucklandia lappa Decne.

The therapeutic composition can include a medicament or a precursor to a medicament. The medicament may be treated camel's milk or HIV-immunized camel's milk, i.e., camel's milk obtained from a camel that was immunized using modified DNA inserts of HIV pWT/BaL (SEQ ID NO.: 3) and HIV pNL4-3 (SEQ ID NO.: 4). The HIV-immunized may include milk obtained from a camel that is caused to digest the herbal composition which comprises solid material or liquid extracts of solid material from one or more of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne, and immunized using modified DNA inserts of HIV pWT/BaL (SEQ ID NO.: 3) and HIV pNL4-3 (SEQ ID NO.: 4). This may be accomplished by supplementing the camel's feed, or by intubating the camel with 500-1000 g, preferably 800 g, of the solid material or liquid extracts of solid material from one or more of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. Preferably, immunization occurs prior to causing the camel to digest the herbal composition. About 300 ml of the HIV-immunized camel's milk can be administered to a patient in need thereof three times a day.

The immunized camel's milk may include anti-HIV heavy chain IgG antibodies (hcIgG). hcIgG antibodies are rare, small antibodies, known to be produced by camels. These antibodies have unique features due to their smaller size, the absence of heavy chain constant regions, and the complete absence of the light chain. Due to their smaller size and unique features, hcIgG antibodies may possess improved bioactivity when compared with normal IgG antibodies.

The therapeutic composition may be used in medical treatment, by administering it to a person in need thereof. The therapeutic composition can be processed and formulated in a form suitable for oral, parenteral, or intravenous administration to a patient.

The medical treatment may be medical treatment for treatment of HIV/AIDS or prevention of human immunodeficiency virus (HIV) infection.

The medical treatment may also be medical treatment for treatment or prevention of any one or more of viral infections including but not limited to Epstein Barr virus, Herpes simplex, Herpes zoster, influenza, and cancer.

The treated camel's milk can be formed by mixing the solid material or liquid extracts of solid material from one or more of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. The treated camel's milk may comprise a concentration of about 800 g of the solid material or liquid extracts of solid material from one or more of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne, per about 5-6 liters of the camel's milk. Other concentrations may also find application.

The medical treatment may involve administering the treated camel's milk to a subject in need thereof. The treated camel's milk can be prepared by mixing about 6.8 g of extract of Saussurea acrophila Diels, about 6.8 g of extract of Saussurea ceratocarpa, and about 34.6 g of extract of Aucklandia lappa Decne with about 300 ml camel's milk. The medical treatment may include administering 300 ml of the treated camel's milk to a subject three times a day.

The dosage of the compositions of the invention to be ingested will vary, depending on factors such as severity of the conditions, age, physical condition and body weight of the patient, diet, etc. Subjects may vary considerably depending on the factors indicated above. However, one benefit of the composition of the present invention is that the dosage is not "critical" since the edible compositions of the present invention are holistic in nature and represent dietary supplements in their own right, "over dosing" is not a problem. The individual patient with a particular body weight and life style may readily determine the proper dosage by starting out the general dosage.

As a general guide, it is expected that a camel with a body weight in the range of about 600 to about 1000 Kg would ingest about 500-1000 g/day of the solid material or liquid extracts from the solid material of at least one of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne. Typically, a camel would ingest about 0.18 g of the solid material or liquid extracts from the solid material of at least one of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne per Kg of body weight. It is to be understood that these consumption amounts are only general guides and the proper amounts for individual camels may vary. The amount ingested by a camel has been found to be most effective when administered at a dosage of about 750 g per day, with an effective range of about 500-800 g per day. This is based on a pilot study where a subject is administered about 300 ml of the medicament or HIV-immunized camel's milk three times a day, in which case the camel was intubated with about 800 g of the solid material or liquid extracts from the solid material of at least one of Saussurea acrophila Diels, Saussurea ceratocarpa, and Aucklandia lappa Decne per day. However, it will be appreciated that subjects may be given a higher dosage according to their status and life style with no expected harm or side effects that may result.

The intubated solution administered to the camel may comprise an ethanolic extract of the stem/bark of Saussurea acrophila Diels and Saussurea ceratocarpa in the range of about 10-20% respectively, and preferably about 14% respectively, in water. The intubated solution administered to the camel may further comprise an aqueous extract of the root of Aucklandia lappa Decne in the range of about 45%-75%, and preferably about 72% in water.

Example 1

Method of Preparing Pharmaceutical Compositions

The stem and bark of Saussurea acrophila Diels and Saussurea ceratocarpa, and the root of Aucklandia lappa Decne were harvested, weighed and thoroughly washed in cold water to remove any dirt, soil and other undesirable contaminants.

The washed, harvested material was pre-treated with distilled or de-ionized water in an about 16:1 dry weight ratio of water:harvested material and subsequently dried in an oven. The dried material was ground into a finely divided particulate, powdered form, to provide a powder mixture comprising about 28% (280 g) of Saussurea acrophila Diels and Saussurea ceratocarpa, present in equal amounts (about 14% each), and about 72% (720 g) of Aucklandia lappa Decne. The powder mixture was soaked in about 99.9% ethanol, as extraction solvent, and homogenized for about 10 minutes to provide a solvent/powder mixture.

The solvent/powder mixture was incubated at room temperature for 4 days. The solvent/powder mixture, after incubation, was filtered twice using vacuum to provide a first filtrate and a first retentate. Ethanol was allowed to evaporate from the first filtrate, or extract solution, at room temperature.

The first filtrate was subjected to re-extraction with a re-extraction solvent of about 99.9% ethanol using a shaking water-bath at about 75.degree. C. for about 6 hours, thereby obtaining a re-extraction mixture of the first filtrate and the re-extraction solvent. The re-extraction mixture was filtered, thereby obtaining a second filtrate, or extract solution, and a second retentate. Ethanol was allowed to evaporate from the second filtrate at room temperature.

The yield of this two-step extraction method provided a filtered herbal extract. The filtered herbal extract was yellowish brown.

The filtered herbal extract was kept at about 4.degree. C. The concentration of this filtered herbal extract was adjusted accordingly, e.g. about 800 g thereof was added to one liter of distilled water.

Example 2

Toxicity Testing

Two different species of animals were the subjects of the experiment: rabbits (group-A) and WKY rats (group-B). The animals were obtained from Small Animal House, SQU, Oman. Group-A Rabbits, included ten (10) male rabbits and five (5) female rabbits, each weighing between 800 g to 1300 g. Group-B WKY rats included nine (9) males and six (6) females, each weighing between 300 g to 600 g. Both rabbits and rats were randomly divided to investigate the lethal dosage.

The filtered herbal extract, prepared as described in Example 1, was administered orally to the animals using antistatic at variable dosages to reach a maximum of about 16 g/Kg for each animal group. The concentration of each dose of fluid containing the filtered herbal extract was about 1 g/ml. Accordingly, the higher dosage group at 15 g/Kg had a concentration of about 5 ml of 15 g/Kg, which could be calibrated to suit bigger animals, including humans.

The animals were observed for behavior continuously for a period of two weeks after administration. Observation was conducted hourly at day 1; and during the following days, observation was conducted four to six (4-6) times per day. At the end of the observation period, animals were sacrificed and dissected to examine the eyes, liver, lung, and spleen for any adverse effects.

A separate experiment was performed in parallel to the above animal groups where, a medicament of camel's milk as described below, was also given to each group of animals to evaluate the toxicity status thereof. The volume of the medicament of camel's milk was given according to the animal's capacity on a daily basis for a period of two weeks.

No abnormal behavior was observed in either groups of animals during the observation period. All animals were alive after the two weeks of having been given 16 g/Kg or the medicament of camel's milk. All animals showed normal body weight increase during the two weeks period. Biochemical analysis showed normal range of ALT, AST, CBC, and GGT. Inspection of the eyes, liver, lung, and spleen (after scarification and dissection) showed no extraordinary signs. The results when compared to a general acute toxicity index were normal and no acute toxicity was observed. Based on the results of the toxicity experiment, the weight range of the filtered herbal extract or diffused composition (i.e. a mixture, or solution, of the composition of the extract in water) is preferred to be 10-20 g/Kg of animal weight for each extract.

In the tables below, "composition" means the filtered herbal extract described in Example 1, on a dry mass basis dissolved in distilled water at about 973.5 g/5000 ml.

TABLE-US-00001 TABLE 1 Rabbits Received 16 g/Kg Orally Rabbit No. Weight (Kg) Dose in grams Dose in mL 1 0.8 12.8 68.2 2 1.4 22.4 119.4 3 1.5 24 127.9 4 1.15 18.4 98.1 5 1.05 16.8 89.5 6 1 16 85.3 7 1.15 18.4 98.1 8 1 16 85.3 9 1.3 20.8 110.9 10 0.9 14.4 76.8

TABLE-US-00002 TABLE 2 WKY Rats Received 16 g/Kg Orally Rat No. Weight (Kg) Dose in grams Dose in mL 1 0.3 4.8 25.6 2 0.25 4 21.3 3 0.25 4 21.3 4 0.4 6.4 34.1 5 0.25 4 21.3 6 0.3 4.8 25.6 7 0.25 4 21.3 8 0.25 4 21.3 9 0.5 8 42.6 10 0.5 8 42.6

Example 3

Developing Modified HIV Provirus Plasmids

For generation of anti-mouse HIV antibody, conventional immunization was performed in parallel with DNA immunization using fifty (50) Balb-C mice. Mice were boosted 3 times at 2 weeks interval. Blood was collected before and after boosting for seropositivity analysis using ELISA kit.

For generation of anti-camel HIV antibody, conventional immunization was performed in parallel with DNA immunization using female camels. Each camel was boosted 6 times at two week intervals, using different delivery routes. Blood was collected before and after boosting for seropositivity analysis using ELISA kit.

For analysis of the generated anti-HIV camel antibodies, anti-HIV camel antibodies secreted in the camel milk and orally taken by HIV/AIDS patients was analyzed. Assessment of the impact of the treatment modality, virus load, and CD4.sup.+T cell count of HIV/AIDS patients was made for three months.

Both HIV/pwT/Bal vector (SEQ ID NO.:2) and HIV/pNL/4-3 vector (SEQ ID NO.:1), were donated from the NIH-AIDS Research and Reference Reagent Program, USA. Prior to the amplification step, both vectors' DNA insets were modified using techniques generally known in the art. All experiments were performed using the modified vectors "HIV/pwT/Bal/Mod" and "HIV/pNL/4-3/Mod" (corresponding to SEQ ID NO.:3 and SEQ ID NO.:4, respectively). Transformed "one shot chemically competent" E. coli colonies were amplified in about 500 ml LB cultures and the plasmid DNA constructs were purified chromatographically using Qiagen Megaprep kits, according to manufacturer's instructions (MegaPrep; Qiagen, Hilden, Germany). The purified DNA was stored at -20.degree. C. until ready for use.

Before proceeding to DNA immunization, the ability of the HIV/pwT/Bal/Mod vector (SEQ ID NO.:3) and HIV/pNL/4-3/Mod vector (SEQ ID NO.:4) to express recombinant HIV Provirus was confirmed in vitro by transfection of mammalian COS-7 and 293 T cells (ATCCC, Germany) with DNA or vector vaccines alone (Control plasmids lacked the HIV inserts). The main reagents used in this transfection/expression procedure were FUGENE 6 (Roche) and/or Lipofectamine 2000 (Invitrogen, CA). The transfection procedure was performed according to manufacturer instructions.

Monitoring of virus production was performed every 2 days by measuring p24 antigen concentration in the culture fluid starting on day 5 using the p24 antigen ELISA kit. Culture medium was changed fully and regularly every two days after day 5 when completing the p24 antigen measurement. Three or more harvests were performed when the p24 concentration reached greater than about 10 ng p24 per milliliter. Each harvested culture fluid sample was saved, filtered through a 0.45-.mu.m pore filter, and treated with about 50 units of Benzonase per ml of filtered HIV viral stock for about 25 minutes at about 37.degree. C. to remove contaminating plasmid DNA. Previous studies proposed that the use of Benzonase has no impact on the viral ability to infect or to bind to its specific targets, and therefore any effect of Benzonase on infectivity would be only slight. Virus stocks were frozen at -80.degree. C. to halt Benzonase activity and/or until use.

Viral titers were calculated using the TCID50 assay, according to the method of Reed and Muench (1938). Samples with less than about 0.2 ng p24/ml were considered negative for the calculation.

Example 4

HIV Provirus Immunization in BALB/c Mice

Female BALB/c Mice [a total of 10 mice] were immunized by intramuscular injection with about 1.0.times.10.sup.8 infection units (equivalent to 1.times.10.sup.6 TCID50 of concentrated vector stocks) of HIV recombinant viruses over a time course of 4 weeks post-immunization and boosted 1 month later with the same dose of recombinant viruses. The control group (a total of 10 BALB/c mice), were immunized with the same dose but with vectors alone. For seroconversion analysis, blood samples were collected via the periorbital route 4 weeks after the first inoculation, then at 4 weeks after boosting.

