# Patent

## Title
Administration of transposon-based vectors to reproductive organs

## Abstract

Methods and compositions for the administration of transposon-based vectors to the reproductive organs of animals and the creation of transgenic animals. Preferred methods involve administration of the transposon-based vectors to the lumen of the oviduct of an avian, expression of a vector derived transgene in the avian, and deposition of the resultant polypeptide in an egg. This invention allows for large amounts of protein to be deposited in the egg.

## Background
This application is a continuation application of U.S. patent application Ser. No. 10/746,149 filed Dec. 24, 2003, now abandoned, and is a continuation-in-part application of 10/609,019 filed Jun. 26, 2003, now U.S. Pat. No. 7,527,966, dated May 5, 2009, which claims benefit to Provisional Patent Application No. 60/441,392 filed Jan. 21, 2003, Provisional Patent Application No. 60/441,377 filed Jan. 21, 2003, Provisional Patent Application No. 60/441,502 filed Jan. 21, 2003, Provisional Patent Application No. 60/441,405 filed Jan. 21, 2003, Provisional Patent Application No. 60/441,447 filed Jan. 21, 2003, Provisional Patent Application No. 60/441,381 filed Jan. 21, 2003, and Provisional Patent Application No. 60/392,415 filed Jun. 26, 2002.


The U.S. Government has certain rights in this invention. The development of this invention was partially funded by the United States Government under a HATCH grant from the United States Department of Agriculture, partially funded by the United States Government with Formula 1433 funds from the United States Department of Agriculture and partially funded by the United States Government under contract DAAD 19-02016 awarded by the Army.


FIELD OF THE INVENTION
The present invention relates generally to administration of a transposon-based vector to the reproductive tract in an animal. The reproductive tract includes an ovary, ova within an ovary, and an oviduct. Such administration results in incorporation of a gene of interest contained in the vector in the ovary, the oviduct or an ovum of the animal. In some embodiments, the present invention further includes production of a protein encoded by the gene in an egg produced by the animal.
BACKGROUND OF THE INVENTION
Transgenic animals are desirable for a variety of reasons, including their potential as biological factories to produce desired molecules for pharmaceutical, diagnostic and industrial uses. This potential is attractive to the industry due to the inadequate capacity in facilities used for recombinant production of desired molecules and the increasing demand by the pharmaceutical industry for use of these facilities. Numerous attempts to produce transgenic animals have met several problems, including low rates of gene incorporation and unstable gene incorporation. Accordingly, improved gene technologies are needed for the development of transgenic animals for the production of desired molecules.
Improved gene delivery technologies are also needed for the treatment of disease in animals and humans. Many diseases and conditions can be treated with gene-delivery technologies, which provide a gene of interest to a patient suffering from the disease or the condition. An example of such disease is Type 1 diabetes. Type 1 diabetes is an autoimmune disease that ultimately results in destruction of the insulin producing β-cells in the pancreas. Although patients with Type 1 diabetes may be treated adequately with insulin injections or insulin pumps, these therapies are only partially effective. Insulin replacement, such as via insulin injection or pump administration, cannot fully reverse the defect in the vascular endothelium found in the hyperglycemic state (Pieper et al., 1996. Diabetes Res. Clin. Pract. Suppl. S157-S162). In addition, hyper- and hypoglycemia occurs frequently despite intensive home blood glucose monitoring. Finally, careful dietary constraints are needed to maintain an adequate ratio of calories consumed. This often causes major psychosocial stress for many diabetic patients. Development of gene therapies providing delivery of the insulin gene into the pancreas of diabetic patients could overcome many of these problems and result in improved life expectancy and quality of life.
Several of the prior art gene delivery technologies employed viruses that are associated with potentially undesirable side effects and safety concerns. The majority of current gene-delivery technologies useful for gene therapy rely on virus-based delivery vectors, such as adeno and adeno-associated viruses, retroviruses, and other viruses, which have been attenuated to no longer replicate. (Kay, M. A., et al. 2001. Nature Medicine 7:33-40).
There are multiple problems associated with the use of viral vectors. Firstly, they are not tissue-specific. In fact, a gene therapy trial using adenovirus was recently halted because the vector was present in the patient's sperm (Gene trial to proceed despite fears that therapy could change child's genetic makeup. The New York Times, Dec. 23, 2001). Secondly, viral vectors are likely to be transiently incorporated, which necessitates re-treating a patient at specified time intervals. (Kay, M. A., et al. 2001. Nature Medicine 7:33-40). Thirdly, there is a concern that a viral-based vector could revert to its virulent form and cause disease. Fourthly, viral-based vectors require a dividing cell for stable integration. Fifthly, viral-based vectors indiscriminately integrate into various cells, which can result in undesirable germline integration. Sixthly, the required high titers needed to achieve the desired effect have resulted in the death of one patient and they are believed to be responsible for induction of cancer in a separate study. (Science, News of the Week, Oct. 4, 2002).
Accordingly, what is needed is a new method to produce transgenic animals and humans with stably incorporated genes, in which the vector containing those genes does not cause disease or other unwanted side effects. There is also a need for DNA constructs that would be stably incorporated into the tissues and cells of animals and humans, including cells in the resting state that are not replicating. There is a further recognized need in the art for DNA constructs capable of delivering genes to specific tissues and cells of animals and humans.
When incorporating a gene of interest into an animal for the production of a desired protein or when incorporating a gene of interest in an animal or human for the treatment of a disease, it is often desirable to selectively activate incorporated genes using inducible promoters. These inducible promoters are regulated by substances either produced or recognized by the transcription control elements within the cell in which the gene is incorporated. In many instances, control of gene expression is desired in transgenic animals or humans so that incorporated genes are selectively activated at desired times and/or under the influence of specific substances. Accordingly, what is needed is a means to selectively activate genes introduced into the genome of cells of a transgenic animal or human. This can be taken a step further to cause incorporation to be tissue-specific, which prevents widespread gene incorporation throughout a patient's body (animal or human). This decreases the amount of DNA needed for a treatment, decreases the chance of incorporation in gametes, and targets gene delivery, incorporation, and expression to the desired tissue where the gene is needed to function. What is also needed is a rapid expression method for rapidly producing a protein or peptide of interest in eggs and milk of transgenic animals.
SUMMARY OF THE INVENTION
The present invention addresses the problems described above by providing new, effective and efficient compositions for producing transgenic animals and for treating disease in animals or humans. Transgenic animals include all egg-laying animals and milk-producing animals. Transgenic animals further include but are not limited to avians, fish, amphibians, reptiles, insects, mammals and humans. In another preferred embodiment, the animal is a milk-producing animal, including but not limited to bovine, porcine, ovine and equine animals. In a preferred embodiment, the animal is an avian animal. In another preferred embodiment, the animal is a mammal. Animals are made transgenic through administration of a composition comprising a transposon-based vector designed for incorporation of a gene of interest for production of a desired protein, together with an acceptable carrier. The compositions of the present invention are introduced into the reproductive system of an animal. The compositions of the present invention are administered to a reproductive organ including, but not limited to, an oviduct, an ovary, or into the duct system of the mammary gland. The compositions of the present invention are may be administered to a reproductive organ of an animal through the cloaca. The compositions of the present invention may be directly administered to a reproductive organ or can be administered to an artery leading to the reproductive organ. In a preferred embodiment, the compositions of the present invention are introduced into the reproductive system of an avian animal. In another preferred embodiment, the compositions of the present invention are introduced into the intramammary duct system of a mammal. A transfection reagent is optionally added to the composition before administration.
The transposon-based vectors of the present invention include a transposase, operably-linked to a first promoter, and a coding sequence for a protein or peptide of interest operably-linked to a second promoter, wherein the coding sequence for the protein or peptide of interest and its operably-linked promoter are flanked by transposase insertion sequences recognized by the transposase. The transposon-based vector also includes the following characteristics: a) one or more modified Kozak sequences at the 3′ end of the first promoter to enhance expression of the transposase; b) modifications of the codons for the first several N-terminal amino acids of the transposase, wherein the nucleotide at the third base position of each codon is changed to an A or a T without changing the corresponding amino acid; c) addition of one or more stop codons to enhance the termination of transposase synthesis; and/or, d) addition of an effective polyA sequence operably-linked to the transposase to further enhance expression of the transposase gene. In some embodiments, the effective polyA sequence is an avian optimized polyA sequence.
The present invention also provides for tissue-specific incorporation and/or expression of a gene of interest. Tissue-specific incorporation of a gene of interest may be achieved by placing the transposase gene under the control of a tissue-specific promoter, whereas tissue-specific expression of a gene of interest may be achieved by placing the gene of interest under the control of a tissue-specific promoter. In some embodiments, the gene of interest is transcribed under the influence of an ovalbumin, or other oviduct specific, promoter. Linking the gene of interest to an oviduct specific promoter in an egg-laying animal results in synthesis of a desired molecule and deposition of the desired molecule in a developing egg.
The present invention advantageously produces a high number of transgenic animals having a gene of interest stably incorporated. In some embodiments wherein the transposon-based vector is administered to the ovary, these transgenic animals successfully pass the desired gene to their progeny. Accordingly, the present invention can be used to obtain transgenic animals having the gene of interest incorporated into the germline through transfection of the ovary or the present invention can be used to obtain transgenic animals having the gene of interest incorporated into the oviduct in a tissue-specific manner. Both types of transgenic animals of the present invention produce large amounts of a desired molecule encoded by the transgene. Transgenic egg-laying animals, particularly avians, produce large amounts of a desired protein that is deposited in the egg for rapid harvest and purification.
Any desired gene may be incorporated into the novel transposon-based vectors of the present invention in order to synthesize a desired molecule in the transgenic animals. Proteins, peptides and nucleic acids are preferred desired molecules to be produced by the transgenic animals of the present invention. Particularly preferred proteins are antibody proteins and other immunopharmecuetical proteins.
This invention provides a composition useful for the production of transgenic hens capable of producing substantially high amounts of a desired protein or peptide. Entire flocks of transgenic birds may be developed very quickly in order to produce industrial amounts of desired molecules. The present invention solves the problems inherent in the inadequate capacity of fermentation facilities used for bacterial production of molecules and provides a more efficient and economical way to produce desired molecules. Accordingly, the present invention provides a means to produce large amounts of therapeutic, diagnostic and reagent molecules.
Transgenic chickens are excellent in terms of convenience and efficiency of manufacturing molecules such as proteins and peptides. Starting with a single transgenic rooster, thousands of transgenic offspring can be produced within a year. (In principle, up to forty million offspring could be produced in just three generations). Each transgenic female is expected to lay at least 250 eggs/year, each potentially containing hundreds of milligrams of the selected protein. Flocks of chickens numbering in the hundreds of thousands are readily handled through established commercial systems. The technologies for obtaining eggs and fractionating them are also well known and widely accepted. Thus, for each therapeutic, diagnostic, or other protein of interest, large amounts of a substantially pure material can be produced at relatively low incremental cost.
A wide range of recombinant peptides and proteins can be produced in transgenic egg-laying animals. Enzymes, hormones, antibodies, growth factors, serum proteins, commodity proteins, biological response modifiers, peptides and designed proteins may all be made through practice of the present invention. For example, rough estimates suggest that it is possible to produce in bulk growth hormone, insulin, or Factor VIII, and deposit them in egg whites, for an incremental cost in the order of one dollar per gram. At such prices it is feasible to consider administering such medical agents by inhalation or even orally, instead of through injection. Even if bioavailability rates through these avenues were low, the cost of a much higher effective-dose would not be prohibitive.
In one embodiment, the egg-laying transgenic animal is an avian. The method of the present invention may be used in avians including Ratites, Psittaciformes, Falconiformes, Piciformes, Strigiformes, Passeriformes, Coraciformes, Ralliformes, Cuculiformes, Columbiformes, Galliformes, Anseriformes, and Herodiones. Preferably, the egg-laying transgenic animal is a poultry bird. More preferably, the bird is a chicken, turkey, duck, goose or quail. Another preferred bird is a ratite, such as, an emu, an ostrich, a rhea, or a cassowary. Other preferred birds are partridge, pheasant, kiwi, parrot, parakeet, macaw, falcon, eagle, hawk, pigeon, cockatoo, song birds, jay bird, blackbird, finch, warbler, canary, toucan, mynah, or sparrow.
Accordingly, it is an object of the present invention to provide novel transposon-based vectors.
It is another object of the present invention to provide novel transposon-based vectors that encode for the production of desired proteins or peptides in cells.
It is an object of the present invention to produce transgenic animals through intraoviduct or intraovarian administration of a transposon-based vector.
Another object of the present invention is to produce transgenic animals through intraoviduct or intraovarian administration of a transposon-based vector, wherein the transgenic animals produce desired proteins or peptides.
It is further an object of the present invention to provide a method to produce transgenic animals through intraovarian administration of a transposon-based vector that are capable of producing transgenic progeny.
Yet another object of the present invention is to provide a method to produce transgenic animals through intraoviduct or intraovarian administration of a transposon-based vector that are capable of producing a desired molecule, such as a protein, peptide or nucleic acid.
Another object of the present invention is to provide a method to produce transgenic animals through intraoviduct or intraovarian administration of a transposon-based vector, wherein such administration results in modulation of endogenous gene expression.
It is yet another object of the present invention to provide a method to produce transgenic avians through intraoviduct or intraovarian administration of a transposon-based vector that are capable of producing proteins, peptides or nucleic acids.
It is another object of the present invention to produce transgenic animals through intraoviduct or intraovarian administration of a transposon-based vector encoding an antibody or a fragment thereof.
Still another object of the present invention is to provide a method to produce transgenic avians through intraoviduct or intraovarian administration of a transposon-based vector that are capable of producing proteins or peptides and depositing these proteins or peptides in the egg.
Another object of the present invention is to provide transgenic avians that contain a stably incorporated transgene.
Still another object of the present invention is to provide eggs containing desired proteins or peptides encoded by a transgene incorporated into the transgenic avian that produces the egg.
It is further an object of the present invention to provide a method to produce transgenic milk-producing animals through administration of a transposon-based vector that are capable of producing proteins, peptides or nucleic acids.
Still another object of the present invention is to provide a method to produce transgenic milk-producing animals through administration of a transposon-based vector that are capable of producing proteins or peptides and depositing these proteins or peptides in their milk.
Another object of the present invention is to provide transgenic milk-producing animals that contain a stably incorporated transgene.
Another object of the present invention is to provide transgenic milk-producing animals that are capable of producing proteins or peptides and depositing these proteins or peptides in their milk.
Yet another object of the present invention is to provide milk containing desired molecules encoded by a transgene incorporated into the transgenic milk-producing animals that produce the milk.
Still another object of the present invention is to provide milk containing desired proteins or peptides encoded by a transgene incorporated into the transgenic milk-producing animals that produce the milk.
An advantage of the present invention is that transgenic animals are produced with higher efficiencies than observed in the prior art.
Another advantage of the present invention is that these transgenic animals possess high copy numbers of the transgene.
Another advantage of the present invention is that the transgenic animals produce large amounts of desired molecules encoded by the transgene.
Still another advantage of the present invention is that desired molecules are produced by the transgenic animals much more efficiently and economically than prior art methods, thereby providing a means for large scale production of desired molecules, particularly proteins and peptides.
Yet another advantage of the present invention is that the desired proteins and peptides are produced rapidly after making animals transgenic through introduction of the vectors of the present invention.
These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and claims.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 depicts schematically a transposon-based vector containing a transposase operably linked to a first promoter and a gene of interest operably-linked to a second promoter, wherein the gene of interest and its operably-linked promoter are flanked by insertion sequences (IS) recognized by the transposase. “Pro” designates a promoter. In this and subsequent figures, the size of the actual nucleotide sequence is not necessarily proportionate to the box representing that sequence.
FIG. 2 depicts schematically a transposon-based vector for targeting deposition of a polypeptide in an egg white wherein Ov pro is the ovalbumin promoter, Ov protein is the ovalbumin protein and PolyA is a polyadenylation sequence. The TAG sequence includes a spacer sequence, the gp41 hairpin loop from HIV I and a protease cleavage site.
FIG. 3 depicts schematically a transposon-based vector for targeting deposition of a polypeptide in an egg white wherein Ovo pro is the ovomucoid promoter and Ovo SS is the ovomucoid signal sequence. The TAG sequence includes a spacer, the gp41 hairpin loop from HIV I and a protease cleavage site.
FIG. 4 depicts schematically a transposon based-vector for expression of an RNAi molecule. “Teti pro” indicates a tetracycline inducible promoter whereas “pro” indicates the pro portion of a prepro sequence as described herein. “Ovgen” indicates approximately 60 base pairs of an ovalbumin gene, “Ovotrans” indicates approximately 60 base pairs of an ovotransferrin gene and “Ovomucin” indicates approximately 60 base pairs of an ovomucin gene.
FIG. 5 is a picture of an SDS-PAGE gel wherein a pooled fraction of an isolated proinsulin fusion protein was run in lanes 4 and 6. Lanes 1 and 10 of the gel contain molecular weight standards, lanes 2 and 8 contain non-transgenic chicken egg white, and lanes 3, 5, 7 and 9 are blank.



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a new, effective and efficient method of producing transgenic animals, particularly egg-laying animals and milk-producing animals, through administration of a composition comprising a transposon-based vector designed for incorporation of a gene of interest and production of a desired molecule. The transposon-based vectors are administered to a reproductive organ including, but not limited to, an oviduct, an ovary, or into the duct system of the mammary gland. The vectors may be directly administered to a reproductive organ or can be administered to an artery leading to the reproductive organ or to a lymph system proximate to the cells to be genetically altered. The vectors may be administered to a reproductive organ of an animal through the cloaca. One method of direct administration is by injection, and in one embodiment, the lumen of the magnum of the oviduct is injected with a transposon-based vector. Another method of direct administration is by injection, and in one embodiment, the lumen of the infundibulum of the oviduct is injected with a transposon-based vector. A preferred intrarterial administration is an administration into an artery that supplies the oviduct or the ovary. In some embodiments, administration of the transposon-based vector to an oviduct or an artery that leads to the oviduct results in incorporation of the vector into the epithelial and/or secretory cells of the oviduct. In other embodiments, administration of the transposon-based vector to an ovary or an artery that leads to the ovary or a lymphatic system proximal to the ovary results in incorporation of the vector into an oocyte or a germinal disk inside the ovary.
Definitions
It is to be understood that as used in the specification and in the claims, “a” or “an” can mean one or more, depending upon the context in which it is used. Thus, for example, reference to “a cell” can mean that at least one cell can be utilized.
The term “antibody” is used interchangeably with the term “immunoglobulin” and is defined herein as a protein synthesized by an animal or a cell of the immune system in response to the presence of a foreign substance commonly referred to as an “antigen” or an “immunogen”. The term antibody includes fragments of antibodies. Antibodies are characterized by specific affinity to a site on the antigen, wherein the site is referred to an “antigenic determinant” or an “epitope”. Antigens can be naturally occurring or artificially engineered. Artificially engineered antigens include, but are not limited to, small molecules, such as small peptides, attached to haptens such as macromolecules, for example proteins, nucleic acids, or polysaccharides. Artificially designed or engineered variants of naturally occurring antibodies and artificially designed or engineered antibodies not occurring in nature are all included in the current definition. Such variants include conservatively substituted amino acids and other forms of substitution as described in the section concerning proteins and polypeptides.
As used herein, the term “egg-laying animal” includes all amniotes such as birds, turtles, lizards and monotremes. Monotremes are egg-laying mammals and include the platypus and echidna. The term “bird” or “fowl,” as used herein, is defined as a member of the Aves class of animals which are characterized as warm-blooded, egg-laying vertebrates primarily adapted for flying. Avians include, without limitation, Ratites, Psittaciformes, Falconiformes, Piciformes, Strigiformes, Passeriformes, Coraciformes, Ralliformes, Cuculiformes, Columbiformes, Galliformes, Anseriformes, and Herodiones. The term “Ratite,” as used herein, is defined as a group of flightless, mostly large, running birds comprising several orders and including the emus, ostriches, kiwis, and cassowaries. The term “Psittaciformes”, as used herein, includes parrots and refers to a monofamilial order of birds that exhibit zygodactylism and have a strong hooked bill. A “parrot” is defined as any member of the avian family Psittacidae (the single family of the Psittaciformes), distinguished by the short, stout, strongly hooked beak. Avians include all poultry birds, especially chickens, geese, turkeys, ducks and quail. The term “chicken” as used herein denotes chickens used for table egg production, such as egg-type chickens, chickens reared for public meat consumption, or broilers, and chickens reared for both egg and meat production (“dual-purpose” chickens). The term “chicken” also denotes chickens produced by primary breeder companies, or chickens that are the parents, grandparents, great-grandparents, etc. of those chickens reared for public table egg, meat, or table egg and meat consumption.
The term “egg” is defined herein as including a large female sex cell enclosed in a porous, calcarous or leathery shell, produced by birds and reptiles. The term “ovum” is defined as a female gamete, and is also known as an egg. Therefore, egg production in all animals other than birds and reptiles, as used herein, is defined as the production and discharge of an ovum from an ovary, or “ovulation”. Accordingly, it is to be understood that the term “egg” as used herein is defined as a large female sex cell enclosed in a porous, calcarous or leathery shell, when a bird or reptile produces it, or it is an ovum when it is produced by all other animals.
The term “milk-producing animal” refers herein to mammals including, but not limited to, bovine, ovine, porcine, equine, and primate animals. Milk-producing animals include but are not limited to cows, llamas, camels, goats, reindeer, zebu, water buffalo, yak, horses, pigs, rabbits, non-human primates, and humans.
The term “gene” is defined herein to include a coding region for a protein, peptide or polypeptide.
The term “transgenic animal” refers to an animal having at least a portion of the transposon-based vector DNA incorporated into its DNA. While a transgenic animal includes an animal wherein the transposon-based vector DNA is incorporated into the germline DNA, a transgenic animal also includes an animal having DNA in one or more cells that contain a portion of the transposon-based vector DNA for any period of time. In a preferred embodiment, a portion of the transposon-based vector comprises a gene of interest. More preferably, the gene of interest is incorporated into the animal's DNA for a period of at least five days, more preferably the reproductive life of the animal, and most preferably the life of the animal. In a further preferred embodiment, the animal is an avian.
The term “vector” is used interchangeably with the terms “construct”, “DNA construct” and “genetic construct” to denote synthetic nucleotide sequences used for manipulation of genetic material, including but not limited to cloning, subcloning, sequencing, or introduction of exogenous genetic material into cells, tissues or organisms, such as birds. It is understood by one skilled in the art that vectors may contain synthetic DNA sequences, naturally occurring DNA sequences, or both. The vectors of the present invention are transposon-based vectors as described herein.
When referring to two nucleotide sequences, one being a regulatory sequence, the term “operably-linked” is defined herein to mean that the two sequences are associated in a manner that allows the regulatory sequence to affect expression of the other nucleotide sequence. It is not required that the operably-linked sequences be directly adjacent to one another with no intervening sequence(s).
The term “regulatory sequence” is defined herein as including promoters, enhancers and other expression control elements such as polyadenylation sequences, matrix attachment sites, insulator regions for expression of multiple genes on a single construct, ribosome entry/attachment sites, introns that are able to enhance expression, and silencers.
Transposon-Based Vectors
While not wanting to be bound by the following statement, it is believed that the nature of the DNA construct is an important factor in successfully producing transgenic animals. The “standard” types of plasmid and viral vectors that have previously been almost universally used for transgenic work in all species, especially avians, have low efficiencies and may constitute a major reason for the low rates of transformation previously observed. The DNA (or RNA) constructs previously used often do not integrate into the host DNA, or integrate only at low frequencies. Other factors may have also played a part, such as poor entry of the vector into target cells. The present invention provides transposon-based vectors that can be administered to an animal that overcome the prior art problems relating to low transgene integration frequencies. Two preferred transposon-based vectors of the present invention in which a tranposase, gene of interest and other polynucleotide sequences may be introduced are termed pTnMCS (SEQ ID NO:2) and pTnMod (SEQ ID NO:3).
The transposon-based vectors of the present invention produce integration frequencies an order of magnitude greater than has been achieved with previous vectors. More specifically, intratesticular injections performed with a prior art transposon-based vector (described in U.S. Pat. No. 5,719,055) resulted in 41% sperm positive roosters whereas intratesticular injections performed with the novel transposon-based vectors of the present invention resulted in 77% sperm positive roosters. Actual frequencies of integration were estimated by either or both comparative strength of the PCR signal from the sperm and histological evaluation of the testes and sperm by quantitative PCR.
The transposon-based vectors of the present invention include a transposase gene operably-linked to a first promoter, and a coding sequence for a desired protein or peptide operably-linked to a second promoter, wherein the coding sequence for the desired protein or peptide and its operably-linked promoter are flanked by transposase insertion sequences recognized by the transposase. The transposon-based vector also includes one or more of the following characteristics: a) one or more modified Kozak sequences comprising ACCATG (SEQ ID NO:1) at the 3′ end of the first promoter to enhance expression of the transposase; b) modifications of the codons for the first several N-terminal amino acids of the transposase, wherein the third base of each codon was changed to an A or a T without changing the corresponding amino acid; c) addition of one or more stop codons to enhance the termination of transposase synthesis; and/or, d) addition of an effective polyA sequence operably-linked to the transposase to further enhance expression of the transposase gene. The transposon-based vector may additionally or alternatively include one or more of the following Kozak sequences at the 3′ end of any promoter, including the promoter operably-linked to the transposase: ACCATGG (SEQ ID NO:4), AAGATGT (SEQ ID NO:5), ACGATGA (SEQ ID NO:6), AAGATGG (SEQ ID NO:7), GACATGA (SEQ ID NO:8), ACCATGA (SEQ ID NO:9), and ACCATGT (SEQ ID NO:52).
FIG. 1 shows a schematic representation of several components of the transposon-based vector. The present invention further includes vectors containing more than one gene of interest, wherein a second or subsequent gene of interest is operably-linked to the second promoter or to a different promoter. It is also to be understood that the transposon-based vectors shown in the Figures are representative of the present invention and that the order of the vector elements may be different than that shown in the Figures, that the elements may be present in various orientations, and that the vectors may contain additional elements not shown in the Figures.
Transposases and Insertion Sequences
In a further embodiment of the present invention, the transposase found in the transposase-based vector is an altered target site (ATS) transposase and the insertion sequences are those recognized by the ATS transposase. However, the transposase located in the transposase-based vectors is not limited to a modified ATS transposase and can be derived from any transposase. Transposases known in the prior art include those found in AC7, Tn5SEQ1, Tn916, Tn951, Tn1721, Tn2410, Tn1681, Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn9, Tn10, Tn30, Tn101, Tn903, Tn501, Tn1000 (γδ), Tn1681, Tn2901, AC transposons, Mp transposons, Spm transposons, En transposons, Dotted transposons, Mu transposons, Ds transposons, dSpm transposons and I transposons. According to the present invention, these transposases and their regulatory sequences are modified for improved functioning as follows: a) the addition one or more modified Kozak sequences comprising ACCATG (SEQ ID NO: 1) at the 3′ end of the promoter operably-linked to the transposase; b) a change of the codons for the first several amino acids of the transposase, wherein the third base of each codon was changed to an A or a T without changing the corresponding amino acid; c) the addition of one or more stop codons to enhance the termination of transposase synthesis; and/or, d) the addition of an effective polyA sequence operably-linked to the transposase to further enhance expression of the transposase gene.
Although not wanting to be bound by the following statement, it is believed that the modifications of the first several N-terminal codons of the transposase gene increase transcription of the transposase gene, in part, by increasing strand dissociation. It is preferable that between approximately 1 and 20, more preferably 3 and 15, and most preferably between 4 and 12 of the first N-terminal codons of the transposase are modified such that the third base of each codon is changed to an A or a T without changing the encoded amino acid. In one embodiment, the first ten N-terminal codons of the transposase gene are modified in this manner. It is also preferred that the transposase contain mutations that make it less specific for preferred insertion sites and thus increases the rate of transgene insertion as discussed in U.S. Pat. No. 5,719,055.
In some embodiments, the transposon-based vectors are optimized for expression in a particular host by changing the methylation patterns of the vector DNA. For example, prokaryotic methylation may be reduced by using a methylation deficient organism for production of the transposon-based vector. The transposon-based vectors may also be methylated to resemble eukaryotic DNA for expression in a eukaryotic host.
Transposases and insertion sequences from other analogous eukaryotic transposon-based vectors that can also be modified and used are, for example, the Drosophila P element derived vectors disclosed in U.S. Pat. No. 6,291,243; the Drosophila mariner element described in Sherman et al. (1998); or the sleeping beauty transposon. See also Hackett et al. (1999); D. Lampe et al., 1999. Proc. Natl. Acad. Sci. USA, 96:11428-11433; S. Fischer et al., 2001. Proc. Natl. Acad. Sci. USA, 98:6759-6764; L. Zagoraiou et al., 2001. Proc. Natl. Acad. Sci. USA, 98:11474-11478; and D. Berg et al. (Eds.), Mobile DNA, Amer. Soc. Microbiol. (Washington, D.C., 1989). However, it should be noted that bacterial transposon-based elements are preferred, as there is less likelihood that a eukaryotic transposase in the recipient species will recognize prokaryotic insertion sequences bracketing the transgene.
Many transposases recognize different insertion sequences, and therefore, it is to be understood that a transposase-based vector will contain insertion sequences recognized by the particular transposase also found in the transposase-based vector. In a preferred embodiment of the invention, the insertion sequences have been shortened to about 70 base pairs in length as compared to those found in wild-type transposons that typically contain insertion sequences of well over 100 base pairs.
While the examples provided below incorporate a “cut and insert” Tn10 based vector that is destroyed following the insertion event, the present invention also encompasses the use of a “rolling replication” type transposon-based vector. Use of a rolling replication type transposon allows multiple copies of the transposon/transgene to be made from a single transgene construct and the copies inserted. This type of transposon-based system thereby provides for insertion of multiple copies of a transgene into a single genome. A rolling replication type transposon-based vector may be preferred when the promoter operably-linked to gene of interest is endogenous to the host cell and present in a high copy number or highly expressed. However, use of a rolling replication system may require tight control to limit the insertion events to non-lethal levels. Tn1, Tn2, Tn3, Tn4, Tn5, Tn9, Tn21, Tn501, Tn551, Tn951, Tn1721, Tn2410 and Tn2603 are examples of a rolling replication type transposon, although Tn5 could be both a rolling replication and a cut and insert type transposon.
Stop Codons and PolyA Sequences
In one embodiment, the transposon-based vector contains two stop codons operably-linked to the transposase and/or to the gene of interest. In an alternate embodiment, one stop codon of UAA or UGA is operably linked to the transposase and/or to the gene of interest.
As used herein an “effective polyA sequence” refers to either a synthetic or non-synthetic sequence that contains multiple and sequential nucleotides containing an adenine base (an A polynucleotide string) and that increases expression of the gene to which it is operably-linked. A polyA sequence may be operably-linked to any gene in the transposon-based vector including, but not limited to, a transposase gene and a gene of interest. A preferred polyA sequence is optimized for use in the host animal or human. In one embodiment, the polyA sequence is optimized for use in an avian species and more specifically, a chicken. An avian optimized polyA sequence generally contains a minimum of 40 base pairs, preferably between approximately 40 and several hundred base pairs, and more preferably approximately 75 base pairs that precede the A polynucleotide string and thereby separate the stop codon from the A polynucleotide string. In one embodiment of the present invention, the polyA sequence comprises a conalbumin polyA sequence as provided in SEQ ID NO:11 and as taken from GenBank accession # Y00407, base pairs 10651-11058. In another embodiment, the polyA sequence comprises a synthetic polynucleotide sequence shown in SEQ ID NO:12. In yet another embodiment, the polyA sequence comprises an avian optimized polyA sequence provided in SEQ ID NO:13. A chicken optimized polyA sequence may also have a reduced amount of CT repeats as compared to a synthetic polyA sequence.