Example 5

DNA Immunization of BALB/c Mice

The HIV/pwT/Bal/Mod (SEQ ID NO.:3) and HIV/pNL/4-3/Mod (SEQ ID NO.:4) vectors and the control pwT/Bal and pNL/4-3 plasmids were precipitated onto 1.6 .mu.m gold beads and loaded into half-inch lengths of plastic tubing according to the manufacturer's instructions (BioRad, Hercules, Calif.). The amount of gold powder and DNA were acclimated to give pieces of tubing (`shots`) containing 1 mg DNA/0.5 mg gold. The abdomens of thirty anaesthetized, 8.+-.10-week-old female BALB/c were shaved and subjected to three `shots` expelled under a burst of helium gas at about 350 psi into the epidermal layer using the Helios GeneGun (BioRad). Mice were divided into three groups of 10 mice in each group, immunized with about 3 mg of the HIV/pwT/Bal/Mod (SEQ ID NO.:3) and HIV/pNL/4-3/Mod (SEQ ID NO.:4) DNA constructs or the vectors alone, on three occasions, two weeks apart and their sera were examined four weeks later.

For each of the HIV/pwT/Bal/Mod (SEQ ID NO.:3) and HIV/pNL/4-3/Mod (SEQ ID NO.:4) DNA constructs and the empty plasmids vectors, about 0.5 mg of each in saline was adjusted to about 100 .mu.g with distilled water and about 25 .mu.l was injected into each rectus femoris muscle of 30 8.+-.10-week-old female BALB/c mice with a 25 gauge needle on three occasions, two weeks apart. Clinical parameters for each animal were inspected, including weight of animals, temperature and swelling of lymph nodes. Blood samples were drawn prior to each vaccination, then at week six and week eight. The immunization sites were examined for any adverse reactions.

Evaluation of antibody seroconversion was conducted using ELISA. Ninety-six-well plates (ICN, Costa Mesa, Calif.) coated with recombinant gp140 (0.5-1 .mu.g/ml in PBS) or gp120 (1 .mu.g/ml) in 0.05 M carbonate were buffered overnight at 4.degree. C. The plates were washed 3 times with TST (Tris (0'01 m, pH 8.5), saline (NaCl, 0.15 m) and Tween 20 (0.1%)) and blocked for 1 hour with 5% fat-free dried milk (Carnation, Wirral, UK) in TST at 37.degree. C. Individual sera from all inoculated BALB/c mice, including the control groups, were diluted 1:500 with 5% milk and applied, in duplicate, to the plates overnight at 4.degree. C. The plates were washed 3 times with TST and horseradish peroxidase-(HRP)-conjugated anti-mouse immunoglobulin reagents (Nordic, Tilburg, The Netherlands), diluted to 1:1000 with TST, and then added for 2 hours at 37.degree. C. The plates were washed 3 times and the assay developed with a 0.02% solution of the chromogenic substrate 2,2'-azino-bis (2-ethylbenzthiazoline-6-sulphonic acid; Sigma, Poole, UK) in phosphate-citrate buffer (pH 4.0) containing 0.015% hydrogen peroxide and the optical density (OD) were read at 405 nm.

Immunoblotting results were analyzed using one and two-dimensional SDS-PAGE immunoblotting assay. Western blot or protein immunoblot from the above gels were used to transfer HIV fractionated recombinant proteins to nitrocellulose and molecular weight markers visualized by reversible staining with Ponceau S.

The filters were blocked with 5% non-fat milk for 1 hour at room temperature, washed 3 times with TST and diluted (5% milk) sera are added overnight at 4.degree. C. The filters were washed 3 times with TST and incubated for 2 hours at room temperature with HRP- or alkaline phosphatase-conjugated goat anti-mouse IgG, or anti-rabbit IgG (1:1000; Sigma Aldrich). Unbound secondary antibody was washed off and the specific antigen-bound antibody was visualized with the appropriate substrate solution.

Seroconversion of the anti-HIV antibodies was confirmed. DNA immunization with the HIV Provirus DNA constructs was found to generate higher titer seroconversion than immunization with the recombinant HIV Provirus of Example 4.

Example 6

Generation of Anti-HIV Camel Antibodies

The methods for DNA immunization of Example 5 were followed to immunize female camels with slight modifications based on the vector/recombinant HIV Provirus and the period of immunization. Seven female well-milking camels (healthy and infection free, as checked by veterinary doctor) were used. The first 5 camels were immunized with about 10 mg each of the HIV/pwT/Bal/Mod (SEQ ID NO. 3) and HIV/pNL/4-3/Mod DNA (SEQ ID NO.: 4) constructs, using the GeneGun protocol, on six occasions, two weeks apart. Their sera and milk were then examined 12 weeks later. The same group was further injected "i.m." with 1 ml of both the HIV/pwT/Bal/Mod (SEQ ID NO. 3) and HIV/pNL/4-3/Mod DNA (SEQ ID NO. 4) constructs adjusted to about 20 mg in distilled water, into each rectus femoris muscle of the same camel with an 18 gauge needle. The same camels were used for the two routes to emphasize the delivery intake of the vector/DNA constructs and to initiate different orientation of the Th1 and Th2 immune responses. The second group comprising the last two female camels was non-immunized and used as a control group.

Confirmation of seroconversion of the anti-HIV camelid antibodies was analyzed in the same manner as in Example 5. HIV neutralization assays were also performed as described below (prior to performing the clinical trial) as an efficacy procedure of the generated anti-HIV camelid antibodies. Both the camel serum and camel milk were used for these analyses. Furthermore, techniques generally known in the art were used to generate a secondary antibody against the generated anti-HIV camel IgG antibodies (for use in the ELISA and immunoblotting assays, conjugated with a proper substrate such as HRP). The secondary antibody was generated by using serum from an unimmunized camel to immunize a goat, and isolating the goat anti-camel antibodies.

Example 7

Sero-Neutralization

In vitro HIV neutralization assessment was performed using the indicator cell line HEK293T. Briefly, HEK293T cells were seeded into 24-well plates (20,000 cells per well) and incubated for 24 hours at 37.degree. C. in DMEM-10% FCS supplemented with hygromycin plus G418 to select and maintain the eukaryotic cells. Culture medium was changed with 30 .mu.l of DMEM-10% FCS-DEAE Dextran (10 .mu.g/ml) and cells were then incubated further for 30 minutes at 37.degree. C. HIV Provirus (about 0.5 ng per well) was then incubated with 1:30 diluted camel milk and/or sera in 30 .mu.l of FCS-free culture medium at 37.degree. C. for 45 minutes and the mixtures virus-sera/milk were added to the cells in duplicate. Two hours later 100 .mu.l of DMEM-10% FCS was added to each well. Subsequently wells were incubated for 48 hours and then .beta.-galactosidase activity was measured using a chemiluminescent reporter gene assay (Roche). The mean neutralization for milk and sera tested were compared and reported to the value recorded in wells of the control containing serum of non-immunized camel at the same dilution that served as reference for 100% infection.

These tests confirmed that (i) DNA immunization is the most efficient DNA-delivery route in contrast with the recombinant HIV immunization and (ii) also showed that satisfactory anti-HIV titers are only achieved after the final immunization. The generated anti-HIV antibodies were found to be significant and showed 100% efficacy to neutralize the HIV Provirus.

Example 8

Clinical Testing

Seventeen (17) patients of Arab, Indian and African origin [excluding +Ve and -Ve controls] were studied and followed up for six months [two months of which are under the invented therapy]: Patients were excluded if they had administered any investigational drug less than 6 weeks prior to the first dose of the medicament of the invention. The selected patients had never been prescribed any HIV/AIDS drugs. Patients' immune function was abnormal (i.e., the CD4.sup.+ cells to CD8.sup.+ cells ratio is under 1, the number of CD4.sup.+ T cells is under 200/mm.sup.3). The average age of each patient is above 16 years. No pregnant women were chosen. The patients agreed voluntarily to use the medicament of the invention.

Patients were required to take an ELISA test to indicate the presence of the HIV virus. Patients underwent testing with monoclonal antibody reagent and flow cytometry (FAC-SCAN) before and subsequently every two, four weeks and then after two and six months and after treatment ceased to calculate the rate of CD4.sup.+ to CD8.sup.+ cells, and the quantity of CD4.sup.+ cells (in cm.sup.3) as index to measure the immune function of patients under the treatment. Complete diagnostic records were recorded, including physical sign and pulse condition.

The criteria for measuring curative effect were: if testing PCR and/or RT-PCR is negative, the immune function increased and/or recovers to normal (i.e., ratio of CD4.sup.+ to CD8.sup.+>1 and/or the percentage of the CD4.sup.+ T cells are increased in correlation to the total WBC, and if there are no symptoms, no physical signs and no opportunistic infections. The criteria for measuring evident effect were: if HIV appears negative or undetectable, the immune function improved dramatically (i.e., ratio of CD4.sup.+ and CD8.sup.+>0.2 but the number of CD4.sup.+ T cells is .gtoreq.200/mm.sup.3), if opportunistic infections were basically removed (disappeared) and the symptoms and physical signs recovered fundamentally back to normal. The criteria for measuring some effect were: if testing PCR for HIV appeared positive, the immune function improved (ratio CD4.sup.+ to CD8.sup.+>0.2, the quantity of CD4.sup.+ T cells is beyond 40/mm.sup.3), opportunistic infections improved and the symptoms and physical signs were relieved. The criteria for measuring no effect were: if there were no dramatic changes to the index of immune function, or if there was a decrease of immune function in the treatment.

A comprehensive analysis of the curative effect according to the clinical symptoms and immune state before and after treatment are studied and recoded. The total number of patients was 17 (4 females and 13 males). Their infection was confirmed by both ELISA and Quantitative PCR. The average age was about 33 years, with the oldest being 55 years old, and the youngest being 24 years old.

Pretreatment status was determined by physical examination. All patients showed common symptoms of depression, weakness, stegnosis and weight loss associated with loss of appetite, and some patients occasionally developed fever.

In accordance with the present techniques, camel's milk was used from a camel which had been DNA immunized against HIV and which had ingested, via intubation, the filtered herbal extract of Example 1. The immunized camel's milk from these camels was the only source of treatment administered to the patients. Each patient consumed about 300 ml of the immunized milk three times a day on a daily basis. Immunized milk was administered until HIV viral load was undetected for two-tested occasions or when a -Ve viral load was shown. Patients were then asked to stop taking the medicament of the invention for 16 weeks and were tested for HIV from the start of the 16 week period. Two patients "as control group 1" were administered the same dose of milk but from an unimmunized camel with no intake of the composition of the invention.

Detailed hematological and biochemical tests were conducted on each patient. Table 2 shows the most significant clinical data obtained from the pilot studies on the effectiveness of the herbal composition and camel milk in protecting HIV/AIDS patients from the debilitating effects of the disease.

Ten (10) ml blood samples were taken from the patients each time before, during, and after the treatment and further tested using ELISA, Viral Load (VL), CD4.sup.+ cells, CD8.sup.+ cells, White Blood cells (WBC) and other screening profile such as full blood count (FBC), liver function, renal function, fasting lipid profile, blood glucose. Moreover, physical examination including recording weight, was performed each time before, during, and after the treatment.

Of the 17 patients, the mean CD4.sup.+/CD8.sup.+ ratio (ratio of helper T cells to cytotoxic T cells) at the commencement of treatment was 0.42.+-.0.05 (the ratio in a healthy population is 1.0-3.5). After a treatment period ranging from 2-8 weeks the CD4.sup.+/CD8.sup.+ cells ratio had significantly increased by over two-fold to 0.90.+-.0.02 (p<0.001). Concurrently with this change, an improvement in the response to skin hypersensitivity tests and a general increase in well-being were observed. This latter effect is demonstrated by the overall restoration of body weight, an increasing average from 67.9 to 88.1 Kg, a weight gain of 10 Kg (p<0.003).

Table 3 shows the percentage elevation in the CD4.sup.+ T cell populations of peripheral blood cells from HIV infected patients upon receiving the immunized camel's milk within the period of 2 months. It also shows the CD4.sup.+ T cells after 6 months from which patients stopped the treatment. The CD4.sup.+ percentages reported in Table 3 represent the percentage of white blood cells that are CD4.sup.+ cells. For HIV negative adults, the average CD4.sup.+ percentage is about 45% (This can range from 24% to 64%). In adults, a CD4.sup.+ percentage of about 12%-15% is considered similar to a CD4.sup.+ count of 200 cells/mm.sup.3. A percentage of about 15%-20% is considered similar to an absolute CD4.sup.+ cell count of 300 cells/mm.sup.3. Missing data points were due to patients lacking the financial means to travel to the checkup location (Sana'a City in the Republic of Yemen).

Table 4 shows the elevation in the Total White Blood Cells population of peripheral blood from HIV infected patients upon receiving the immunized camel's milk within the period of 2 months. The data reported in Table 4 represents the total white blood cells per cubic millimeter (mm.sup.3). Normal white blood cell counts of healthy adults are within the range of about 5,000-10,000 mm.sup.3. Missing data points were again due to patients lacking the financial means to travel to the checkup location (Sana'a City in the Republic of Yemen).

Table 5 shows the monitoring of "HIV Viral Load" throughout the treatment period. Moreover, it also illustrates the screening profile of the patients' viral load after 6 months from which patients stopped the treatment. Viral load is reported as copies of HIV in one cubic millimeter of blood.

TABLE-US-00003 TABLE 3 CD4.sup.+ T Cell Populations in Peripheral Blood P1f P2f P3f P4f P5m P6m P7m P8m P9m P10m P11m P12m P13m P14m P15m P16m P1- 7m Pc1m Pc2m Pc1f Pc2f Pre 38 19 31 8 8 21 42 41 31 34 36 29 31 49 22 19 13 11 22 23 19 2 Wks 27 10 23 3 3 11 21 32 24 18 28 17 22 26 11 16 6 4 Wks 39 21 26 11 11 23 38 28 42 39 31 11 15 32 17 21 19 2 M 53 48 43 36 36 23* 61 39 55 50 37 32 47 63 36 29 25 13 19 22 23 6 M 43 39 31 29 29 29 30 P = patient; 1-17 = patient number; m = male; f = female; c = control; *Patient experienced diarrhea and had a sexual relationship with an infected individual during the course of treatment.

TABLE-US-00004 TABLE 4 Total White Blood Cells in Peripheral Blood (In Thousands) P1f P2f P3f P4f P5m P6m P7m P8m P9m P10m P11m P12m P13m P14m P15m P16m P1- 7m Pc1m Pc2m Pc1f Pc2f Pre 2.7 3.5 4.0 5.2 2.8 1.953 2.7 3.1 4.1 0.82 3.6 1.953 2.08 3.1 2.7 3.1 - 4.1 3.25 2.89 3.1 3.2 2 Wks 2.6 3.4 3.3 5.4 3.2 1.88 2.3 5.7 5.1 2.04 4.0 1.88 1.984 3.0 2.3 5.7- 5.1 4 Wks 2.8 4.3 3.4 5.9 3.8 0.82 1.01 6.1 5.8 1.96 4.4 2.89 3.4 2.66 1.01 6.- 1 5.8 2 M 3.5 6.8 5.1 6.5 3.95 1.01* 3.0 8.322 5.6 3.2 3.9 3.981 3.9 3.45 3.2 4.- 5 6.366 3.0 3.143 3.5 3.01 6 M 4.1 5.6 4.8 5.2 4.0 4.2 5.1 P = patient; 1-17 = patient number; m = male; f = female; c = control; *Patient experienced diarrhea and had a sexual relationship with an infected individual during the course of treatment.

TABLE-US-00005 TABLE 5 HIV Viral Load in Copies per Cubic Milliliter of Blood (In Thousands) 1f 2f 3f 4f 5m 6m 7m 8m 9m 10m 11m Pre 8.3 1.1 4.6 10.04 0.759 0.65* 27 19 0.72 18.0 4.0 2 Wks 4.1 0 0.397 2.613 1.01 47.3* 0 2.1 1.223 18.7 5.52 4 Wks 0 0 0 0.18 2.99 18.7* 0 0 0 0.02 0 2 M Ve.sup.- Ve.sup.- Ve.sup.- Ve.sup.- 0 0* Ve.sup.- Ve.sup.- Ve.sup.- Ve.sup.- Ve.sup.- 6 M Ve.sup.- Ve.sup.- Ve.sup.- Ve.sup.- Ve.sup.- 12m 13m 14m 15m 16m 17m c1m c2m c1f c2f Pre 219 2.5 18.7 30.3 2.5 12.5 56 333 10 121 2 Wks 133 10.7 15.8 37.1 0 7.6 4 Wks 0 0 0 0 0 2 M Ve.sup.- Ve.sup.- 0 Ve.sup.- Ve.sup.- Ve.sup.- 62 320 14.3 193 6 M Ve.sup.- Ve.sup.- 1-17 = patient number; m = male; f = female; c = control; *Patient experienced diarrhea and had a sexual relationship with an infected individual during the course of treatment.

All patients regardless of their lifestyle, race, age and sex showed that their case of HIV/AIDS to be improved and 0 cases became worse. According to the criteria of curative effect the 17 cases are effective and 0 cases were ineffective in this study.

No case in the study showed side effects in general, except that namely 1 case felt diarrhea and tired for a short period. All of the other 16 cases continued to take the medicine in the prescribed quantity.

This therapeutic composition was very effective to suppress HIV replication as well as to stimulate its eradication from the blood stream. During the treatment process, all patients had positive response with no side effects, except that namely 1 case felt diarrhea and tired for a short period. However, the symptoms of the patients were noticeably improved after two weeks of the treatment including alleviation of weakness, depression, and stegnosis. The abdominal region pain and uncomfortable feeling also disappeared. Those patients who lost weight had about 10 Kg increase of body weight after two months of treatment. In the treatment, most if not all of the symptoms of the cases showed improvement after taking the HIV-immunized camel's milk. This was also associated with immune function improvement as well as increased in the number of the CD4+ T cells. The HIV-immunized camel's milk for all cases was found to be effective in the eradication of the HIV from the patients' blood streams. This was confirmed after four months after patients stopped taking the medication and were screened by Real Time PCR.

The therapeutic composition was therefore found to be highly effective to quickly decrease HIV viral load, and has the potential to eradicate HIV infection. The therapeutic composition also has the potential to stimulate and increase WBC significantly.

Two similar and independent investigations were performed where two groups of patients were given either (i) the medicament milk from camels intubated with the herb alone (without DNA immunization), or (ii) the medicament milk from camels that had been DNA immunized, but had not been fed the herbal composition. Both of these groups demonstrated reduction of viral load after treatment; however, neither of these experimental groups demonstrated full eradication of the HIV infection, as the virus was detected four months from the time that treatment was stopped (Table 6 & Table 7).

TABLE-US-00006 TABLE 6 Camel Milk Medicament from HIV DNA Immunized Camels (viral load in copies per cubic ml blood) P1m P2m P3f P4m P5f P6m P7m Pc1f Pc2m Pre 1200 4300 1500 2100 1800 700 9100 1000 5600 2 Wks 2100 5100 9800 3400 1300 390* 10900 1100 5200 4 Wks 650 1100 3100 430 600 50* 3200 2 M 0 0 100 0 0 2000 300 6 M 2800 1200 3100 3100 2800 9000 43000 1400 4700 1-7 = patient number; m = male, f = female, c = control; *Patient showed no adherence with the medicament regimen protocol and had a sexual relationship with an infected individual during the course of treatment.

TABLE-US-00007 TABLE 7 Camel Intubated with Herbal Extracts Only (viral load in copier per cubic ml blood) P1f P2f P3f P4m P5m P6f P7m P8m P9f Pc1f Pc2m Pre 17,000 21,000 1,200 3,200 210 1,900 6,500 3,600 7,450 10,000 76,000 2 Wks 3,000 3,400 250 1,800 0 400 590 2,000 1,800 8,700 48,000 4 Wks 1,500* 800** 0 430 0 0 0 210 190 2 M 0 0 0 0 0 0 0 6 M 3,000 98,000 4,200 7,000 4,200 56,000 115000 9,400 41,000 1-9 = patient number; m = male; f = female; c = control; *Patient withdrawn from the study at week six. **Patient showed no adherence with the medicament regiment protocol during the second month of the course of treatment.

It is to be understood that the present invention is not limited to the embodiments described above, but encompasses any and all embodiments within the scope of the following claims.