It is a surprising discovery of the present invention that such an avian optimized poly A sequence increases expression of a polynucleotide to which it is operably-linked in an avian as compared to a non-avian optimized polyA sequence. Accordingly, the present invention includes methods of or increasing incorporation of a gene of interest wherein the gene of interest resides in a transposon-based vector containing a transposase gene and wherein the transposase gene is operably linked to an avian optimized polyA sequence. The present invention also includes methods of increasing expression of a gene of interest in an avian that includes administering a gene of interest to the avian, wherein the gene of interest is operably-linked to an avian optimized polyA sequence. An avian optimized polyA nucleotide string is defined herein as a polynucleotide containing an A polynucleotide string and a minimum of 40 base pairs, preferably between approximately 40 and several hundred base pairs, and more preferably approximately 60 base pairs that precede the A polynucleotide string. The present invention further provides transposon-based vectors containing a gene of interest or transposase gene operably linked to an avian optimized polyA sequence.
Promoters and Enhancers
The first promoter operably-linked to the transposase gene and the second promoter operably-linked to the gene of interest can be a constitutive promoter or an inducible promoter. Constitutive promoters include, but are not limited to, immediate early cytomegalovirus (CMV) promoter, herpes simplex virus 1 (HSV1) immediate early promoter, SV40 promoter, lysozyme promoter, early and late CMV promoters, early and late HSV promoters, β-actin promoter, tubulin promoter, Rous-Sarcoma virus (RSV) promoter, and heat-shock protein (HSP) promoter. Inducible promoters include tissue-specific promoters, developmentally-regulated promoters and chemically inducible promoters. Examples of tissue-specific promoters include the glucose 6 phosphate (G6P) promoter, vitellogenin promoter, ovalbumin promoter, ovomucoid promoter, conalbumin promoter, ovotransferrin promoter, prolactin promoter, kidney uromodulin promoter, and placental lactogen promoter. In one embodiment, the vitellogenin promoter includes a polynucleotide sequence of SEQ ID NO: 14. The G6P promoter sequence may be deduced from a rat G6P gene untranslated upstream region provided in GenBank accession number U57552.1. Examples of developmentally-regulated promoters include the homeobox promoters and several hormone induced promoters. Examples of chemically inducible promoters include reproductive hormone induced promoters and antibiotic inducible promoters such as the tetracycline inducible promoter and the zinc-inducible metallothionine promoter.
Other inducible promoter systems include the Lac operator repressor system inducible by IPTG (isopropyl beta-D-thiogalactoside) (Cronin, A. et al. 2001. Genes and Development, v. 15), ecdysone-based inducible systems (Hoppe, U. C. et al. 2000. Mol. Ther. 1:159-164); estrogen-based inducible systems (Braselmann, S. et al. 1993. Proc. Natl. Acad. Sci. 90:1657-1661); progesterone-based inducible systems using a chimeric regulator, GLVP, which is a hybrid protein consisting of the GAL4 binding domain and the herpes simplex virus transcriptional activation domain, VP16, and a truncated form of the human progesterone receptor that retains the ability to bind ligand and can be turned on by RU486 (Wang, et al. 1994. Proc. Natl. Acad. Sci. 91:8180-8184); CID-based inducible systems using chemical inducers of dimerization (CIDs) to regulate gene expression, such as a system wherein rapamycin induces dimerization of the cellular proteins FKBP12 and FRAP (Belshaw, P. J. et al. 1996. J. Chem. Biol. 3:731-738; Fan, L. et al. 1999. Hum. Gene Ther. 10:2273-2285; Shariat, S. F. et al. 2001. Cancer Res. 61:2562-2571; Spencer, D. M. 1996. Curr. Biol. 6:839-847). Chemical substances that activate the chemically inducible promoters can be administered to the animal containing the transgene of interest via any method known to those of skill in the art.
Other examples of cell or tissue-specific and constitutive promoters include but are not limited to smooth-muscle SM22 promoter, including chimeric SM22alpha/telokin promoters (Hoggatt A. M. et al., 2002. Circ Res. 91(12):1151-9); ubiquitin C promoter (Biochim Biophys Acta, 2003. Jan. 3; 1625(1):52-63); Hsf2 promoter; murine COMP (cartilage oligomeric matrix protein) promoter; early B cell-specific mb-1 promoter (Sigvardsson M., et al., 2002. Mol. Cell. Biol. 22(24):8539-51); prostate specific antigen (PSA) promoter (Yoshimura I. et al., 2002, J. Urol. 168(6):2659-64); exorh promoter and pineal expression-promoting element (Asaoka Y., et al., 2002. Proc. Natl. Acad. Sci. 99(24):15456-61); neural and liver ceramidase gene promoters (Okino N. et al., 2002. Biochem. Biophys. Res. Commun. 299(1):160-6); PSP94 gene promoter/enhancer (Gabril M. Y. et al., 2002. Gene Ther. 9(23):1589-99); promoter of the human FAT/CD36 gene (Kuriki C., et al., 2002. Biol. Pharm. Bull. 25(11):1476-8); VL30 promoter (Staplin W. R. et al., 2002. Blood Oct. 24, 2002); and, IL-10 promoter (Brenner S., et al., 2002. J. Biol. Chem. Dec. 18, 2002).
Examples of avian promoters include, but are not limited to, promoters controlling expression of egg white proteins, such as ovalbumin, ovotransferrin (conalbumin), ovomucoid, lysozyme, ovomucin, g2 ovoglobulin, g3 ovoglobulin, ovoflavoprotein, ovostatin (ovomacroglobin), cystatin, avidin, thiamine-binding protein, glutamyl aminopeptidase minor glycoprotein 1, minor glycoprotein 2; and promoters controlling expression of egg-yolk proteins, such as vitellogenin, very low-density lipoproteins, low density lipoprotein, cobalamin-binding protein, riboflavin-binding protein, biotin-binding protein (Awade, 1996. Z. Lebensm. Unters. Forsch. 202:1-14). An advantage of using the vitellogenin promoter is that it is active during the egg-laying stage of an animal's life-cycle, which allows for the production of the protein of interest to be temporally connected to the import of the protein of interest into the egg yolk when the protein of interest is equipped with an appropriate targeting sequence. In some embodiments, the avian promoter is an oviduct-specific promoter. As used herein, the term “oviduct-specific promoter” includes, but is not limited to, ovalbumin; ovotransferrin (conalbumin); ovomucoid; 01, 02, 03, 04 or 05 avidin; ovomucin; g2 ovoglobulin; g3 ovoglobulin; ovoflavoprotein; and ovostatin (ovomacroglobin) promoters.
When germline transformation occurs via intraovarian administration, liver-specific promoters may be operably-linked to the gene of interest to achieve liver-specific expression of the transgene. Liver-specific promoters of the present invention include, but are not limited to, the following promoters, vitellogenin promoter, G6P promoter, cholesterol-7-alpha-hydroxylase (CYP7A) promoter, phenylalanine hydroxylase (PAH) promoter, protein C gene promoter, insulin-like growth factor I (IGF-I) promoter, bilirubin UDP-glucuronosyltransferase promoter, aldolase B promoter, furin promoter, metallothioneine promoter, albumin promoter, and insulin promoter.
Also included in the present invention are promoters that can be used to target expression of a protein of interest into the milk of a milk-producing animal including, but not limited to, β lactoglobin promoter, whey acidic protein promoter, lactalbumin promoter and casein promoter.
When germline transformation occurs via intraovarian administration, immune system-specific promoters may be operably-linked to the gene of interest to achieve immune system-specific expression of the transgene. Accordingly, promoters associated with cells of the immune system may also be used. Acute phase promoters such as interleukin (IL)-1 and IL-2 may be employed. Promoters for heavy and light chain Ig may also be employed. The promoters of the T cell receptor components CD4 and CD8, B cell promoters and the promoters of CR2 (complement receptor type 2) may also be employed. Immune system promoters are preferably used when the desired protein is an antibody protein.
Also included in this invention are modified promoters/enhancers wherein elements of a single promoter are duplicated, modified, or otherwise changed. In one embodiment, steroid hormone-binding domains of the ovalbumin promoter are moved from about −6.5 kb to within approximately the first 1000 base pairs of the gene of interest. Modifying an existing promoter with promoter/enhancer elements not found naturally in the promoter, as well as building an entirely synthetic promoter, or drawing promoter/enhancer elements from various genes together on a non-natural backbone, are all encompassed by the current invention.
Accordingly, it is to be understood that the promoters contained within the transposon-based vectors of the present invention may be entire promoter sequences or fragments of promoter sequences. For example, in one embodiment, the promoter operably linked to a gene of interest is an approximately 900 base pair fragment of a chicken ovalbumin promoter (SEQ ID NO:15). The constitutive and inducible promoters contained within the transposon-based vectors may also be modified by the addition of one or more modified Kozak sequences of ACCATG (SEQ ID NO:1).
As indicated above, the present invention includes transposon-based vectors containing one or more enhancers. These enhancers may or may not be operably-linked to their native promoter and may be located at any distance from their operably-linked promoter. A promoter operably-linked to an enhancer and a promoter modified to eliminate repressive regulatory effects are referred to herein as an “enhanced promoter.” The enhancers contained within the transposon-based vectors are preferably enhancers found in birds, and more preferably, an ovalbumin enhancer, but are not limited to these types of enhancers. In one embodiment, an approximately 675 base pair enhancer element of an ovalbumin promoter is cloned upstream of an ovalbumin promoter with 300 base pairs of spacer DNA separating the enhancer and promoter. In one embodiment, the enhancer used as a part of the present invention comprises base pairs 1-675 of a chicken ovalbumin enhancer from GenBank accession #S82527.1. The polynucleotide sequence of this enhancer is provided in SEQ ID NO:16.
Also included in some of the transposon-based vectors of the present invention are cap sites and fragments of cap sites. In one embodiment, approximately 50 base pairs of a 5′ untranslated region wherein the capsite resides are added on the 3′ end of an enhanced promoter or promoter. An exemplary 5′ untranslated region is provided in SEQ ID NO:17. A putative cap-site residing in this 5′ untranslated region preferably comprises the polynucleotide sequence provided in SEQ ID NO:18.
In one embodiment of the present invention, the first promoter operably-linked to the transposase gene is a constitutive promoter and the second promoter operably-linked to the gene of interest is a tissue-specific promoter. In the second embodiment, use of the first constitutive promoter allows for constitutive activation of the transposase gene and incorporation of the gene of interest into virtually all cell types, including the germline of the recipient animal. Although the gene of interest is incorporated into the germline generally, the gene of interest may only be expressed in a tissue-specific manner. A transposon-based vector having a constitutive promoter operably-linked to the transposase gene can be administered by any route, and in one embodiment, the vector is administered to an ovary, to an artery leading to the ovary or to a lymphatic system or fluid proximal to the ovary.
It should be noted that cell- or tissue-specific expression as described herein does not require a complete absence of expression in cells or tissues other than the preferred cell or tissue. Instead, “cell-specific” or “tissue-specific” expression refers to a majority of the expression of a particular gene of interest in the preferred cell or tissue, respectively.
When incorporation of the gene of interest into the germline is not preferred, the first promoter operably-linked to the transposase gene can be a tissue-specific promoter. For example, transfection of a transposon-based vector containing a transposase gene operably-linked to an oviduct specific promoter such as the ovalbumin promoter provides for activation of the transposase gene and incorporation of the gene of interest in the cells of the oviduct but not into the germline and other cells generally. In this embodiment, the second promoter operably-linked to the gene of interest can be a constitutive promoter or an inducible promoter. In a preferred embodiment, both the first promoter and the second promoter are an ovalbumin promoter. In embodiments wherein tissue-specific expression or incorporation is desired, it is preferred that the transposon-based vector is administered directly to the tissue of interest, to an artery leading to the tissue of interest or to fluids surrounding the tissue of interest. In a preferred embodiment, the tissue of interest is the oviduct and administration is achieved by direct injection into the oviduct or an artery leading to the oviduct. In a further preferred embodiment, administration is achieved by direct injection into the lumen of the magnum or the infundibulum of the oviduct. Indirect administration to the oviduct may occur through the cloaca.
Accordingly, cell specific promoters may be used to enhance transcription in selected tissues. In birds, for example, promoters that are found in cells of the fallopian tube, such as ovalbumin, conalbumin, ovomucoid and/or lysozyme, are used in the vectors to ensure transcription of the gene of interest in the epithelial cells and tubular gland cells of the fallopian tube, leading to synthesis of the desired protein encoded by the gene and deposition into the egg white. In mammals, promoters specific for the epithelial cells of the alveoli of the mammary gland, such as prolactin, insulin, beta lactoglobin, whey acidic protein, lactalbumin, casein, and/or placental lactogen, are used in the design of vectors used for transfection of these cells for the production of desired proteins for deposition into the milk. In liver cells, the G6P promoter may be employed to drive transcription of the gene of interest for protein production. Proteins made in the liver of birds may be delivered to the egg yolk.
In order to achieve higher or more efficient expression of the transposase gene, the promoter and other regulatory sequences operably-linked to the transposase gene may be those derived from the host. These host specific regulatory sequences can be tissue specific as described above or can be of a constitutive nature. For example, an avian actin promoter and its associated polyA sequence can be operably-linked to a transposase in a transposase-based vector for transfection into an avian. Examples of other host specific promoters that could be operably-linked to the transposase include the myosin and DNA or RNA polymerase promoters.
Directing Sequences
In some embodiments of the present invention, the gene of interest is operably-linked to a directing sequence or a sequence that provides proper conformation to the desired protein encoded by the gene of interest. As used herein, the term “directing sequence” refers to both signal sequences and targeting sequences. An egg directing sequence includes, but is not limited to, an ovomucoid signal sequence, an ovalbumin signal sequence, a cecropin pre pro signal sequence, and a vitellogenin targeting sequence. The term “signal sequence” refers to an amino acid sequence, or the polynucleotide sequence that encodes the amino acid sequence, that directs the protein to which it is linked to the endoplasmic reticulum in a eukaryote, and more preferably the translocational pores in the endoplasmic reticulum, or the plasma membrane in a prokaryote, or mitochondria, such as for the purpose of gene therapy for mitochondrial diseases. Signal and targeting sequences can be used to direct a desired protein into, for example, the milk, when the transposon-based vectors are administered to a milk-producing animal.
Signal sequences can also be used to direct a desired protein into, for example, a secretory pathway for incorporation into the egg yolk or the egg white, when the transposon-based vectors are administered to a bird or other egg-laying animal. One example of such a transposon-based vector is provided in FIG. 3 wherein the gene of interest is operably linked to the ovomucoid signal sequence. The present invention also includes a gene of interest operably-linked to a second gene containing a signal sequence. An example of such an embodiment is shown in FIG. 2 wherein the gene of interest is operably-linked to the ovalbumin gene that contains an ovalbumin signal sequence. Other signal sequences that can be included in the transposon-based vectors include, but are not limited to the ovotransferrin and lysozyme signal sequences. In one embodiment, the signal sequence is an ovalbumin signal sequence including a sequence shown in SEQ ID NO:19. In another embodiment, the signal sequence is a modified ovalbumin signal sequence including a sequence shown in SEQ ID NO:20 or SEQ ID NO:21.
As also used herein, the term “targeting sequence” refers to an amino acid sequence, or the polynucleotide sequence encoding the amino acid sequence, which amino acid sequence is recognized by a receptor located on the exterior of a cell. Binding of the receptor to the targeting sequence results in uptake of the protein or peptide operably-linked to the targeting sequence by the cell. One example of a targeting sequence is a vitellogenin targeting sequence that is recognized by a vitellogenin receptor (or the low density lipoprotein receptor) on the exterior of an oocyte. In one embodiment, the vitellogenin targeting sequence includes the polynucleotide sequence of SEQ ID NO:22. In another embodiment, the vitellogenin targeting sequence includes all or part of the vitellogenin gene. Other targeting sequences include VLDL and Apo E, which are also capable of binding the vitellogenin receptor. Since the ApoE protein is not endogenously expressed in birds, its presence may be used advantageously to identify birds carrying the transposon-based vectors of the present invention.