SEQUENCE LISTINGS

1

4112256DNAArtificial SequencePlasmid 1gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 60ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 120tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 180agtggcgccc gaacagggac atgaaagcga aagggaaacc agaggagctc tctcgacgca 240ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc 300caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta 360agcgggggaa aattagatcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa 420tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct 480ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt 540cagacaggat cagaagaacg tagatcatta tataatacag tagcaaccct ctattgtgtg 600catcaaagga tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa 660aacaaaagta agaaaaaagc acagcaagca gcagctgaca caggacacag cagccaggtc 720agccaaaatt accctatagt gcagaacatc caggggcaaa tggtacatca ggccatatca 780cctagaactt taaatgcatg ggtaaaagta gtagaagaga aggctttcag cccagaagtg 840atacccatgt tttcagcatt atcagaagga gccaccccac aagatttaaa caccatgcta 900aacacagtgg ggggacatca agcagccatg caaatgttaa aagagaccat caatgaggaa 960gctgcagaat gggatagagt gcatccagtg catgcagggc ctattgcacc aggccagatg 1020agagaaccaa ggggaagtga catagcagga actactagta cccttcagga acaaatagga 1080tggatgacac ataatccacc tatcccagta ggagaaatct ataaaagatg gataatcctg 1140ggattaaata aaatagtaag aatgtatagc cctaccagca ttctggacat aagacaagga 1200ccaaaggaac cctttagaga ctatgtagac cgattctata aaactctaag agccgagcaa 1260gcttcacaag aggtaaaaaa ttggatgaca gaaaccttgt tggtccaaaa tgcgaaccca 1320gattgtaaga ctattttaaa agcattggga ccaggagcga cactagaaga aatgatgaca 1380gcatgtcagg gagtgggggg acccggccat aaagcaagag ttttggctga agcaatgagc 1440caagtaacaa atccagctac cataatgata cagaaaggca attttaggaa ccaaagaaag 1500actgttaagt gtttcaattg tggcaaagaa gggcacatag ccaaaaattg cagggcccct 1560aggaaaaagg gctgttggaa atgtggaaag gaaggacacc aaatgaaaga ttgtactgag 1620agacaggcta attttttagg gaagatctgg ccttcccaca agggaaggcc agggaatttt 1680cttcagagca gaccagagcc aacagcccca ccagaagaga gcttcaggtt tggggaagag 1740acaacaactc cctctcagaa gcaggagccg atagacaagg aactgtatcc tttagcttcc 1800ctcagatcac tctttggcag cgacccctcg tcacaataaa gatagggggg caattaaagg 1860aagctctatt agatacagga gcagatgata cagtattaga agaaatgaat ttgccaggaa 1920gatggaaacc aaaaatgata gggggaattg gaggttttat caaagtaaga cagtatgatc 1980agatactcat agaaatctgc ggacataaag ctataggtac agtattagta ggacctacac 2040ctgtcaacat aattggaaga aatctgttga ctcagattgg ctgcacttta aattttccca 2100ttagtcctat tgagactgta ccagtaaaat taaagccagg aatggatggc ccaaaagtta 2160aacaatggcc attgacagaa gaaaaaataa aagcattagt agaaatttgt acagaaatgg 2220aaaaggaagg aaaaatttca aaaattgggc ctgaaaatcc atacaatact ccagtatttg 2280ccataaagaa aaaagacagt actaaatgga gaaaattagt agatttcaga gaacttaata 2340agagaactca agatttctgg gaagttcaat taggaatacc acatcctgca gggttaaaac 2400agaaaaaatc agtaacagta ctggatgtgg gcgatgcata tttttcagtt cccttagata 2460aagacttcag gaagtatact gcatttacca tacctagtat aaacaatgag acaccaggga 2520ttagatatca gtacaatgtg cttccacagg gatggaaagg atcaccagca atattccagt 2580gtagcatgac aaaaatctta gagcctttta gaaaacaaaa tccagacata gtcatctatc 2640aatacatgga tgatttgtat gtaggatctg acttagaaat agggcagcat agaacaaaaa 2700tagaggaact gagacaacat ctgttgaggt ggggatttac cacaccagac aaaaaacatc 2760agaaagaacc tccattcctt tggatgggtt atgaactcca tcctgataaa tggacagtac 2820agcctatagt gctgccagaa aaggacagct ggactgtcaa tgacatacag aaattagtgg 2880gaaaattgaa ttgggcaagt cagatttatg cagggattaa agtaaggcaa ttatgtaaac 2940ttcttagggg aaccaaagca ctaacagaag tagtaccact aacagaagaa gcagagctag 3000aactggcaga aaacagggag attctaaaag aaccggtaca tggagtgtat tatgacccat 3060caaaagactt aatagcagaa atacagaagc aggggcaagg ccaatggaca tatcaaattt 3120atcaagagcc atttaaaaat ctgaaaacag gaaagtatgc aagaatgaag ggtgcccaca 3180ctaatgatgt gaaacaatta acagaggcag tacaaaaaat agccacagaa agcatagtaa 3240tatggggaaa gactcctaaa tttaaattac ccatacaaaa ggaaacatgg gaagcatggt 3300ggacagagta ttggcaagcc acctggattc ctgagtggga gtttgtcaat acccctccct 3360tagtgaagtt atggtaccag ttagagaaag aacccataat aggagcagaa actttctatg 3420tagatggggc agccaatagg gaaactaaat taggaaaagc aggatatgta actgacagag 3480gaagacaaaa agttgtcccc ctaacggaca caacaaatca gaagactgag ttacaagcaa 3540ttcatctagc tttgcaggat tcgggattag aagtaaacat agtgacagac tcacaatatg 3600cattgggaat cattcaagca caaccagata agagtgaatc agagttagtc agtcaaataa 3660tagagcagtt aataaaaaag gaaaaagtct acctggcatg ggtaccagca cacaaaggaa 3720ttggaggaaa tgaacaagta gataaattgg tcagtgctgg aatcaggaaa gtactatttt 3780tagatggaat agataaggcc caagaagaac atgagaaata tcacagtaat tggagagcaa 3840tggctagtga ttttaaccta ccacctgtag tagcaaaaga aatagtagcc agctgtgata 3900aatgtcagct aaaaggggaa gccatgcatg gacaagtaga ctgtagccca ggaatatggc 3960agctagattg tacacattta gaaggaaaag ttatcttggt agcagttcat gtagccagtg 4020gatatataga agcagaagta attccagcag agacagggca agaaacagca tacttcctct 4080taaaattagc aggaagatgg ccagtaaaaa cagtacatac agacaatggc agcaatttca 4140ccagtactac agttaaggcc gcctgttggt gggcggggat caagcaggaa tttggcattc 4200cctacaatcc ccaaagtcaa ggagtaatag aatctatgaa taaagaatta aagaaaatta 4260taggacaggt aagagatcag gctgaacatc ttaagacagc agtacaaatg gcagtattca 4320tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag 4380acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa 4440attttcgggt ttattacagg gacagcagag atccagtttg gaaaggacca gcaaagctcc 4500tctggaaagg tgaaggggca gtagtaatac aagataatag tgacataaaa gtagtgccaa 4560gaagaaaagc aaagatcatc agggattatg gaaaacagat ggcaggtgat gattgtgtgg 4620caagtagaca ggatgaggat taacacatgg aaaagattag taaaacacca tatgtatatt 4680tcaaggaaag ctaaggactg gttttataga catcactatg aaagtactaa tccaaaaata 4740agttcagaag tacacatccc actaggggat gctaaattag taataacaac atattggggt 4800ctgcatacag gagaaagaga ctggcatttg ggtcagggag tctccataga atggaggaaa 4860aagagatata gcacacaagt agaccctgac ctagcagacc aactaattca tctgcactat 4920tttgattgtt tttcagaatc tgctataaga aataccatat taggacgtat agttagtcct 4980aggtgtgaat atcaagcagg acataacaag gtaggatctc tacagtactt ggcactagca 5040gcattaataa aaccaaaaca gataaagcca cctttgccta gtgttaggaa actgacagag 5100gacagatgga acaagcccca gaagaccaag ggccacagag ggagccatac aatgaatgga 5160cactagagct tttagaggaa cttaagagtg aagctgttag acattttcct aggatatggc 5220tccataactt aggacaacat atctatgaaa cttacgggga tacttgggca ggagtggaag 5280ccataataag aattctgcaa caactgctgt ttatccattt cagaattggg tgtcgacata 5340gcagaatagg cgttactcga cagaggagag caagaaatgg agccagtaga tcctagacta 5400gagccctgga agcatccagg aagtcagcct aagactgctt gtaccacttg ctattgtaaa 5460aagtgttgct ttcattgcca agtttgcttc ataacaaaag gcttaggcat ctcctatggc 5520aggaagaagc ggagacagcg acgaagagct cctcaagaca gtgagactca tcaagtttct 5580ctatcaaagc agtaagtagt acatgtaatg caagctttac aaatatcagc aatagtagga 5640ttagtagtag cagcaataat agcaatagtt gtgtggacca tagtattcat agaatatagg 5700aaaatattaa ggcaaagaaa aatagacagg ataattgata gaataataga aagagcagaa 5760gacagtggca atgagagtga cggagatcag gaagagttat cagcactggt ggagatgggg 5820catcatgctc cttgggatgt taatgatctg taatgctgag gaaaaattgt gggtcacagt 5880ctattatggg gtacctgtgt ggaaagaagc aaccaccact ctattttgtg catcagatgc 5940taaagcatat gatacagagg tacataatgt ttgggccaca catgcctgtg tacccacaga 6000ccccaaccca caagaagtag aattggaaaa tgtgacagaa aattttaaca tgtggaaaaa 6060taacatggta gaacagatgc atgaggatat aatcagttta tgggatcaaa gcctaaagcc 6120atgtgtaaaa ttaactccac tctgtgttac tttaaattgc actgatttga ggaatgctac 6180taatgggaat gacactaata ccactagtag tagcagggaa atgatggggg gaggagaaat 6240gaaaaattgc tctttcaaaa tcaccacaaa cataagaggt aaggtgcaga aagaatatgc 6300acttttttat gaacttgata tagtaccaat agataataat agtaataata gatataggtt 6360gataagttgt aacacctcag tcattacaca ggcctgtcca aagatatcct ttgagccaat 6420tcccatacat tattgtgccc cggctggttt tgcgattcta aagtgtaaag ataagaagtt 6480caatggaaaa ggaccatgtt caaatggcag cacagtacaa tgtacacatg ggattaggcc 6540agtagtatca actcaactgc tgttaaatgg cagtctagca gaagaagagg tagtaattag 6600atccgaaaat ttcgcggaca atgctaaaac cataatagta cagctgaatg aatctgtaga 6660aattaattgt acaagaccca acaacaatac aagaaaaagt atacatatag gaccaggcag 6720agcattatat acaacaggag aaataatagg agatataaga caagcacatt gtaaccttag 6780tagagcaaag tggaatgaca ctttaaataa gatagttata aaattaagag aacaatttgg 6840gaataaaaca atagtcttta agcattcctc aggaggggac ccagaaattg tgacgcacag 6900ttttaattgt ggaggggaat ttttctactg taattcaaca caactgttta atagtacttg 6960gaatgttact gaagagtcaa ataacactgt agaaaataac acaatcacac tcccatgcag 7020aataaaacaa attataaaca tgtggcagaa agtaggaaga gcaatgtatg cccctcccat 7080cagaggacaa attagatgtt catcaaatat tacagggctg ctattaacaa gagatggtgg 7140tccagaggac aacaagaccg aggtcttcag acctggagga ggagatatga gggacaattg 7200gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac 7260caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctgtgtt 7320ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt 7380acaggccaga ctattattgt ctggtatagt gcaacagcag aacaatctgc tgagggctat 7440tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag 7500agtcctggct gtggaaagat acctaaggga tcaacagctc ctgggaattt ggggttgctc 7560tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct 7620gaataagatt tgggataaca tgacctggat ggagtgggac agagaaatta acaattacac 7680aagcataata tatagcttaa ttgaagaatc gcagaaccaa caagaaaaga atgaacaaga 7740attattagaa ttagacaaat gggcaagttt gtggaattgg tttgacataa cagaatggct 7800gtggtatata aaaatattca taatgataat aggaggcttg ataggtttaa gaatagtttt 7860ttctgtactt tctataatga atagagttag gcagggatac tcaccattat cgtttcagac 7920ccacctccca gcctcgaggg gacccgacag gcccggagga atcgaagaag aaggtggaga 7980gagagacaga gacagatccg gtcgattagt gaacggatcc ttagcactta tctgggacga 8040tctgcggagc ctgtgcctct tcagctacca ccgcttgaga gacttactct tgattgtaac 8100gaggattgtg gaacttctgg gacgcagggg gtgggaagcc ctcaaatatt ggtggaatct 8160cctacaatat tggagtcagg agctaaagaa tagtgctgtt agcttgctca atgccacagc 8220tatagcagta gctgagggga cagatagggt tatagaagta gtacaagaag cttatagagc 8280tattcgccac atacctagaa gaataagaca gggcttggaa aggattttgc tataagatgg 8340gtggcaagtg gtcaaaaagt agtgtggttg gatggcctgc tgtaagggaa agaatgagac 8400gagctgagcc agcagcagat ggggtgggag cagcatctcg agacctagaa aaacatggag 8460caatcacaag tagcaacaca gcagctaaca atgctgcttg tgcctggcta gaagcacaag 8520aggaggagaa ggtgggtttt ccagtcacac ctcaggtacc tttaagacca atgacttaca 8580aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc 8640actcccaacg aagacaagat atccttgatc tgtggatcta ccacacacaa ggctacttcc 8700ctgattggca gaactacaca ccaggaccag ggatcagata tccactgacc tttggatggc 8760gctacaagct agtaccagtt gagccagaga agttagaaga agccaacaaa ggagagaaca 8820ccagcttgtt acaccctgtg agcctgcatg gaatggatga cccggagaga gaagtgttag 8880agtggaggtt tgacagccgc ctagcatttc atcacgtggc ccgagagctg catccggagt 8940acttcaagaa ctgctgatat cgagcttgct acaagggact ttccgctggg gactttccag 9000ggaggcgtgg cctgggcggg actggggagt ggcgagccct cagatcctgc atataagcag 9060ctgctttttg cctgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct 9120ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta 9180gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca 9240gtgtggaaaa tctctagcag tcacgtaact ctgaaaaggt caaagatatt gatgcttgat 9300acagaaggaa gggaatacca gtgagttgtc tggactacac taaacatacc tgaaatctga 9360gttcgagaag tataatagaa ctggactcca aattaagtct cagttgcaaa ctggattaat 9420gggggataga tttctatcta gaggggggca ggtgaccttc agaccttggc actggaggtg 9480gcccggcaga agcgcggcat cgtggatcag tgctgcacca gcatctgctc tctctaccaa 9540ctggagaact actgcaacta ggcccaccac taccctgtcc acccctctgc aatgaataaa 9600acctttgaaa gagcactaca agttgtgtgt acatgcgtgc atgtgcatat gtggtgcggg 9660gggaacatga gtggggctgg ctggagtggc gatgataagc tgtcaaacat gagaattctt 9720gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 9780tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 9840ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 9900aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 9960tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 10020atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 10080agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 10140tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca 10200tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 10260atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 10320ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 10380tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 10440acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa 10500ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 10560aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 10620ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 10680cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 10740gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 10800actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggctg 10860ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 10920accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct 10980tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 11040cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 11100gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 11160gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 11220gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 11280cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 11340tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 11400ggggcggagc ctatggaaaa acgccagcaa cggagatgcg ccgcgtgcgg ctgctggaga 11460tggcggacgc gatggatatg ttctgccaag ggttggtttg cgcattcaca gttctccgca 11520agaattgatt ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc 11580cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag 11640gtatagggcg gcgcctacaa tccatgccaa cccgttccat gtgctcgccg aggcggcata 11700aatcgccgtg acgatcagcg gtccaatgat cgaagttagg ctggtaagag ccgcgagcga 11760tccttgaagc tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc 11820gggcatcccg atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg 11880cgtcggggag ctttttgcaa aagcctaggc ctccaaaaaa gcctcctcac tacttctgga 11940atagctcaga ggccgaggcg gcctcggcct ctgcataaat aaaaaaaatt agtcagccat 12000ggggcggaga atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg 12060ggactatggt tgctgactaa ttgagatgca tgttcgaatc gcgaccgcgg tccggagtac 12120ttcaagaact gctgatatcg agcttgctac aagggacttt ccgctgggga ctttccaggg 12180aggcgtggcc tgggcgggac tggggagtgg cgagccctca gatcctgcat ataagcagct 12240gctttttgcc tgtact 12256214825DNAArtificial SequencePlasmid 2tggaagggct aatttggtcc caaaaaagac aagagatcct tgatctgtgg atctaccaca 60cacaaggcta cttccctgat tggcagaact acacaccagg gccagggatc agatatccac 120tgacctttgg atggtgcttc aagttagtac cagttgaacc agagcaagta gaagaggcca 180atgaaggaga gaacaacagc ttgttacacc ctatgagcca gcatgggatg gaggacccgg 240agggagaagt attagtgtgg aagtttgaca gcctcctagc atttcgtcac atggcccgag 300agctgcatcc ggagtactac aaagactgct gacatcgagc tttctacaag ggactttccg 360ctggggactt tccagggagg tgtggcctgg gcgggactgg ggagtggcga gccctcagat 420gctacatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540tgagtgctca aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660cgaaagtaaa gccagaggag atctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780aggagagaga tgggtgcgag agcgtcggta ttaagcgggg gagaattaga taaatgggaa 840aaaattcggt taaggccagg gggaaagaaa caatataaac taaaacatat agtatgggca 900agcagggagc tagaacgatt cgcagttaat cctggccttt tagagacatc agaaggctgt 960agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca 1020ttatataata caatagcagt cctctattgt gtgcatcaaa ggatagatgt aaaagacacc 1080aaggaagcct tagataagat agaggaagag caaaacaaaa gtaagaaaaa ggcacagcaa 1140gcagcagctg acacaggaaa caacagccag gtcagccaaa attaccctat agtgcagaac 1200ctccaggggc aaatggtaca tcaggccata tcacctagaa ctttaaatgc atgggtaaaa 1260gtagtagaag agaaggcttt cagcccagaa gtaataccca tgttttcagc attatcagaa 1320ggagccaccc cacaagattt aaataccatg ctaaacacag tggggggaca tcaagcagcc 1380atgcaaatgt taaaagagac catcaatgag gaagctgcag aatgggatag attgcatcca 1440gtgcatgcag ggcctattgc accaggccag atgagagaac caaggggaag tgacatagca 1500ggaactacta gtacccttca ggaacaaata ggatggatga cacataatcc acctatccca 1560gtaggagaaa tctataaaag atggataatc ctgggattaa ataaaatagt aagaatgtat 1620agccctacca gcattctgga cataagacaa ggaccaaagg aaccctttag agactatgta 1680gaccgattct ataaaactct aagagccgag caagcttcac aagaggtaaa aaattggatg 1740acagaaacct tgttggtcca aaatgcgaac ccagattgta agactatttt aaaagcattg 1800ggaccaggag cgacactaga agaaatgatg acagcatgtc agggagtggg gggacccggc 1860cataaagcaa gagttttggc tgaagcaatg agccaagtaa caaatccagc taccataatg 1920atacagaaag gcaattttag gaaccaaaga aagactgtta agtgtttcaa ttgtggcaaa 1980gaagggcaca tagccaaaaa ttgcagggcc cctaggaaaa agggctgttg gaaatgtgga 2040aaggaaggac accaaatgaa agattgtact gagagacagg ctaatttttt agggaagatc 2100tggccttccc acaagggaag gccagggaat tttcttcaga gcagaccaga gccaacagcc 2160ccaccagaag agagcttcag gtttggggaa gagacaacaa ctccctctca gaagcaggag 2220ccgatagaca aggaactgta tcctttagct tccctcagat cactctttgg cagcgacccc 2280tcgtcacaat aaagataggg gggcaattaa aggaagctct attagataca ggagcagatg 2340atacagtatt agaagaaatg aatttgccag gaagatggaa accaaaaatg atagggggaa 2400ttggaggttt tatcaaagta agacagtatg atcagatact catagaaatc tgcggacata 2460aagctatagg tacagtatta gtaggaccta cacctgtcaa cataattgga agaaatctgt 2520tgactcagat tggctgcact ttaaattttc ccattagtcc tattgagact gtaccagtaa 2580aattaaagcc aggaatggat ggcccaaaag ttaaacaatg gccattgaca gaagaaaaaa 2640taaaagcatt agtagaaatt tgtacagaaa tggaaaagga aggaaaaatt tcaaaaattg

2700ggcctgaaaa tccatacaat actccagtat ttgccataaa gaaaaaagac agtactaaat 2760ggagaaaatt agtagatttc agagaactta ataagagaac tcaagatttc tgggaagttc 2820aattaggaat accacatcct gcagggttaa aacagaaaaa atcagtaaca gtactggatg 2880tgggcgatgc atatttttca gttcccttag ataaagactt caggaagtat actgcattta 2940ccatacctag tataaacaat gagacaccag ggattagata tcagtacaat gtgcttccac 3000agggatggaa aggatcacca gcaatattcc agtgtagcat gacaaaaatc ttagagcctt 3060ttagaaaaca aaatccagac atagtcatct atcaatacat ggatgatttg tatgtaggat 3120ctgacttaga aatagggcag catagaacaa aaatagagga actgagacaa catctgttga 3180ggtggggatt taccacacca gacaaaaaac atcagaaaga acctccattc ctttggatgg 3240gttatgaact ccatcctgat aaatggacag tacagcctat agtgctgcca gaaaaggaca 3300gctggactgt caatgacata cagaaattag tgggaaaatt gaattgggca agtcagattt 3360atgcagggat taaagtaagg caattatgta aacttcttag gggaaccaaa gcactaacag 3420aagtagtacc actaacagaa gaagcagagc tagaactggc agaaaacagg gagattctaa 3480aagaaccggt acatggagtg tattatgacc catcaaaaga cttaatagca gaaatacaga 3540agcaggggca aggccaatgg acatatcaaa tttatcaaga gccatttaaa aatctgaaaa 3600caggaaagta tgcaagaatg aagggtgccc acactaatga tgtgaaacaa ttaacagagg 3660cagtacaaaa aatagccaca gaaagcatag taatatgggg aaagactcct aaatttaaat 3720tacccataca aaaggaaaca tgggaagcat ggtggacaga gtattggcaa gccacctgga 3780ttcctgagtg ggagtttgtc aatacccctc ccttagtgaa gttatggtac cagttagaga 3840aagaacccat aataggagca gaaactttct atgtagatgg ggcagccaat agggaaacta 3900aattaggaaa agcaggatat gtaactgaca gaggaagaca aaaagttgtc cccctaacgg 3960acacaacaaa tcagaagact gagttacaag caattcatct agctttgcag gattcgggat 4020tagaagtaaa catagtgaca gactcacaat atgcattggg aatcattcaa gcacaaccag 4080ataagagtga atcagagtta gtcagtcaaa taatagagca gttaataaaa aaggaaaaag 4140tctacctggc atgggtacca gcacacaaag gaattggagg aaatgaacaa gtagataaat 4200tggtcagtgc tggaatcagg aaagtactat ttttagatgg aatagataag gcccaagaag 4260aacatgagaa atatcacagt aattggagag caatggctag tgattttaac ctaccacctg 4320tagtagcaaa agaaatagta gccagctgtg ataaatgtca gctaaaaggg gaagccatgc 4380atggacaagt agactgtagc ccaggaatat ggcagctaga ttgtacacat ttagaaggaa 4440aagttatctt ggtagcagtt catgtagcca gtggatatat agaagcagaa gtaattccag 4500cagagacagg gcaagaaaca gcatacttcc tcttaaaatt agcaggaaga tggccagtaa 4560aaacagtaca tacagacaat ggcagcaatt tcaccagtac tacagttaag gccgcctgtt 4620ggtgggcggg gatcaagcag gaatttggca ttccctacaa tccccaaagt caaggagtaa 4680tagaatctat gaataaagaa ttaaagaaaa ttataggaca ggtaagagat caggctgaac 4740atcttaagac agcagtacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga 4800ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta 4860aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca 4920gagatccagt ttggaaagga ccagcaaagc tcctctggaa aggtgaaggg gcagtagtaa 4980tacaagataa tagtgacata aaagtagtgc caagaagaaa agcaaagatc atcagggatt 5040atggaaaaca gatggcaggt gatgattgtg tggcaagtag acaggatgag gattaacaca 5100tggaaaagat tagtaaaaca ccatatgtat atttcaagga aagctaagga ctggttttat 5160agacatcact atgaaagtac taatccaaaa ataagttcag aagtacacat cccactaggg 5220gatgctaaat tagtaataac aacatattgg ggtctgcata caggagaaag agactggcat 5280ttgggtcagg gagtctccat agaatggagg aaaaagagat atagcacaca agtagaccct 5340gacctagcag accaactaat tcatctgcac tattttgatt gtttttcaga atctgctata 5400agaaatacca tattaggacg tatagttagt cctaggtgtg aatatcaagc aggacataac 5460aaggtaggat ctctacagta cttggcacta gcagcattaa taaaaccaaa acagataaag 5520ccacctttgc ctagtgttag gaaactgaca gaggacagat ggaacaagcc ccagaagacc 5580aagggccaca gagggagcca tacaatgaat ggacactaga gcttttagag gaacttaaga 5640gtgaagctgt tagacatttt cctaggatat ggctccataa cttaggacaa catatctatg 5700aaacttacgg ggatacttgg gcaggagtgg aagccataat aagaattctg caacaactgc 5760tgtttatcca tttcagaatt gggtgtcgac atagcagaat aggcgttact cgacagagga 5820gagcaagaaa tggagccagt agatcctaga ctagagccct ggaagcatcc aggaagtcag 5880cctaaaactg cttgtaccaa ttgctattgt aaaaagtgtt gctttcattg ccaagtttgt 5940ttcatgacaa aagccttagg catctcctat ggcaggaaga agcggagaca gcgacgaaga 6000gctcatcaga acagtcagac tcatcaagct tctctatcaa agcagtaagt agtacatgta 6060atgcaaccta taatagtagc aatagtagca ttagtagtag caataataat agcaatagtt 6120gtgtggtcca tagtaatcat agaatatagg aaaatattaa gacaaagaaa aatagacagg 6180ttaattgata gactaataga aagagcagaa gacagtggca atgagagtga aggagaagta 6240tcagcacttg tggagatggg ggtggaaatg gggcaccatg ctccttggga tattgatgat 6300ctgtagtgct acagaaaaat tgtgggtcac agtctattat ggggtacctg tgtggaagga 6360agcaaccacc actctatttt gtgcatcaga tgctaaagca tatgatacag aggtacataa 6420tgtttgggcc acacatgcct gtgtacccac agaccccaac ccacaagaag tagtattggt 6480aaatgtgaca gaaaatttta acatgtggaa aaatgacatg gtagaacaga tgcatgagga 6540tataatcagt ttatgggatc aaagcctaaa gccatgtgta aaattaaccc cactctgtgt 6600tagtttaaag tgcactgatt tgaagaatga tactaatacc aatagtagta gcgggagaat 6660gataatggag aaaggagaga taaaaaactg ctctttcaat atcagcacaa gcataagaga 6720taaggtgcag aaagaatatg cattctttta taaacttgat atagtaccaa tagataatac 6780cagctatagg ttgataagtt gtaacacctc agtcattaca caggcctgtc caaaggtatc 6840ctttgagcca attcccatac attattgtgc cccggctggt tttgcgattc taaaatgtaa 6900taataagacg ttcaatggaa caggaccatg tacaaatgtc agcacagtac aatgtacaca 6960tggaatcagg ccagtagtat caactcaact gctgttaaat ggcagtctag cagaagaaga 7020tgtagtaatt agatctgcca atttcacaga caatgctaaa accataatag tacagctgaa 7080cacatctgta gaaattaatt gtacaagacc caacaacaat acaagaaaaa gtatccgtat 7140ccagagggga ccagggagag catttgttac aataggaaaa ataggaaata tgagacaagc 7200acattgtaac attagtagag caaaatggaa tgccacttta aaacagatag ctagcaaatt 7260aagagaacaa tttggaaata ataaaacaat aatctttaag caatcctcag gaggggaccc 7320agaaattgta acgcacagtt ttaattgtgg aggggaattt ttctactgta attcaacaca 7380actgtttaat agtacttggt ttaatagtac ttggagtact gaagggtcaa ataacactga 7440aggaagtgac acaatcacac tcccatgcag aataaaacaa tttataaaca tgtggcagga 7500agtaggaaaa gcaatgtatg cccctcccat cagtggacaa attagatgtt catcaaatat 7560tactgggctg ctattaacaa gagatggtgg taataacaac aatgggtccg agatcttcag 7620acctggagga ggcgatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt 7680aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga 7740aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac 7800tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctgatatagt 7860gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac 7920agtctggggc atcaaacagc tccaggcaag aatcctggct gtggaaagat acctaaagga 7980tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc 8040ttggaatgct agttggagta ataaatctct ggaacagatt tggaataaca tgacctggat 8100ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc 8160gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt 8220gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt 8280aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag 8340gcagggatat tcaccattat cgtttcagac ccacctccca atcccgaggg gacccgacag 8400gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt 8460gaacggatcc ttagcactta tctgggacga tctgcggagc ctgtgcctct tcagctacca 8520ccgcttgaga gacttactct tgattgtaac gaggattgtg gaacttctgg gacgcagggg 8580gtgggaagcc ctcaaatatt ggtggaatct cctacagtat tggagtcagg aactaaagaa 8640tagtgctgtt aacttgctca atgccacagc catagcagta gctgagggga cagatagggt 8700tatagaagta ttacaagcag cttatagagc tattcgccac atacctagaa gaataagaca 8760gggcttggaa aggattttgc tataagatgg gtggcaagtg gtcaaaaagt agtgtgattg 8820gatggcctgc tgtaagggaa agaatgagac gagctgagcc agcagcagat ggggtgggag 8880cagtatctcg agacctagaa aaacatggag caatcacaag tagcaataca gcagctaaca 8940atgctgcttg tgcctggcta gaagcacaag aggaggaaga ggtgggtttt ccagtcacac 9000ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa 9060aagaaaaggg gggactggaa gggctaattc actcccaaag aagacaagat atccttgatc 9120tgtggatcta ccacacacaa ggctacttcc ctgattggca gaactacaca ccagggccag 9180gggtcagata tccactgacc tttggatggt gctacaagct agtaccagtt gagccagata 9240aggtagaaga ggccaataaa ggagagaaca ccagcttgtt acaccctgtg agcctgcatg 9300gaatggatga ccctgagaga gaagtgttag agtggaggtt tgacagccgc ctagcatttc 9360atcacgtggc ccgagagctg catccggagt acttcaagaa ctgctgacat cgagcttgct 9420acaagggact ttccgctggg gactttccag ggaggcgtgg cctgggcggg actggggagt 9480ggcgagccct cagatgctgc atataagcag ctgctttttg cctgtactgg gtctctctgg 9540ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct 9600caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt 9660aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcac ccaggaggta 9720gaggttgcag tgagccaaga tcgcgccact gcattccagc ctgggcaaga aaacaagact 9780gtctaaaata ataataataa gttaagggta ttaaatatat ttatacatgg aggtcataaa 9840aatatatata tttgggctgg gcgcagtggc tcacacctgc gcccggccct ttgggaggcc 9900gaggcaggtg gatcacctga gtttgggagt tccagaccag cctgaccaac atggagaaac 9960cccttctctg tgtattttta gtagatttta ttttatgtgt attttattca caggtatttc 10020tggaaaactg aaactgtttt tcctctactc tgataccaca agaatcatca gcacagagga 10080agacttctgt gatcaaatgt ggtgggagag ggaggttttc accagcacat gagcagtcag 10140ttctgccgca gactcggcgg gtgtccttcg gttcagttcc aacaccgcct gcctggagag 10200aggtcagacc acagggtgag ggctcagtcc ccaagacata aacacccaag acataaacac 10260ccaacaggtc caccccgcct gctgcccagg cagagccgat tcaccaagac gggaattagg 10320atagagaaag agtaagtcac acagagccgg ctgtgcggga gaacggagtt ctattatgac 10380tcaaatcagt ctccccaagc attcggggat cagagttttt aaggataact tagtgtgtag 10440ggggccagtg agttggagat gaaagcgtag ggagtcgaag gtgtcctttt gcgccgagtc 10500agttcctggg tgggggccac aagatcggat gagccagttt atcaatccgg gggtgccagc 10560tgatccatgg agtgcagggt ctgcaaaata tctcaagcac tgattgatct taggttttac 10620aatagtgatg ttaccccagg aacaatttgg ggaaggtcag aatcttgtag cctgtagctg 10680catgactcct aaaccataat ttcttttttg tttttttttt tttatttttg agacagggtc 10740tcactctgtc acctaggctg gagtgcagtg gtgcaatcac agctcactgc agcctcaacg 10800tcgtaagctc aagcgatcct cccacctcag cctgcctggt agctgagact acaagcgacg 10860ccccagttaa tttttgtatt tttggtagag gcagcgtttt gccgtgtggc cctggctggt 10920ctcgaactcc tgggctcaag tgatccagcc tcagcctccc aaagtgctgg gacaaccggg 10980gccagtcact gcacctggcc ctaaaccata atttctaatc ttttggctaa tttgttagtc 11040ctacaaaggc agtctagtcc ccaggcaaaa agggggtttg tttcgggaaa gggctgttac 11100tgtctttgtt tcaaactata aactaagttc ctcctaaact tagttcggcc tacacccagg 11160aatgaacaag gagagcttgg aggttagaag cacgatggaa ttggttaggt cagatctctt 11220tcactgtctg agttataatt ttgcaatggt ggttcaaaga ctgcccgctt ctgacaccag 11280tcgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 11340tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 11400gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 11460atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 11520ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 11580cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 11640tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 11700gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 11760aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 11820tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 11880aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 11940aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 12000ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 12060ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 12120atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 12180atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 12240tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 12300gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 12360tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 12420gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 12480cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 12540gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 12600atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 12660aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 12720atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 12780aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 12840aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 12900gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 12960gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 13020gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 13080ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 13140ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 13200atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 13260gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 13320atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg 13380cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt 13440cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag 13500cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga 13560aaataccgca tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg 13620gtgcgggcct cttcgctatt acgccagggg aggcagagat tgcagtaagc tgagatcgca 13680gcactgcact ccagcctggg cgacagagta agactctgtc tcaaaaataa aataaataaa 13740tcaatcagat attccaatct tttcctttat ttatttattt attttctatt ttggaaacac 13800agtccttcct tattccagaa ttacacatat attctatttt tctttatatg ctccagtttt 13860ttttagacct tcacctgaaa tgtgtgtata caaaatctag gccagtccag cagagcctaa 13920aggtaaaaaa taaaataata aaaaataaat aaaatctagc tcactccttc acatcaaaat 13980ggagatacag ctgttagcat taaataccaa ataacccatc ttgtcctcaa taattttaag 14040cgcctctctc caccacatct aactcctgtc aaaggcatgt gccccttccg ggcgctctgc 14100tgtgctgcca accaactggc atgtggactc tgcagggtcc ctaactgcca agccccacag 14160tgtgccctga ggctgcccct tccttctagc ggctgccccc actcggcttt gctttcccta 14220gtttcagtta cttgcgttca gccaaggtct gaaactaggt gcgcacagag cggtaagact 14280gcgagagaaa gagaccagct ttacaggggg tttatcacag tgcaccctga cagtcgtcag 14340cctcacaggg ggtttatcac attgcaccct gacagtcgtc agcctcacag ggggtttatc 14400acagtgcacc cttacaatca ttccatttga ttcacaattt ttttagtctc tactgtgcct 14460aacttgtaag ttaaatttga tcagaggtgt gttcccagag gggaaaacag tatatacagg 14520gttcagtact atcgcatttc aggcctccac ctgggtcttg gaatgtgtcc cccgaggggt 14580gatgactacc tcagttggat ctccacaggt cacagtgaca caagataacc aagacacctc 14640ccaaggctac cacaatgggc cgccctccac gtgcacatgg ccggaggaac tgccatgtcg 14700gaggtgcaag cacacctgcg catcagagtc cttggtgtgg agggagggac cagcgcagct 14760tccagccatc cacctgatga acagaaccta gggaaagccc cagttctact tacaccagga 14820aaggc 1482539774DNAArtificial SequencePlasmid 3tgggttaatt tactccaaga aaagacaaga gatccttgat ctgtgggtct ataacacaca 60aggattcttc ccagattggc agaactacac accaggacca ggggttagat acccactgac 120ctttgggtgg tgcttcaaac tagtaccaat ggatccagca gagatagaga aagccaatga 180agaagagaac aactgtttat tacatcccat ctgccagcat ggaatggagg acgaagacag 240agaagtgctg gtctggaagt ttgacagtcg cctggcactc aaacacatag ctcgagagaa 300acatccggag ttttacaaag actgctgaca cagaagttgc tgacaaggga ctttccgctg 360gggactttcc gagggaggtg tggtttggga ggagttgggg agtggctagc cctcagatgc 420tgcatataag cagctgcttc tcgcctgtac tgggtctctc ttgctagacc agatttgagc 480ctgggagctc tctggctagt agagaaccca ctgcttaagc ctcaataaag cttgccttga 540gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga 600ccactttaga ctgtgtaaaa atctctagca gtggcgcccg aacagggact tgaagttaat 660agggactcga aagcgaaagt tccagagaag atctctcgac gcacggactc ggcttgctga 720ggtgcacaca gcaagaggcg agagcggcga ctggtgagta cgccaatttt tgactagcgg 780aggctagaag gagagagatg ggtgcgagag cgtcagtgtt aagtggggga agattagatg 840catgggagaa aattcggtta aggccagggg gaaagaaaaa atatagacta aaacatttag 900tatgggcaag cagggagttg gaaagattcg cacttaaccc tggcctttta gaaacagcag 960aaggatgtca acaaataata gaacagttac aatcaactct caagacagga tcagaagaac 1020ttaaatcatt atataatgca gtagcagtcc tttactgcgt acatcaaagg atagaggtaa 1080aagacaccaa ggaagcttta gataaaatag aggaactgca aaacaagagt aagcaaaaaa 1140cacagcaggc agcagcaaag acacagcagg cagcagctgg cacaggatgc agccaaaatt 1200accctatagt gcaaaatgca caagggcaaa tgacacacca gaatctgtca cctaggactt 1260taaatgcatg ggtgaaggta atagaagaga aaggtttcag cccagaagta atacccatgt 1320tctcagcatt atcagaggga gccaccccac aagatttaaa tatgatgcta aacatagtgg 1380ggggacacca ggcagcaatg cagatgttga aagataccat caatgaggaa gctgcagaat 1440gggacaggac acatccagta catgcagggc ctattccacc aggtcagatg agagaaccaa 1500ggggaagtga catagcagga actactagta cccttcaaga acaaatgggc tggatgacaa 1560gcaatccacc tatcccagtg ggagacatct ataaaagatg gataatccta ggattaaata 1620aaatagtaag aatgtatagc cctgtcagca tcttggacat aaaacaaggg ccaaaagaac 1680cctttagaga ttatgtagat aggttcttta aaactttgag agctgaacaa gctacgcagg 1740aggtaaaaaa ctggatgaca gaaaccttgc tggtccaaaa tgcaaatcca gactgtaagt 1800ccattttaaa agcattagga gtaggggcca cattagaaga aatgatgaca gcatgccagg 1860gagtaggagg acctggccat aaagcaaggg ttctggctga ggcaatgagt caagtacaac 1920aggcaggagt aatgatgcag agaagcaatt ttaggggcca gagaacaata aagtgtttca 1980actgtggcaa agaaggacac ctcgctagaa attgtaaggc ccctaggaaa agaggctgtt 2040ggaaatgtgg gaaggaggga caccaaatga aagactgcac tgaaagacag gcgaattttt 2100tagggagaat ctggccttcc agcaagggga ggccaggcaa ttttcctcag agcagaccag 2160agccaacagc cccaccagca gagagcttgg ggatggggga agagataccc tcctccccga 2220agcaggaacc gagggacaag gaactatacc ctcctttagc ttccctcaaa tcactctttg 2280gcaacgaccc ttagttacag taaaattagg gggacagctg atagaagcct tattagacac 2340aggggcagat gatacagtat tagaagaaat agatttacca ggaaaatgga aaccaaaaat 2400gataggggga attggaggtt ttatcaaagt aagacaatat gatcagatac ttatagaaat 2460ttgtggaaaa aaggctatag gtacagtgtt agtaggacct acacctgtca acataattgg 2520acgaaatatg ttgactcaga ttggttgcac tctaaatttc ccaattagtc ctattgaaac 2580tgcaccagta aaactaaagc caggaatgga tggcccaaag gttaaacaat ggccattgac 2640agaagaaaaa ataaaggcat taacagacat ttgtacagag atggaaaagg aagggaaaat 2700ttcaaaaatt gggcctgaaa atccatacaa tactccagta tttgccataa agaaaaaaga 2760tagtactaaa tggagaaaat tagtcgactt cagagaactc aataaaagaa ctcaagactt 2820ctgggaggtc caattaggaa tacctcatcc