Genes of Interest Encoding Desired Proteins
A gene of interest selected for stable incorporation is designed to encode any desired protein or peptide or to regulate any cellular response. In some embodiments, the desired proteins or peptides are deposited in an egg or in milk. It is to be understood that the present invention encompasses transposon-based vectors containing multiple genes of interest. The multiple genes of interest may each be operably-linked to a separate promoter and other regulatory sequence(s) or may all be operably-linked to the same promoter and other regulatory sequences(s). In one embodiment, multiple gene of interest are linked to a single promoter and other regulatory sequence(s) and each gene of interest is separated by a cleavage site or a pro portion of a signal sequence. A gene of interest may contain modifications of the codons for the first several N-terminal amino acids of the gene of interest, wherein the third base of each codon is changed to an A or a T without changing the corresponding amino acid.
Protein and peptide hormones are a preferred class of proteins in the present invention. Such protein and peptide hormones are synthesized throughout the endocrine system and include, but are not limited to, hypothalamic hormones and hypophysiotropic hormones, anterior, intermediate and posterior pituitary hormones, pancreatic islet hormones, hormones made in the gastrointestinal system, renal hormones, thymic hormones, parathyroid hormones, adrenal cortical and medullary hormones. Specifically, hormones that can be produced using the present invention include, but are not limited to, chorionic gonadotropin, corticotropin, erythropoietin, glucagons, IGF-1, oxytocin, platelet-derived growth factor, calcitonin, follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone, insulin, gonadotropin-releasing hormone and its analogs, vasopressin, octreotide, somatostatin, prolactin, adrenocorticotropic hormone, antidiuretic hormone, thyrotropin-releasing hormone (TRH), growth hormone-releasing hormone (GHRH), dopamine, melatonin, thyroxin (T4), parathyroid hormone (PTH), glucocorticoids such as cortisol, mineralocorticoids such as aldosterone, androgens such as testosterone, adrenaline (epinephrine), noradrenaline (norepinephrine), estrogens such as estradiol, progesterone, glucagons, calcitrol, calciferol, atrial-natriuretic peptide, gastrin, secretin, cholecystokinin (CCK), neuropeptide Y, ghrelin, PYY3-36, angiotensinogen, thrombopoietin, and leptin. By using appropriate polynucleotide sequences, species-specific hormones may be made by transgenic animals.
In one embodiment of the present invention, the gene of interest is a proinsulin gene and the desired molecule is insulin. Proinsulin consists of three parts: a C-peptide and two strands of amino acids (the alpha and beta chains) that later become linked together to form the insulin molecule. FIGS. 2 and 3 are schematics of transposon-based vector constructs containing a proinsulin gene operably-linked to an ovalbumin promoter and ovalbumin protein or an ovomucoid promoter and ovomucoid signal sequence, respectively. In these embodiments, proinsulin is expressed in the oviduct tubular gland cells and then deposited in the egg white. One example of a proinsulin polynucleotide sequence is shown in SEQ ID NO:23, wherein the C-peptide cleavage site spans from Arg at position 31 to Arg at position 65.
Serum proteins including lipoproteins such as high density lipoprotein (HDL), HDL-Milano and low density lipoprotein, albumin, clotting cascade factors, factor VIII, factor IX, fibrinogen, and globulins are also included in the group of desired proteins of the present invention. Immunoglobulins are one class of desired globulin molecules and include but are not limited to IgG, IgM, IgA, IgD, IgE, IgY, lambda chains, kappa chains and fragments thereof; Fc fragments, and Fab fragments. Desired antibodies include, but are not limited to, naturally occurring antibodies, human antibodies, humanized antibodies, and hybrid antibodies. Genes encoding modified versions of naturally occurring antibodies or fragments thereof and genes encoding artificially designed antibodies or fragments thereof may be incorporated into the transposon-based vectors of the present invention. Desired antibodies also include antibodies with the ability to bind specific ligands, for example, antibodies against proteins associated with cancer-related molecules, such as anti-her 2, or anti-CA125. Accordingly, the present invention encompasses a transposon-based vector containing one or more genes encoding a heavy immunoglobulin (Ig) chain and a light Ig chain. Further, more than one gene encoding for more than one antibody may be administered in one or more transposon-based vectors of the present invention. In this manner, an egg may contain more than one type of antibody in the egg white, the egg yolk or both. In one embodiment, a transposon-based vector contains a heavy Ig chain and a light Ig chain, both operably linked to a promoter.
Antibodies used as therapeutic reagents include but are not limited to antibodies for use in cancer immunotherapy against specific antigens, or for providing passive immunity to an animal or a human against an infectious disease or a toxic agent. Antibodies used as diagnostic reagents include, but are not limited to antibodies that may be labeled and detected with a detector, for example antibodies with a fluorescent label attached that may be detected following exposure to specific wavelengths. Such labeled antibodies may be primary antibodies directed to a specific antigen, for example, rhodamine-labeled rabbit anti-growth hormone, or may be labeled secondary antibodies, such as fluorescein-labeled goat-anti chicken IgG. Such labeled antibodies are known to one of ordinary skill in the art. Labels useful for attachment to antibodies are also known to one of ordinary skill in the art. Some of these labels are described in the “Handbook of Fluorescent Probes and Research Products”, ninth edition, Richard P. Haugland (ed) Molecular Probes, Inc. Eugene, Oreg.), which is incorporated herein in its entirety.
Antibodies produced with using the present invention may be used as laboratory reagents for numerous applications including radioimmunoassay, western blots, dot blots, ELISA, immunoaffinity columns and other procedures requiring antibodies as known to one of ordinary skill in the art. Such antibodies include primary antibodies, secondary antibodies and tertiary antibodies, which may be labeled or unlabeled.
Antibodies that may be made with the practice of the present invention include, but are not limited to primary antibodies, secondary antibodies, designer antibodies, anti-protein antibodies, anti-peptide antibodies, anti-DNA antibodies, anti-RNA antibodies, anti-hormone antibodies, anti-hypophysiotropic peptides, antibodies against non-natural antigens, anti-anterior pituitary hormone antibodies, anti-posterior pituitary hormone antibodies, anti-venom antibodies, anti-tumor marker antibodies, antibodies directed against epitopes associated with infectious disease, including, anti-viral, anti-bacterial, anti-protozoal, anti-fungal, anti-parasitic, anti-receptor, anti-lipid, anti-phospholipid, anti-growth factor, anti-cytokine, anti-monokine, anti-idiotype, and anti-accessory (presentation) protein antibodies. Antibodies made with the present invention, as well as light chains or heavy chains, may also be used to inhibit enzyme activity.
Antibodies that may be produced using the present invention include, but are not limited to, antibodies made against the following proteins: Bovine γ-Globulin, Serum; Bovine IgG, Plasma; Chicken γ-Globulin, Serum; Human γ-Globulin, Serum; Human IgA, Plasma; Human IgA1, Myeloma; Human IgA2, Myeloma; Human IgA2, Plasma; Human IgD, Plasma; Human IgE, Myeloma; Human IgG, Plasma; Human IgG, Fab Fragment, Plasma; Human IgG, F(ab′)2 Fragment, Plasma; Human IgG, Fc Fragment, Plasma; Human IgG1, Myeloma; Human IgG2, Myeloma; Human IgG3, Myeloma; Human IgG4, Myeloma; Human IgM, Myeloma; Human IgM, Plasma; Human Immunoglobulin, Light Chain κ, Urine; Human Immunoglobulin, Light Chains κ and λ, Plasma; Mouse γ-Globulin, Serum; Mouse IgG, Serum; Mouse IgM, Myeloma; Rabbit γ-Globulin, Serum; Rabbit IgG, Plasma; and Rat γ-Globulin, Serum. In one embodiment, the transposon-based vector comprises the coding sequence of light and heavy chains of a murine monoclonal antibody that shows specificity for human seminoprotein (GenBank Accession numbers AY129006 and AY129304 for the light and heavy chains, respectively).
A further non-limiting list of antibodies that recognize other antibodies is as follows: Anti-Chicken IgG, heavy (H) & light (L) Chain Specific (Sheep); Anti-Goat γ-Globulin (Donkey); Anti-Goat IgG, Fc Fragment Specific (Rabbit); Anti-Guinea Pig γ-Globulin (Goat); Anti-Human Ig, Light Chain, Type κ Specific; Anti-Human Ig, Light Chain, Type λ Specific; Anti-Human IgA, α-Chain Specific (Goat); Anti-Human IgA, Fab Fragment Specific; Anti-Human IgA, Fc Fragment Specific; Anti-Human IgA, Secretory; Anti-Human IgE, ε-Chain Specific (Goat); Anti-Human IgE, Fc Fragment Specific; Anti-Human IgG, Fc Fragment Specific (Goat); Anti-Human IgG, γ-Chain Specific (Goat); Anti-Human IgG, Fc Fragment Specific; Anti-Human IgG, Fd Fragment Specific; Anti-Human IgG, H & L Chain Specific (Goat); Anti-Human IgG, Fc Fragment Specific; Anti-Human IgG2, Fc Fragment Specific; Anti-Human IgG2, Fd Fragment Specific; Anti-Human IgG3, Hinge Specific; Anti-Human IgG4, Fc Fragment Specific; Anti-Human IgM, Fc Fragment Specific; Anti-Human IgM, μ-Chain Specific; Anti-Mouse IgE, ε-Chain Specific; Anti-Mouse γ-Globulin (Goat); Anti-Mouse IgG, γ-Chain Specific (Goat); Anti-Mouse IgG, γ-Chain Specific (Goat) F(ab′)2 Fragment; Anti-Mouse IgG, H & L Chain Specific (Goat); Anti-Mouse IgM, μ-Chain Specific (Goat); Anti-Mouse IgM, H & L Chain Specific (Goat); Anti-Rabbit γ-Globulin (Goat); Anti-Rabbit IgG, Fc Fragment Specific (Goat); Anti-Rabbit IgG, H & L Chain Specific (Goat); Anti-Rat γ-Globulin (Goat); Anti-Rat IgG, H & L Chain Specific; Anti-Rhesus Monkey γ-Globulin (Goat); and, Anti-Sheep IgG, H & L Chain Specific.
Another non-limiting list of the antibodies that may be produced using the present invention is provided in product catalogs of companies such as Phoenix Pharmaceuticals, Inc. (530 Harbor Boulevard, Belmont, Calif.), Peninsula Labs (San Carlos Calif.), SIGMA (St. Louis, Mo.), Cappel ICN (Irvine, Calif.), Calbiochem (La Jolla, Calif.), which are all available electronically via the internet and which are incorporated herein by reference in their entirety. The polynucleotide sequences encoding these antibodies may be obtained from the scientific literature, from patents, and from databases such as GenBank. Alternatively, one of ordinary skill in the art may design the polynucleotide sequence to be incorporated into the genome by choosing the codons that encode for each amino acid in the desired antibody. Antibodies made by the transgenic animals of the present invention include antibodies that may be used as therapeutic reagents, for example in cancer immunotherapy against specific antigens, as diagnostic reagents and as laboratory reagents for numerous applications including immunoneutralization, radioimmunoassay, western blots, dot blots, ELISA, immunoprecipitation and immunoaffinity columns. Some of these antibodies include, but are not limited to, antibodies which bind the following ligands: adrenomedulin, amylin, calcitonin, amyloid, calcitonin gene-related peptide, cholecystokinin, gastrin, gastric inhibitory peptide, gastrin releasing peptide, interleukin, interferon, cortistatin, somatostatin, endothelin, sarafotoxin, glucagon, glucagon-like peptide, insulin, atrial natriuretic peptide, BNP, CNP, neurokinin, substance P, leptin, neuropeptide Y, melanin concentrating hormone, melanocyte stimulating hormone, orphanin, endorphin, dynorphin, enkephalin, enkephalin, leumorphin, peptide F, PACAP, PACAP-related peptide, parathyroid hormone, urocortin, corticotrophin releasing hormone, PHM, PHI, vasoactive intestinal polypeptide, secretin, ACTH, angiotensin, angiostatin, bombesin, endostatin, bradykinin, FMRF amide, galanin, gonadotropin releasing hormone (GnRH) associated peptide, GnRH, growth hormone releasing hormone, inhibin, granulocyte-macrophage colony stimulating factor (GM-CSF), motilin, neurotensin, oxytocin, vasopressin, osteocalcin, pancreastatin, pancreatic polypeptide, peptide YY, proopiomelanocortin, transforming growth factor, vascular endothelial growth factor, vesicular monoamine transporter, vesicular acetylcholine transporter, ghrelin, NPW, NPB, C3d, prokinetican, thyroid stimulating hormone, luteinizing hormone, follicle stimulating hormone, prolactin, growth hormone, beta-lipotropin, melatonin, kallikriens, kinins, prostaglandins, erythropoietin, p146 (SEQ ID NO:24 amino acid sequence, SEQ ID NO:25, nucleotide sequence), estrogen, testosterone, corticosteroids, mineralocorticoids, thyroid hormone, thymic hormones, connective tissue proteins, nuclear proteins, actin, avidin, activin, agrin, albumin, and prohormones, propeptides, splice variants, fragments and analogs thereof.
The following is yet another non-limiting list of antibodies that can be produced by the methods of present invention: abciximab (ReoPro), abciximab anti-platelet aggregation monoclonal antibody, anti-CD11a (hu1124), anti-CD18 antibody, anti-CD20 antibody, anti-cytomegalovirus (CMV) antibody, anti-digoxin antibody, anti-hepatitis B antibody, anti-HER-2 antibody, anti-idiotype antibody to GD3 glycolipid, anti-IgE antibody, anti-IL-2R antibody, antimetastatic cancer antibody (mAb 17-1A), anti-rabies antibody, anti-respiratory syncytial virus (RSV) antibody, anti-Rh antibody, anti-TCR, anti-TNF antibody, anti-VEGF antibody and fab fragment thereof, rattlesnake venom antibody, black widow spider venom antibody, coral snake venom antibody, antibody against very late antigen-4 (VLA-4), C225 humanized antibody to EGF receptor, chimeric (human & mouse) antibody against TNFα, antibody directed against GPIIb/IIIa receptor on human platelets, gamma globulin, anti-hepatitis B immunoglobulin, human anti-D immunoglobulin, human antibodies against S. aureus, human tetanus immunoglobulin, humanized antibody against the epidermal growth receptor-2, humanized antibody against the a subunit of the interleukin-2 receptor, humanized antibody CTLA4IG, humanized antibody to the IL-2 R α-chain, humanized anti-CD40-ligand monoclonal antibody (5c8), humanized mAb against the epidermal growth receptor-2, humanized mAb to rous sarcoma virus, humanized recombinant antibody (IgG1k) against respiratory syncytial virus (RSV), lymphocyte immunoglobulin (anti-thymocyte antibody), lymphocyte immunoglobulin, mAb against factor VII, MDX-210 bi-specific antibody against HER-2, MDX-22, MDX-220 bi-specific antibody against TAG-72 on tumors, MDX-33 antibody to FcγR1 receptor, MDX-447 bi-specific antibody against EGF receptor, MDX-447 bispecific humanized antibody to EGF receptor, MDX-RA immunotoxin (ricin A linked) antibody, Medi-507 antibody (humanized form of BTI-322) against CD2 receptor on T-cells, monoclonal antibody LDP-02, muromonab-CD3(OKT3) antibody, OKT3 (“muromomab-CD3”) antibody, PRO542 antibody, ReoPro (“abciximab”) antibody, and TNF-IgG fusion protein.
The antibodies prepared using the methods of the present invention may also be designed to possess specific labels that may be detected through means known to one of ordinary skill in the art. The antibodies may also be designed to possess specific sequences useful for purification through means known to one of ordinary skill in the art. Specialty antibodies designed for binding specific antigens may also be made in transgenic animals using the transposon-based vectors of the present invention.
Production of a monoclonal antibody using the transposon-based vectors of the present invention can be accomplished in a variety of ways. In one embodiment, two vectors may be constructed: one that encodes the light chain, and a second vector that encodes the heavy chain of the monoclonal antibody. These vectors may then be incorporated into the genome of the target animal by methods disclosed herein. In an alternative embodiment, the sequences encoding light and heavy chains of a monoclonal antibody may be included on a single DNA construct. For example, the coding sequence of light and heavy chains of a murine monoclonal antibody that show specificity for human seminoprotein can be expressed using transposon-based constructs of the present invention (GenBank Accession numbers AY129006 and AY129304 for the light and heavy chains, respectively).
Further included in the present invention are proteins and peptides synthesized by the immune system including those synthesized by the thymus, lymph nodes, spleen, and the gastrointestinal associated lymph tissues (GALT) system. The immune system proteins and peptides proteins that can be made in transgenic animals using the transposon-based vectors of the present invention include, but are not limited to, alpha-interferon, beta-interferon, gamma-interferon, alpha-interferon A, alpha-interferon 1, G-CSF, GM-CSF, interlukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TNF-α, and TNF-β. Other cytokines included in the present invention include cardiotrophin, stromal cell derived factor, macrophage derived chemokine (MDC), melanoma growth stimulatory activity (MGSA), macrophage inflammatory proteins 1 alpha (MIP-1 alpha), 2, 3 alpha, 3 beta, 4 and 5.
Lytic peptides such as p146 are also included in the desired molecules of the present invention. In one embodiment, the p146 peptide comprises an amino acid sequence of SEQ ID NO:24. The present invention also encompasses a transposon-based vector comprising a p146 nucleic acid comprising a polynucleotide sequence of SEQ ID NO:25.