cgcgggatta aaaaagaaaa aatcggtaac 2880agtactagat gtgggggatg catatttttc agttccctta gatgaagact ttagaaaata 2940tactgcattc actataccta gtgtaaataa tgagacacca ggaattagat atcagtacaa 3000tgtacttcca cagggatgga aaggatcacc agcaatattt caggctagca tgacaaaaat 3060cttagagccc tttagaacag aaaatccaga gatagtgatt taccaatata tggatgattt 3120atatgtagga tctgacttag agataggaca gcatagggca aaaatagaag agttaagaga 3180acatctactg agatgggggt ttaccacacc agacaaaaaa catcagaaag aacctccatt 3240cctttggatg ggatatgaac tccatcctga caaatggaca gtccagccca tacagctgcc 3300agaaaaagac agctggactg tcaatgatat acagaaatta gtgggaaaac taaattgggc 3360aagccagatt tatccaggaa ttaaagtaaa gcaactgtgt aaactcctca ggggagccaa 3420agcactaaca gatataataa cactgactga ggaagcagaa ttagaattgg cagagaacag 3480ggagattcta aaagaacctg tacatggagt ctattatgac ccagcaaaag gcctagtagc 3540agaaatacag aagcaaggac aagaccaatg gacatatcaa atttatcaag agccatttaa 3600aaatctaaaa acaggaaaat atgcaaaaaa gaggtctgct cacactaatg atgtaaaaca 3660attaacagag gtagtgcaaa aagtggccac agaaagtgta gtaatatggg gaaagacccc 3720taaatttaga ctacccatac aaaaagaaac atgggaagca tggtggatgg agtattggca 3780ggccacctgg attcctgaat gggagtttgt caatacccct cctctagtaa aattatggta 3840ccagttagag aaagatccca tagtaggggc agaaactttc tatgtagatg gggcagctaa 3900tagggaaact aagctaggaa aagcagggta tgtcactgac agaggaagac aaaaagttgt 3960ctccataact gagacaacaa atcagaagac tgagttacat gcaatttatc tagccttgca 4020ggattcagga tcagaagtaa atatagtaac agactcacag tatgcattag gaatcattca 4080ggcacaacca gacaagagtg aatcagagtt agtcaatcta ataatagaga agctaataga 4140aaaggacaaa gtctacctgt catgggtacc agcacacaaa ggaattggag gaaatgaaca 4200agtagataaa ttagtcagta gtggagtcag gaaagtacta tttttagatg gcatagataa 4260agcccaagaa gagcatgaaa aatatcacag caattggaga gcaatggcta gtgattttaa 4320cctgccacct atagtagcaa aagaaatagt ggccagctgt gataaatgtc aactaaaagg 4380ggaagccatg catggacaag tagactgtag tccaggaata tggcaattag attgtacaca 4440tttagaagga aaaattatca tagtagcagt ccatgtagcc agtggctata tagaagcaga 4500agttatccca gcagaaacag gacaggagac agcatacttt atattaaaat tagcaggaag 4560atggccagtg aaagtaatac acacagataa tggcagtaat tttaccagtg ctgcagtaaa 4620ggcagcatgt tggtgggcaa atgtcacaca agaatttgga attccctaca atccccaaag 4680tcaaggagta gtagaatcta tgaataaaga attaaagaaa attatagggc aggtcaggga 4740tcaagctgaa caccttaaga cagcagtaca gatggcagta ttcatccaca attttaaaag 4800aaaagggggg attggggggt acagtgcagg ggagagaata atagacataa tagcatcaga 4860catacaaact aaagaactac aaaaacaaat tatacaaatt caaaattttc gggtttatta 4920cagggacagc agagatccaa tttggaaagg accagcaaaa ctactctgga aaggtgaagg 4980ggcagtagta atacaggaca atagtgatat aaaggtagta ccaagaagaa aagcaaaaat 5040cattagggat tatggaaaac agatggcagg taatgattgt gtggcaggta gacaggatga 5100ggattagaac atggaacagt ttagtaaagc atcatatgta tgtctctaag aaagctggaa 5160agtggtttta tagacatcac tatgaaagta gacatccaaa agtaagttca gaagtacaca 5220ttccactagg agatgctaca ttggtaataa gaacatattg gggtctgcaa acaggagaaa 5280gagactggca attgggtcat ggggtctcca tagaatggag actgagaaga tatagcacac 5340aaatagatcc tgacctagca gaccaactaa ttcacctgca ttattttaac tgtttttcag 5400aatctgccat aaggagagcc atattaggac aagtagttag acctaggtgt gaatatcaag 5460caggacataa tcaggtagga tctctacaat atttagcatt gaaagcatta gtaacaccaa 5520caaggacaag gctacctttg cctagtgtta agaaattaac agaagacaga tggaacaagc 5580cccagaagac caggggccac aaagggagcc gttcagtgaa tggacactag aattgttaga 5640agagcttaaa catgaagctg ttagacattt tcctaggcca tggctccatg gattagggca 5700acatatctac aacacatatg gggatacttg ggaaggggtt ggagctataa taagaatgtt 5760gcaataacta ctgtttgttc atttcagaat tgggtgccaa catagcagaa taggcattat 5820tcgagggaga agaggcagga atggagctgg tagatcctaa cctagagccc tggaaacacc 5880cgggaagtca gcctacaact gcttgtagca attgctattg taaaaagtgc tgctggcatt 5940gccaattatg ctttctgaac aagggcttag gtatctccta tggcaggaag aagcggagac 6000gccgacgagg aactcctcag agccgtcagg atcatcaaaa tcctgtacca aagcagtgag 6060tagcaataat tagtatatgt gatgcaacct ttagaaatat ctgcaatagt aggactagta 6120gtagctttca tagcagctgt agttgtgtgg accatagtat ttatagaata taggaaaata 6180agaaaacaga agaaaataga taggttactt gaaagaataa aggaaagagc agaagatagt 6240ggcaatgaga gtgatgggga cacagaggaa ttatccaccc ttattgaggt gggtgactat 6300catcttgtgg ataatcataa tttgtagtgc tgaaaagttg tgggttacag tatactatgg 6360ggtaccggtg tggagagacg cagagaccac tctattttgt gcatcagagg ctagggcata 6420tgatccagaa gcacataatg tctgggctac acatgcctgt gtacccacag accccaaccc 6480acaagaatta cctttagaaa atgtaacaga agagtttaac atgtggaaaa ataacatggt 6540agagcagatg catgaagata taattagtct atgggaccaa agcctaaagc catgtgtaaa 6600gctaacccct ctctgcgtta cattaaattg ttctgacgtc accttcaaca gaacctttga 6660tagtgaaatg aaaaaggaaa taaaaaactg cactttcaat acgaccacag aaagaataga 6720taaaaaagag aaagcatatg cactttttta taaacttgat ataaaagaac ttgagggaag 6780taaggataat agtagtaata gtagccagtt tatactaata cattgtaata cctcgaccat 6840tgcacaggct tgtccaaagg tgtcctttga gccaattccc atacattatt gtgccccagc 6900tggttttgcg attctggagt gtaaagagaa gaatttcaat ggaacagggc catgcaagaa 6960tgtcagtaca gtacaatgca cacatggaat caaaccagta gtgtcaactc aactgctact 7020aaatggcagt ctagcagaag aagaggtaat gattagatct gaaaatatca caaacaatgc 7080caaaaccata atagtacagc tggttactcc tgtaaaaatt aattgtacca gacctggcaa 7140tcctataaga aaaagggtag gtataggacc aggacaagca ttccatgcaa caggtaatat 7200aataggagac ataagacgag cacagtgtaa tgtcagtgga acagaatgga aggaagcttt 7260acaaaaggta actgaacaat tagggaagca ctttaatgtt agcacaataa actttactaa 7320atcctcagga ggggatgtag aaattacaac acatagtttt aattgtggag gagagttttt 7380ctattgcaat acatcaggac tatttaatag cacttggagt aggaatagca ctgataacag 7440cacttggaat atcaatgaca ctgtcagctc aaatcacaca ggaaatataa ctctccaatg 7500cagaataagg caagttgtaa gaatgtggca gagggtagga caagcaatgt atgcccctcc 7560catcccagga gaaattaagt gtaaatcaaa cattacagga atactattga caagagatgg 7620agggacaaaa aatcagaatg caagtgagga atttaataca actgaggtat tcagacctgg 7680aggaggagac atgagggaca attggagaag tgaattatat aaatataagg tagtacaaat 7740tgaaccaata ggtgtggcac ccacccgtgc aaaaagaaga gtggtgcaga aagaaaaaag 7800agcagttgga ctgggagctt tcttccttgg gttcttagga gttgcaggaa gcactatggg 7860cgcagcgtca ataacgctga cggtacaggc cagacaatta ttgtctggta tagtgcaaca 7920gcagagcaat ctgctgaagg ctatagaggc tcaacaacat atattgagac tcacggtctg 7980gggcattaaa cagctccagg caagagtcct ggctctggaa agatacctaa aggatcaaca 8040gctcctagga atttggggct gctctggaaa actcatctgc accactaatg tgccctggaa 8100ctctagttgg agtaataaaa cttatgataa gatatgggac aatatgacct ggatgcaatg 8160ggatagagaa attagcaatt acacagagat aatatatgat ctaattgaag aatcgcagaa 8220gcagcaagaa aagaatgaac aagaattatt ggcattggac aagtggacaa gtctgtggaa 8280ttggtttagc atatcaaact ggctatggta tataagaata ttcataatga tagtaggagg 8340cttgataggc ttaagaatag tttgtgctgt gcttaatgta ataaatagag ttaggcaggg 8400atactcacct ttgtcattcc agacccttac ccaccaccag agggaacccg acagacccga 8460aagaatcgaa gaaggaggtg gcgagcgaga cagagacaga tccgtgcgct tagtgagcgg 8520attcttagca cttgcctggg acgatctgag gagcctgtgc ctcttcagct accaccaatt 8580gagagacttt gtattgattc tgggacacag cagtctcaag ggactgagac tggggtggga 8640agcactcaaa cttctggggg atcttctgtt atactggggt cgggaactaa agaatagtgc 8700tattaatttg cttgatacag tagcaatagc agtagctaac tggacagata gagttataga 8760aatagggcaa agagttggta gagctattct taacatacct acaagaatca gacagggatt 8820tgaaagggct ttgctataac atgggtggca agtggtcaaa aagtagcata gtgggatggc 8880ctcgggttag ggaaagaatg agacaaaacc ctccagaagg ggttagggaa agaatgagac 8940aaaaccctcc agcagcagca gaaggagtag gagcagcatc tcaagattta gctaaacatg 9000gagcaatcac aagcagtaac acagcagaaa ctaatgctga ctgtgcctgg ctagaagcac 9060aagagggaga ggaggtaggc tttccagtca ggccgcaagt acccttgaga ccaatgactt 9120ataagggagc ttttgatctc agccactttt taaaagaaaa ggggggactg gatgggttaa 9180tttactccaa gaaaaggcaa gagatccttg atctgtgggt ctataacaca caaggattct 9240tcccagattg gcagaactac acaccaggac caggggttag atacccactg acctttgggt 9300ggtgcttcaa actagtacca atggatccag cagagataga gaaagccaat gaagaagaga 9360acaactgttt attacatccc atctgccagc atggaatgga ggacgaagac agagaagtgc 9420tggtctggaa gtttgacagt cgcctggcac tcaaacacat agctcgagag aaacatccgg 9480agttttacaa agactgctga cacagaagtt gctgacaagg gactttccgc tggggacttt 9540ccgagggagg tgtggtttgg gaggagttgg ggagtggcta gccctcagat gctgcatata 9600agcagccgct tctcgcttgt actgggtctc tcttgctaga ccagatttga gcctgggagc 9660tctctggcta gtagagaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 9720agtagtgtgt gcccgtctgt tgtgtgactc tggttaacta gagatccctc agac 9774412255DNAArtificial SequencePlasmid 4gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 60ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 120tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 180agtggcgccc gaacagggac atgaaagcga aagggaaacc agaggagctc tctcgacgca 240ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc 300caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta 360agcgggggaa aattagatcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa 420tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct 480ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt 540cagacaggat cagaagaacg tagatcatta tataatacag tagcaaccct ctattgtgtg 600catcaaagga tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa 660aacaaaagta agaaaaaagc acagcaagca gcagctgaca caggacacag cagccaggtc 720agccaaaatt accctatagt gcagaacatc caggggcaaa tggtacatca ggccatatca 780cctagaactt taaatgcatg ggtaaaagta gtagaagaga aggctttcag cccagaagtg 840atacccatgt tttcagcatt atcagaagga gccaccccac aagatttaaa caccatgcta 900aacacagtgg ggggacatca agcagccatg caaatgttaa aagagaccat caatgaggaa 960gctgcagaat gggatagagt gcatccagtg catgcagggc ctattgcacc aggccagatg 1020agagaaccaa ggggaagtga catagcagga actactagta cccttcagga acaaatagga 1080tggatgacac ataatccacc tatcccagta ggagaaatct ataaaagatg gataatcctg 1140ggattaaata aaatagtaag aatgtatagc cctaccagca ttctggacat aagacaagga 1200ccaaaggaac cctttagaga ctatgtagac cgattctata aaactctaag agccgagcaa 1260gcttcacaag aggtaaaaaa ttggatgaca gaaaccttgt tggtccaaaa tgcgaaccca 1320gattgtaaga ctattttaaa agcattggga ccaggagcga cactagaaga aatgatgaca 1380gcatgtcagg gagtgggggg acccggccat aaagcaagag ttttggctga agcaatgagc 1440caagtaacaa atccagctac cataatgata cagaaaggca attttaggaa ccaaagaaag 1500actgttaagt gtttcaattg tggcaaagaa gggcacatag ccaaaaattg cagggcccct 1560aggaaaaagg gctgttggaa atgtggaaag gaaggacacc aaatgaaaga ttgtactgag 1620agacaggcta attttttagg gaagatctgg ccttcccaca agggaaggcc agggaatttt 1680cttcagagca gaccagagcc aacagcccca ccagaagaga gcttcaggtt tggggaagag 1740acaacaactc cctctcagaa gcaggagccg atagacaagg aactgtatcc tttagcttcc 1800ctcagatcac tctttggcag cgacccctcg tcacaataaa gatagggggg caattaaagg 1860aagctctatt agatacagga gcagatgata cagtattaga agaaatgaat ttgccaggaa 1920gatggaaacc aaaaatgata gggggaattg gaggttttat caaagtaaga cagtatgatc 1980agatactcat agaaatctgc ggacataaag ctataggtac agtattagta ggacctacac 2040ctgtcaacat aattggaaga aatctgttga ctcagattgg ctgcacttta aattttccca 2100ttagtcctat tgagactgta ccagtaaaat taaagccagg aatggatggc ccaaaagtta 2160aacaatggcc attgacagaa gaaaaaataa aagcattagt agaaatttgt acagaaatgg 2220aaaaggaagg aaaaatttca aaaattgggc ctgaaaatcc atacaatact ccagtatttg 2280ccataaagaa aaaagacagt actaaatgga gaaaattagt agatttcaga gaacttaata 2340agagaactca agatttctgg gaagttcaat taggaatacc acatcctgca gggttaaaac 2400agaaaaaatc agtaacagta ctggatgtgg gcgatgcata tttttcagtt cccttagata 2460aagacttcag gaagtatact gcatttacca tacctagtat aaacaatgag acaccaggga 2520ttagatatca gtacaatgtg cttccacagg gatggaaagg atcaccagca atattccagt 2580gtagcatgac aaaaatctta gagcctttta gaaaacaaaa tccagacata gtcatctatc 2640aatacatgga tgatttgtat gtaggatctg acttagaaat agggcagcat agaacaaaaa 2700tagaggaact gagacaacat ctgttgaggt ggggatttac cacaccagac aaaaaacatc 2760agaaagaacc tccattcctt tggatgggtt atgaactcca tcctgataaa tggacagtac 2820agcctatagt gctgccagaa aaggacagct ggactgtcaa tgacatacag aaattagtgg 2880gaaaattgaa ttgggcaagt cagatttatg cagggattaa agtaaggcaa ttatgtaaac 2940ttcttagggg aaccaaagca ctaacagaag tagtaccact aacagaagaa gcagagctag 3000aactggcaga aaacagggag attctaaaag aaccggtaca tggagtgtat tatgacccat 3060caaaagactt aatagcagaa atacagaagc aggggcaagg ccaatggaca tatcaaattt 3120atcaagagcc atttaaaaat ctgaaaacag gaaagtatgc aagaatgaag ggtgcccaca 3180ctaatgatgt gaaacaatta acagaggcag tacaaaaaat agccacagaa agcatagtaa 3240tatggggaaa gactcctaaa tttaaattac ccatacaaaa ggaaacatgg gaagcatggt 3300ggacagagta ttggcaagcc acctggattc ctgagtggga gtttgtcaat acccctccct 3360tagtgaagtt atggtaccag ttagagaaag aacccataat aggagcagaa actttctatg 3420tagatggggc agccaatagg gaaactaaat taggaaaagc aggatatgta actgacagag 3480gaagacaaaa agttgtcccc ctaacggaca caacaaatca gaagactgag ttacaagcaa 3540ttcatctagc tttgcaggat tcgggattag aagtaaacat agtgacagac tcacaatatg 3600cattgggaat cattcaagca caaccagata agagtgaatc agagttagtc agtcaaataa 3660tagagcagtt aataaaaaag gaaaaagtct acctggcatg ggtaccagca cacaaaggaa 3720ttggaggaaa tgaacaagta gataaattgg tcagtgctgg aatcaggaaa gtactatttt 3780tagatggaat agataaggcc caagaagaac atgagaaata tcacagtaat tggagagcaa 3840tggctagtga ttttaaccta ccacctgtag tagcaaaaga aatagtagcc agctgtgata 3900aatgtcagct aaaaggggaa gccatgcatg gacaagtaga ctgtagccca ggaatatggc 3960agctagattg tacacattta gaaggaaaag ttatcttggt agcagttcat gtagccagtg 4020gatatataga agcagaagta attccagcag agacagggca agaaacagca tacttcctct 4080taaaattagc aggaagatgg ccagtaaaaa cagtacatac agacaatggc agcaatttca 4140ccagtactac agttaaggcc gcctgttggt gggcggggat caagcaggaa tttggcattc 4200cctacaatcc ccaaagtcaa ggagtaatag aatctatgaa taaagaatta aagaaaatta 4260taggacaggt aagagatcag gctgaacatc ttaagacagc agtacaaatg gcagtattca 4320tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag 4380acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa 4440attttcgggt ttattacagg gacagcagag atccagtttg gaaaggacca gcaaagctcc 4500tctggaaagg tgaaggggca gtagtaatac aagataatag tgacataaaa gtagtgccaa 4560gaagaaaagc aaagatcatc agggattatg gaaaacagat ggcaggtgat gattgtgtgg 4620caagtagaca ggatgaggat taacacatgg aaaagattag taaaacacca tatgtatatt 4680tcaaggaaag ctaaggactg gttttataga catcactatg aaagtactaa tccaaaaata 4740agttcagaag tacacatccc actaggggat gctaaattag taataacaac atattggggt 4800ctgcatacag gagaaagaga ctggcatttg ggtcagggag tctccataga atggaggaaa 4860aagagatata gcacacaagt agaccctgac ctagcagacc aactaattca tctgcactat 4920tttgattgtt tttcagaatc tgctataaga aataccatat taggacgtat agttagtcct 4980aggtgtgaat atcaagcagg acataacaag gtaggatctc tacagtactt ggcactagca 5040gcattaataa aaccaaaaca gataaagcca cctttgccta gtgttaggaa actgacagag 5100gacagatgga acaagcccca gaagaccaag ggccacagag ggagccatac aatgaatgga 5160cactagagct tttagaggaa cttaagagtg aagctgttag acattttcct aggatatggc 5220tccataactt aggacaacat atctatgaaa cttacgggga tacttgggca ggagtggaag 5280ccataataag aattctgcaa caactgctgt ttatccattt cagaattggg tgtcgacata 5340gcagaatagg cgttactcga cagaggagag caagaaatgg agccagtaga tcctagacta 5400gagccctgga agcatccagg aagtcagcct aaaactgctt gtaccaattg ctattgtaaa 5460aagtgttgct ttcattgcca agtttgcttc ataacaaaag gcttaggcat ctcctatggc 5520aggaagaagc ggagacagcg acgaagacct cctcaaggca gtcagactca tcaagtttct 5580ctatcaaagc agtaagtaat acatgtaatg caacctctac aaatagcaat agtagcatta 5640gtagtagcaa taataatagc aatagttgtg tggtccatag taatcataga atataggaaa 5700atattaagac aaagaaaaat agacaggtta attgatagac taatagaaag agcagaagac 5760agtggcaatg agagtgaagg agaatatcaa gtcttatagc actgtggggg agatggggca 5820ccatgctcct tgggatgttg atgatctgta gtgctacaga aaaattgtgg gtcacagtct 5880attatggggt acctgtgtgg aagagaagca accaccactc tattttgtgc atcagatgct 5940aaagcatatg atacagaggt acataatgtt tgggccacac atgcctgtgt acccacagac 6000cccaacccac aagaagtaga attggaaaat gtgacagaaa attttaacat gtggaaaaat 6060aacatggtag aacagatgca tgaggatata atcagtttat gggatcaaag cctaaagcca 6120tgtgtaaaat taaccccact ctgtgttagt ttaaatggca ctgatttgaa actaatgata 6180ctaataccaa tagtagtagc gggagaatga taatgggaaa ggagacatga aaaattgctc 6240tttcaaaatc accacaaaca taagaggtaa ggtgcagaaa gaatatgcac ttttttataa 6300acttgatata ataccaatag ataataatag taccagatat acgttgacaa gttgtaacac 6360ctcagtcatt acacaggcct gtccaaaggt atcctttgag ccaattccca tacattattg 6420tgccccggct ggttttgcga ttctaaaatg taataataag acgttcaatg gaaaaggacc 6480atgttcaaat ggcagcacag tacaatgtac acatgggatt aggccagtag tatcaactca 6540actgctgtta aatggcagtc tagcagaaga agaggtagta attagatccg aaaatttcgc 6600ggacaatgct aaaaccataa tagtacagct gaacacatct gtagaaatta attgtacaag 6660acccaacaac aatacaagaa aaaaaatcca gtatccagag gggaccaggg agagcatttg 6720ttacaatagg agaaataata ggaataggac aagcacattg taacattagt agagcaaaat 6780ggaatgccac ttacaataag atagctagca aattaagaga acaatttgga aataaaacaa 6840tatgctttaa gcatacctca ggaggggacc cagaaattgt aacgcacagt tttaattgtg 6900gaggggaatt tttctactgt aattcaacac aactgtttaa tagtacttgg tttaatagta 6960cttggagtac tgaagggtca aataacactg aaggaagtaa cacaatcaca ctcccatgca 7020gaataaaaca atttataaac atgtggcagg aagtaggaaa agcaatgtat gcccctccca 7080tcagcggaca aattagatgt tcatcaaata ttacagggct gctattaaca agagatggtg 7140gtaataacaa caatggtccg agacttcaga cctggaggag gagatatgag ggacaattgg 7200agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc 7260aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc 7320cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta 7380caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt 7440gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga 7500atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct 7560ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg 7620gaacagattt ggaatcacac ggacctggat ggagtgggac agagaaatta acaattacac 7680aagcttaata tatctcttaa ttgaagaatc gcaaaaccaa aaagaaaaga atgaacaaga 7740attattagaa ttagacaaat gggcaagttt gtggaattgg tttacataac aaaatggctg 7800tggtatataa aattattcat aatgataata ggaggcttgg taggtttaag aatagttttt 7860gctgtacttt ctgtagtgaa tagagttagg cagggatacc caccattatc gtttcagacc 7920cacctccatg ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag 7980agagacagag acagatccat tcgattagtg aacggatcct tagcacttat ctgggacgat 8040ctgcggagcc tgtgcctctt cagctaccac cgcttgagag acttactctt