Enzymes are another class of proteins that may be made through the use of the transposon-based vectors of the present invention. Such enzymes include but are not limited to adenosine deaminase, alpha-galactosidase, cellulase, collagenase, dnaseI, hyaluronidase, lactase, L-asparaginase, pancreatin, papain, streptokinase B, subtilisin, superoxide dismutase, thrombin, trypsin, urokinase, fibrinolysin, glucocerebrosidase and plasminogen activator. In some embodiments wherein the enzyme could have deleterious effects, additional amino acids and a protease cleavage site are added to the carboxy end of the enzyme of interest in order to prevent expression of a functional enzyme. Subsequent digestion of the enzyme with a protease results in activation of the enzyme.
Extracellular matrix proteins are one class of desired proteins that may be made through the use of the present invention. Examples include but are not limited to collagen, fibrin, elastin, laminin, and fibronectin and subtypes thereof. Intracellular proteins and structural proteins are other classes of desired proteins in the present invention.
Growth factors are another desired class of proteins that may be made through the use of the present invention and include, but are not limited to, transforming growth factor-α (“TGF-α”), transforming growth factor-β (TGF-β), platelet-derived growth factors (PDGF), fibroblast growth factors (FGF), including FGF acidic isoforms 1 and 2, FGF basic form 2 and FGF 4, 8, 9 and 10, nerve growth factors (NGF) including NGF 2.5s, NGF 7.0s and beta NGF and neurotrophins, brain derived neurotrophic factor, cartilage derived factor, growth factors for stimulation of the production of red blood cells, growth factors for stimulation of the production of white blood cells, bone growth factors (BGF), basic fibroblast growth factor, vascular endothelial growth factor (VEGF), granulocyte colony stimulating factor (G-CSF), insulin like growth factor (IGF) I and II, hepatocyte growth factor, glial neurotrophic growth factor (GDNF), stem cell factor (SCF), keratinocyte growth factor (KGF), transforming growth factors (TGF), including TGFs alpha, beta, beta1, beta2, beta3, skeletal growth factor, bone matrix derived growth factors, bone derived growth factors, erythropoietin (EPO) and mixtures thereof.
Another desired class of proteins that may be made may be made through the use of the present invention include, but are not limited to, leptin, leukemia inhibitory factor (LIF), tumor necrosis factor alpha and beta, ENBREL, angiostatin, endostatin, thrombospondin, osteogenic protein-1, bone morphogenetic proteins 2 and 7, osteonectin, somatomedin-like peptide, and osteocalcin.
Yet another desired class of proteins are blood proteins or clotting cascade protein including albumin, Prekallikrein, High molecular weight kininogen (HMWK) (contact activation cofactor; Fitzgerald, Flaujeac Williams factor), Factor I (Fibrinogen), Factor II (prothrombin), Factor III (Tissue Factor), Factor IV (calcium), Factor V (proaccelerin, labile factor, accelerator (Ac-) globulin), Factor VI (Va) (accelerin), Factor VII (proconvertin), serum prothrombin conversion accelerator (SPCA), cothromboplastin), Factor VIII (antihemophiliac factor A, antihemophilic globulin (AHG)), Factor IX (Christmas Factor, antihemophilic factor B, plasma thromboplastin component (PTC)), Factor X (Stuart-Prower Factor), Factor XI (Plasma thromboplastin antecedent (PTA)), Factor XII (Hageman Factor), Factor XIII (rotransglutaminase, fibrin stabilizing factor (FSF), fibrinoligase), von Willebrand factor, Protein C, Protein S, Thrombomodulin, Antithrombin III.
A non-limiting list of the peptides and proteins that may be made may be made through the use of the present invention is provided in product catalogs (electronically available over the internet) of companies such as Phoenix Pharmaceuticals, Inc. (530 Harbor Boulevard, Belmont, Calif.), Peninsula Labs (San Carlos Calif.), SIGMA (St. Louis, Mo.), Cappel ICN (Irvine, Calif.), and Calbiochem (La Jolla, Calif.). The polynucleotide sequences encoding these proteins and peptides of interest may be obtained from the scientific literature, from patents, and from databases such as GenBank. Alternatively, one of ordinary skill in the art may design the polynucleotide sequence to be incorporated into the genome by choosing the codons that encode for each amino acid in the desired protein or peptide.
Some of these desired proteins or peptides that may be made through the use of the present invention include but are not limited to the following: adrenomedulin, amylin, calcitonin, amyloid, calcitonin gene-related peptide, cholecystokinin, gastrin, gastric inhibitory peptide, gastrin releasing peptide, interleukin, interferon, cortistatin, somatostatin, endothelin, sarafotoxin, glucagon, glucagon-like peptide, insulin, atrial natriuretic peptide, BNP, CNP, neurokinin, substance P, leptin, neuropeptide Y, melanin concentrating hormone, melanocyte stimulating hormone, orphanin, endorphin, dynorphin, enkephalin, leumorphin, peptide F, PACAP, PACAP-related peptide, parathyroid hormone, urocortin, corticotrophin releasing hormone, PHM, PHI, vasoactive intestinal polypeptide, secretin, ACTH, angiotensin, angiostatin, bombesin, endostatin, bradykinin, FMRF amide, galanin, gonadotropin releasing hormone (GnRH) associated peptide, GnRH, growth hormone releasing hormone, inhibin, granulocyte-macrophage colony stimulating factor (GM-CSF), motilin, neurotensin, oxytocin, vasopressin, osteocalcin, pancreastatin, pancreatic polypeptide, peptide YY, proopiomelanocortin, transforming growth factor, vascular endothelial growth factor, vesicular monoamine transporter, vesicular acetylcholine transporter, ghrelin, NPW, NPB, C3d, prokinetican, thyroid stimulating hormone, luteinizing hormone, follicle stimulating hormone, prolactin, growth hormone, beta-lipotropin, melatonin, kallikriens, kinins, prostaglandins, erythropoietin, p146 (SEQ ID NO:24, amino acid sequence, SEQ ID NO:25, nucleotide sequence), thymic hormones, connective tissue proteins, nuclear proteins, actin, avidin, activin, agrin, albumin, apolipoproteins, apolipoprotein A, apolipoprotein B, and prohormones, propeptides, splice variants, fragments and analogs thereof.
Other desired proteins that may be made by the transgenic animals of the present invention include bacitracin, polymixin b, vancomycin, cyclosporine, anti-RSV antibody, alpha-1 antitrypsin (AAT), anti-cytomegalovirus antibody, anti-hepatitis antibody, anti-inhibitor coagulant complex, anti-rabies antibody, anti-Rh(D) antibody, adenosine deaminase, anti-digoxin antibody, antivenin crotalidae (rattlesnake venom antibody), antivenin latrodectus (black widow spider venom antibody), antivenin micrurus (coral snake venom antibody), aprotinin, corticotropin (ACTH), diphtheria antitoxin, lymphocyte immune globulin (anti-thymocyte antibody), protamine, thyrotropin, capreomycin, α-galactosidase, gramicidin, streptokinase, tetanus toxoid, tyrothricin, IGF-1, proteins of varicella vaccine, anti-TNF antibody, anti-IL-2r antibody, anti-HER-2 antibody, OKT3 (“muromonab-CD3”) antibody, TNF-IgG fusion protein, ReoPro (“abciximab”) antibody, ACTH fragment 1-24, desmopressin, gonadotropin-releasing hormone, histrelin, leuprolide, lypressin, nafarelin, peptide that binds GPIIb/MPIIIa on platelets (integrilin), goserelin, capreomycin, colistin, anti-respiratory syncytial virus, lymphocyte immune globulin (Thymoglovin, Atgam), panorex, alpha-antitrypsin, botulinin, lung surfactant protein, tumor necrosis receptor-IgG fusion protein (enbrel), gonadorelin, proteins of influenza vaccine, proteins of rotavirus vaccine, proteins of haemophilus b conjugate vaccine, proteins of poliovirus vaccine, proteins of pneumococcal conjugate vaccine, proteins of meningococcal C vaccine, proteins of influenza vaccine, megakaryocyte growth and development factor (MGDF), neuroimmunophilin ligand-A (NIL-A), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), leptin (native), leptin B, leptin C, IL-IRA (interleukin-IRA), R-568, novel erythropoiesis-stimulating protein (NESP), humanized mAb to rous sarcoma virus (MEDI-493), glutamyl-tryptophan dipeptide IM862, LFA-3TIP immunosuppressive, humanized anti-CD40-ligand monoclonal antibody (5c8), gelsonin enzyme, tissue factor pathway inhibitor (TFPI), proteins of meningitis B vaccine, antimetastatic cancer antibody (mAb 17-1A), chimeric (human & mouse) mAb against TNFα, mAb against factor VII, relaxin, capreomycin, glycopeptide (LY333328), recombinant human activated protein C (rhAPC), humanized mAb against the epidermal growth receptor-2, altepase, anti-CD20 antigen, C2B8 antibody, insulin-like growth factor-1, atrial natriuretic peptide (anaritide), tenectaplase, anti-CD11a antibody (hu 1124), anti-CD18 antibody, mAb LDP-02, anti-VEGF antibody, fab fragment of anti-VEGF Ab, APO2 ligand (tumor necrosis factor-related apoptosis-inducing ligand), rTGF-β (transforming growth factor-β), alpha-antitrypsin, ananain (a pineapple enzyme), humanized mAb CTLA4IG, PRO542 (mAb), D2E7 (mAb), calf intestine alkaline phosphatase, α-L-iduronidase, α-L-galactosidase (humanglutamic acid decarboxylase, acid sphingomyelinase, bone morphogenetic protein-2 (rhBMP-2), proteins of HIV vaccine, T cell receptor (TCR) peptide vaccine, TCR peptides, V beta 3 and V beta 13.1. (IR502), (IR501), BI 1050/1272 mAb against very late antigen-4 (VLA-4), C225 humanized mAb to EGF receptor, anti-idiotype antibody to GD3 glycolipid, antibacterial peptide against H. pylori, MDX-447 bispecific humanized mAb to EGF receptor, anti-cytomegalovirus (CMV), Medi-491 B 19 parvovirus vaccine, humanized recombinant mAb (IgG1k) against respiratory syncytial virus (RSV), urinary tract infection vaccine (against “pili” on Escherechia coli strains), proteins of lyme disease vaccine against B. burgdorferi protein (DbpA), proteins of Medi-501 human papilloma virus-11 vaccine (HPV), Streptococcus pneumoniae vaccine, Medi-507 mAb (humanized form of BTI-322) against CD2 receptor on T-cells, MDX-33 mAb to FcγR1 receptor, MDX-RA immunotoxin (ricin A linked) mAb, MDX-210 bi-specific mAb against HER-2, MDX-447 bi-specific mAb against EGF receptor, MDX-22, MDX-220 bi-specific mAb against TAG-72 on tumors, colony-stimulating factor (CSF) (molgramostim), humanized mAb to the IL-2 R α-chain (basiliximab), mAb to IgE (IGE 025A), myelin basic protein-altered peptide (MSP771A), humanized mAb against the epidermal growth receptor-2, humanized mAb against the α subunit of the interleukin-2 receptor, low molecular weight heparin, anti-hemophillic factor, and bactericidal/permeability-increasing protein (r-BPI).
The peptides and proteins made using the present invention may be labeled using labels and techniques known to one of ordinary skill in the art. Some of these labels are described in the “Handbook of Fluorescent Probes and Research Products”, ninth edition, Richard P. Haugland (ed) Molecular Probes, Inc. Eugene, Oreg.), which is incorporated herein in its entirety. Some of these labels may be genetically engineered into the polynucleotide sequence for the expression of the selected protein or peptide. The peptides and proteins may also have label-incorporation “handles” incorporated to allow labeling of an otherwise difficult or impossible to label protein.
It is to be understood that the various classes of desired peptides and proteins, as well as specific peptides and proteins described in this section may be modified as described below by inserting selected codons for desired amino acid substitutions into the gene incorporated into the transgenic animal.
The present invention may also be used to produce desired molecules other than proteins and peptides including, but not limited to, lipoproteins such as high density lipoprotein (HDL), HDL-Milano, and low density lipoprotein, lipids, carbohydrates, siRNA and ribozymes. In these embodiments, a gene of interest encodes a nucleic acid molecule or a protein that directs production of the desired molecule.
The present invention further encompasses the use of inhibitory molecules to inhibit endogenous (i.e., non-vector) protein production. These inhibitory molecules include antisense nucleic acids, siRNA and inhibitory proteins. In a preferred embodiment, the endogenous protein whose expression is inhibited is an egg white protein including, but not limited to ovalbumin, ovotransferrin, and ovomucin. In one embodiment, a transposon-based vector containing an ovalbumin DNA sequence, that upon transcription forms a double stranded RNA molecule, is transfected into an animal such as a bird and the bird's production of endogenous ovalbumin protein is reduced by the interference RNA mechanism (RNAi). In other embodiments, a transposon-based vector encodes an inhibitory RNA molecule that inhibits the expression of more than one egg white protein. One exemplary construct is provided in FIG. 4 wherein “Ovgen” indicates approximately 60 base pairs of an ovalbumin gene, “Ovotrans” indicates approximately 60 base pairs of an ovotransferrin gene and “Ovomucin” indicates approximately 60 base pairs of an ovomucin gene. These ovalbumin, ovotransferrin and ovomucin can be from any avian species, and in some embodiments, are from a chicken or quail. The term “pro” indicates the pro portion of a prepro sequence. One exemplary prepro sequence is that of cecropin and comprising base pairs 563-733 of the Cecropin cap site and Prepro provided in Genbank accession number X07404. Additional cecropin prepro and pro sequences are provided in SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51. Additionally, inducible knockouts or knockdowns of the endogenous protein may be created to achieve a reduction or inhibition of endogenous protein production. Endogenous egg white production can be inhibited in an avian at any time, but is preferably inhibited preceding, or immediately preceding, the harvest of eggs.
Modified Desired Proteins and Peptides
“Proteins”, “peptides,” “polypeptides” and “oligopeptides” are chains of amino acids (typically L-amino acids) whose alpha carbons are linked through peptide bonds formed by a condensation reaction between the carboxyl group of the alpha carbon of one amino acid and the amino group of the alpha carbon of another amino acid. The terminal amino acid at one end of the chain (i.e., the amino terminal) has a free amino group, while the terminal amino acid at the other end of the chain (i.e., the carboxy terminal) has a free carboxyl group. As such, the term “amino terminus” (abbreviated N-terminus) refers to the free alpha-amino group on the amino acid at the amino terminal of the protein, or to the alpha-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the protein. Similarly, the term “carboxy terminus” (abbreviated C-terminus) refers to the free carboxyl group on the amino acid at the carboxy terminus of a protein, or to the carboxyl group of an amino acid at any other location within the protein.
Typically, the amino acids making up a protein are numbered in order, starting at the amino terminal and increasing in the direction toward the carboxy terminal of the protein. Thus, when one amino acid is said to “follow” another, that amino acid is positioned closer to the carboxy terminal of the protein than the preceding amino acid.
The term “residue” is used herein to refer to an amino acid (D or L) or an amino acid mimetic that is incorporated into a protein by an amide bond. As such, the amino acid may be a naturally occurring amino acid or, unless otherwise limited, may encompass known analogs of natural amino acids that function in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics). Moreover, an amide bond mimetic includes peptide backbone modifications well known to those skilled in the art.
Furthermore, one of skill will recognize that, as mentioned above, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than about 5%, more typically less than about 1%) in an encoded sequence are conservatively modified variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
A conservative substitution is a substitution in which the substituting amino acid (naturally occurring or modified) is structurally related to the amino acid being substituted, i.e., has about the same size and electronic properties as the amino acid being substituted. Thus, the substituting amino acid would have the same or a similar functional group in the side chain as the original amino acid. A “conservative substitution” also refers to utilizing a substituting amino acid which is identical to the amino acid being substituted except that a functional group in the side chain is protected with a suitable protecting group.
Suitable protecting groups are described in Green and Wuts, “Protecting Groups in Organic Synthesis”, John Wiley and Sons, Chapters 5 and 7, 1991, the teachings of which are incorporated herein by reference. Preferred protecting groups are those which facilitate transport of the peptide through membranes, for example, by reducing the hydrophilicity and increasing the lipophilicity of the peptide, and which can be cleaved, either by hydrolysis or enzymatically (Ditter et al., 1968. J. Pharm. Sci. 57:783; Ditter et al., 1968. J. Pharm. Sci. 57:828; Ditter et al., 1969. J. Pharm. Sci. 58:557; King et al., 1987. Biochemistry 26:2294; Lindberg et al., 1989. Drug Metabolism and Disposition 17:311; Tunek et al., 1988. Biochem. Pharm. 37:3867; Anderson et al., 1985 Arch. Biochem. Biophys. 239:538; and Singhal et al., 1987. FASEB J. 1:220). Suitable hydroxyl protecting groups include ester, carbonate and carbamate protecting groups. Suitable amine protecting groups include acyl groups and alkoxy or aryloxy carbonyl groups, as described above for N-terminal protecting groups. Suitable carboxylic acid protecting groups include aliphatic, benzyl and aryl esters, as described below for C-terminal protecting groups. In one embodiment, the carboxylic acid group in the side chain of one or more glutamic acid or aspartic acid residues in a peptide of the present invention is protected, preferably as a methyl, ethyl, benzyl or substituted benzyl ester, more preferably as a benzyl ester.
Provided below are groups of naturally occurring and modified amino acids in which each amino acid in a group has similar electronic and steric properties. Thus, a conservative substitution can be made by substituting an amino acid with another amino acid from the same group. It is to be understood that these groups are non-limiting, i.e. that there are additional modified amino acids which could be included in each group.