gattgtaacg 8100aggattgtgg aacttctggg acgcaggggg tgggaagccc tcaaatattg gtggaatctc 8160ctacaatatt ggagtcagga gctaaagaat agtgctgtta gcttgctcaa tgccacagct 8220atagcagtag ctgaggggac agatagggtt atagaagtag tacaagaagc ttatagagct 8280attcgccaca tacctagaag aataagacag ggcttggaaa ggattttgct ataagatggg 8340tggcaagtgg tcaaaaagta gtgtggttgg atggcctgct gtaagggaaa gaatgagacg 8400agctgagcca gcagcagatg gggtgggagc agcatctcga gacctagaaa aacatggagc 8460aatcacaagt agcaacacag cagctaacaa tgctgcttgt gcctggctag aagcacaaga 8520ggaggagaag gtgggttttc cagtcacacc tcaggtacct ttaagaccaa tgacttacaa 8580ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca 8640ctcccaacga agacaagata tccttgatct gtggatctac cacacacaag gctacttccc 8700tgattggcag aactacacac caggaccagg gatcagatat ccactgacct ttggatggcg 8760ctacaagcta gtaccagttg agccagagaa gttagaagaa gccaacaaag gagagaacac 8820cagcttgtta caccctgtga gcctgcatgg aatggatgac ccggagagag aagtgttaga 8880gtggaggttt gacagccgcc tagcatttca tcacgtggcc cgagagctgc atccggagta 8940cttcaagaac tgctgatatc gagcttgcta caagggactt tccgctgggg actttccagg 9000gaggcgtggc ctgggcggga ctggggagtg gcgagccctc agatcctgca tataagcagc 9060tgctttttgc ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg 9120gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag 9180tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag 9240tgtggaaaat ctctagcagt cacgtaactc tgaaaaggtc aaagatattg atgcttgata 9300cagaaggaag ggaataccag tgagttgtct ggactacact aaacatacct gaaatctgag 9360ttcgagaagt ataatagaac tggactccaa attaagtctc agttgcaaac tggattaatg 9420ggggatagat ttctatctag aggggggcag gtgaccttca gaccttggca ctggaggtgg 9480cccggcagaa gcgcggcatc gtggatcagt gctgcaccag catctgctct ctctaccaac 9540tggagaacta ctgcaactag gcccaccact accctgtcca cccctctgca atgaataaaa 9600cctttgaaag agcactacaa gttgtgtgta catgcgtgca tgtgcatatg tggtgcgggg 9660ggaacatgag tggggctggc tggagtggcg atgataagct gtcaaacatg agaattcttg 9720aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt 9780ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 9840tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 9900ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 9960ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 10020tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 10080gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct 10140gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat 10200acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 10260tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 10320caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 10380gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 10440cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac 10500tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 10560agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 10620tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 10680ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 10740acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 10800ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggctgc 10860tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 10920ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 10980ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 11040gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 11100ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 11160tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 11220ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 11280agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 11340agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 11400gggcggagcc tatggaaaaa cgccagcaac ggagatgcgc cgcgtgcggc tgctggagat 11460ggcggacgcg atggatatgt tctgccaagg gttggtttgc gcattcacag ttctccgcaa 11520gaattgattg gctccaattc ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc 11580attcaggtcg aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg 11640tatagggcgg cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa 11700atcgccgtga cgatcagcgg tccaatgatc gaagttaggc tggtaagagc cgcgagcgat 11760ccttgaagct gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg 11820ggcatcccga tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc 11880gtcggggagc tttttgcaaa agcctaggcc tccaaaaaag cctcctcact acttctggaa 11940tagctcagag gccgaggcgg cctcggcctc tgcataaata aaaaaaatta gtcagccatg 12000gggcggagaa tgggcggaac tgggcggagt taggggcggg atgggcggag ttaggggcgg 12060gactatggtt gctgactaat tgagatgcat gttcgaatcg cgaccgcggt ccggagtact 12120tcaagaactg ctgatatcga gcttgctaca agggactttc cgctggggac tttccaggga 12180ggcgtggcct gggcgggact ggggagtggc gagccctcag atcctgcata taagcagctg 12240ctttttgcct gtact 12255