    Group I includes leucine, isoleucine, valine, methionine and modified amino acids having the following side chains: ethyl, n-propyl n-butyl. Preferably, Group I includes leucine, isoleucine, valine and methionine.
    Group II includes glycine, alanine, valine and a modified amino acid having an ethyl side chain. Preferably, Group II includes glycine and alanine.
    Group III includes phenylalanine, phenylglycine, tyrosine, tryptophan, cyclohexylmethyl glycine, and modified amino residues having substituted benzyl or phenyl side chains. Preferred substituents include one or more of the following: halogen, methyl, ethyl, nitro, —NH2, methoxy, ethoxy and —CN. Preferably, Group III includes phenylalanine, tyrosine and tryptophan.
    Group IV includes glutamic acid, aspartic acid, a substituted or unsubstituted aliphatic, aromatic or benzylic ester of glutamic or aspartic acid (e.g., methyl, ethyl, n-propyl iso-propyl, cyclohexyl, benzyl or substituted benzyl), glutamine, asparagine, —CO—NH— alkylated glutamine or asparagines (e.g., methyl, ethyl, n-propyl and iso-propyl) and modified amino acids having the side chain —(CH2)3—COOH, an ester thereof (substituted or unsubstituted aliphatic, aromatic or benzylic ester), an amide thereof and a substituted or unsubstituted N-alkylated amide thereof. Preferably, Group IV includes glutamic acid, aspartic acid, methyl aspartate, ethyl aspartate, benzyl aspartate and methyl glutamate, ethyl glutamate and benzyl glutamate, glutamine and asparagine.
    Group V includes histidine, lysine, ornithine, arginine, N-nitroarginine, β-cycloarginine,γ-hydroxyarginine, N-amidinocitruline and 2-amino-4-guanidinobutanoic acid, homologs of lysine, homologs of arginine and homologs of ornithine. Preferably, Group V includes histidine, lysine, arginine and ornithine. A homolog of an amino acid includes from 1 to about 3 additional or subtracted methylene units in the side chain.
    Group VI includes serine, threonine, cysteine and modified amino acids having C1-C5 straight or branched alkyl side chains substituted with —OH or —SH, for example, —CH2CH2OH, —CH2CH2CH2OH or —CH2CH2OHCH3. Preferably, Group VI includes serine, cysteine or threonine.


In another aspect, suitable substitutions for amino acid residues include “severe” substitutions. A “severe substitution” is a substitution in which the substituting amino acid (naturally occurring or modified) has significantly different size and/or electronic properties compared with the amino acid being substituted. Thus, the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted. Examples of severe substitutions of this type include the substitution of phenylalanine or cyclohexylmethyl glycine for alanine, isoleucine for glycine, a D amino acid for the corresponding L amino acid, or —NH—CH[(—CH2)5—COOH]—CO— for aspartic acid. Alternatively, a functional group may be added to the side chain, deleted from the side chain or exchanged with another functional group. Examples of severe substitutions of this type include adding of valine, leucine or isoleucine, exchanging the carboxylic acid in the side chain of aspartic acid or glutamic acid with an amine, or deleting the amine group in the side chain of lysine or ornithine. In yet another alternative, the side chain of the substituting amino acid can have significantly different steric and electronic properties that the functional group of the amino acid being substituted. Examples of such modifications include tryptophan for glycine, lysine for aspartic acid and —(CH2)4COOH for the side chain of serine. These examples are not meant to be limiting.
In another embodiment, for example in the synthesis of a peptide 26 amino acids in length, the individual amino acids may be substituted according in the following manner:
AA1 is serine, glycine, alanine, cysteine or threonine;
AA2 is alanine, threonine, glycine, cysteine or serine;
AA3 is valine, arginine, leucine, isoleucine, methionine, ornithine, lysine, N-nitroarginine, β-cycloarginine, γ-hydroxyarginine, N-amidinocitruline or 2-amino-4-guanidinobutanoic acid;
AA4 is proline, leucine, valine, isoleucine or methionine;
AA5 is tryptophan, alanine, phenylalanine, tyrosine or glycine;
AA6 is serine, glycine, alanine, cysteine or threonine;
AA7 is proline, leucine, valine, isoleucine or methionine;
AA8 is alanine, threonine, glycine, cysteine or serine;
AA9 is alanine, threonine, glycine, cysteine or serine;
AA10 is leucine, isoleucine, methionine or valine;
AA11 is serine, glycine, alanine, cysteine or threonine;
AA12 is leucine, isoleucine, methionine or valine;
AA13 is leucine, isoleucine, methionine or valine;
AA14 is glutamine, glutamic acid, aspartic acid, asparagine, or a substituted or unsubstituted aliphatic or aryl ester of glutamic acid or aspartic acid;
AA15 is arginine, N-nitroarginine, β-cycloarginine, γ-hydroxy-arginine, N-amidinocitruline or 2-amino-4-guanidino-butanoic acid
AA16 is proline, leucine, valine, isoleucine or methionine;
AA17 is serine, glycine, alanine, cysteine or threonine;
AA18 is glutamic acid, aspartic acid, asparagine, glutamine or a substituted or unsubstituted aliphatic or aryl ester of glutamic acid or aspartic acid;
AA19 is aspartic acid, asparagine, glutamic acid, glutamine, leucine, valine, isoleucine, methionine or a substituted or unsubstituted aliphatic or aryl ester of glutamic acid or aspartic acid;
AA20 is valine, arginine, leucine, isoleucine, methionine, ornithine, lysine, N-nitroarginine, β-cycloarginine, γ-hydroxyarginine, N-amidinocitruline or 2-amino-4-guanidinobutanoic acid;
AA21 is alanine, threonine, glycine, cysteine or serine;
AA22 is alanine, threonine, glycine, cysteine or serine;
AA23 is histidine, serine, threonine, cysteine, lysine or ornithine;
AA24 is threonine, aspartic acid, serine, glutamic acid or a substituted or unsubstituted aliphatic or aryl ester of glutamic acid or aspartic acid;
AA25 is asparagine, aspartic acid, glutamic acid, glutamine, leucine, valine, isoleucine, methionine or a substituted or unsubstituted aliphatic or aryl ester of glutamic acid or aspartic acid; and
AA26 is cysteine, histidine, serine, threonine, lysine or ornithine.
It is to be understood that these amino acid substitutions may be made for longer or shorter peptides than the 26 mer in the preceding example above, and for proteins.
In one embodiment of the present invention, codons for the first several N-terminal amino acids of the transposase are modified such that the third base of each codon is changed to an A or a T without changing the corresponding amino acid. It is preferable that between approximately 1 and 20, more preferably 3 and 15, and most preferably between 4 and 12 of the first N-terminal codons of the gene of interest are modified such that the third base of each codon is changed to an A or a T without changing the corresponding amino acid. In one embodiment, the first ten N-terminal codons of the gene of interest are modified in this manner.
When several desired proteins, protein fragments or peptides are encoded in the gene of interest to be incorporated into the genome, one of skill in the art will appreciate that the proteins, protein fragments or peptides may be separated by a spacer molecule such as, for example, a peptide, consisting of one or more amino acids. Generally, the spacer will have no specific biological activity other than to join the desired proteins, protein fragments or peptides together, or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of the spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity. The spacer may also be contained within a nucleotide sequence with a purification handle or be flanked by cleavage sites, such as proteolytic cleavage sites.
Such polypeptide spacers may have from about 5 to about 40 amino acid residues. The spacers in a polypeptide are independently chosen, but are preferably all the same. The spacers should allow for flexibility of movement in space and are therefore typically rich in small amino acids, for example, glycine, serine, proline or alanine. Preferably, peptide spacers contain at least 60%, more preferably at least 80% glycine or alanine. In addition, peptide spacers generally have little or no biological and antigenic activity. Preferred spacers are (Gly-Pro-Gly-Gly)x (SEQ ID NO:26) and (Gly4-Ser)y, wherein x is an integer from about 3 to about 9 and y is an integer from about 1 to about 8. Specific examples of suitable spacers include







(Gly-Pro-Gly-Gly)3


 


SEQ ID NO: 27


Gly Pro Gly Gly Gly Pro Gly Gly Gly Pro Gly Gly


 


(Gly4-Ser)3


 


SEQ ID NO: 28


Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly


 


Gly Gly Ser


or


 


(Gly4-Ser)4


 


SEQ ID NO: 29


Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly


 


Gly Gly Ser Gly Gly Gly Gly Ser.






Nucleotide sequences encoding for the production of residues which may be useful in purification of the expressed recombinant protein may also be built into the vector. Such sequences are known in the art and include the glutathione binding domain from glutathione S-transferase, polylysine, hexa-histidine or other cationic amino acids, thioredoxin, hemagglutinin antigen and maltose binding protein.
Additionally, nucleotide sequences may be inserted into the gene of interest to be incorporated so that the protein or peptide can also include from one to about six amino acids that create signals for proteolytic cleavage. In this manner, if a gene is designed to make one or more peptides or proteins of interest in the transgenic animal, specific nucleotide sequences encoding for amino acids recognized by enzymes may be incorporated into the gene to facilitate cleavage of the large protein or peptide sequence into desired peptides or proteins or both. For example, nucleotides encoding a proteolytic cleavage site can be introduced into the gene of interest so that a signal sequence can be cleaved from a protein or peptide encoded by the gene of interest. Nucleotide sequences encoding other amino acid sequences which display pH sensitivity or chemical sensitivity may also be added to the vector to facilitate separation of the signal sequence from the peptide or protein of interest.
Proteolytic cleavage sites include cleavage sites recognized by exopeptidases such as carboxypeptidase A, carboxypeptidase B, aminopeptidase I, and dipeptidylaminopeptidase; endopeptidases such as trypsin, V8-protease, enterokinase, factor Xa, collagenase, endoproteinase, subtilisin, and thombin; and proteases such as Protease 3C IgA protease (Igase) Rhinovirus 3C(preScission) protease. Chemical cleavage sites are also included in the definition of cleavage site as used herein. Chemical cleavage sites include, but are not limited to, site cleaved by cyanogen bromide, hydroxylamine, formic acid, and acetic acid.
In one embodiment of the present invention, a TAG sequence is linked to the gene of interest. The TAG sequence serves three purposes: 1) it allows free rotation of the peptide or protein to be isolated so there is no interference from the native protein or signal sequence, i.e. vitellogenin, 2) it provides a “purification handle” to isolate the protein using column purification, and 3) it includes a cleavage site to remove the desired protein from the signal and purification sequences. Accordingly, as used herein, a TAG sequence includes a spacer sequence, a purification handle and a cleavage site. The spacer sequences in the TAG proteins contain one or more repeats shown in SEQ ID NO:30. A preferred spacer sequence comprises the sequence provided in SEQ ID NO:31. One example of a purification handle is the gp41 hairpin loop from HIV I. Exemplary gp41 polynucleotide and polypeptide sequences are provided in SEQ ID NO:32 and SEQ ID NO:33, respectively. However, it should be understood that any antigenic region may be used as a purification handle, including any antigenic region of gp41. Preferred purification handles are those that elicit highly specific antibodies. Additionally, the cleavage site can be any protein cleavage site known to one of ordinary skill in the art and includes an enterokinase cleavage site comprising the Asp Asp Asp Asp Lys sequence (SEQ ID NO:34) and a furin cleavage site. Constructs containing a TAG sequence are shown in FIGS. 2 and 3. In one embodiment of the present invention, the TAG sequence comprises a polynucleotide sequence of SEQ ID NO:35.
Methods of Administering Transposon-Based Vectors
In addition to the transposon-based vectors described above, the present invention also includes methods of administering the transposon-based vectors to an animal, methods of producing a transgenic animal wherein a gene of interest is incorporated into the germline of the animal and methods of producing a transgenic animal wherein a gene of interest is incorporated into cells other than the germline cells (somatic cells) of the animal. The transposon-based vectors of the present invention are administered to a reproductive organ of an animal via any method known to those of skill in the art. Preferred reproductive organs include an ovary, an oviduct, a mammary gland, and a fallopian tube.
In some embodiments, a transposon-based vector is directly administered to the reproductive organ. Direct administration encompasses injection into the organ, and in a preferred embodiment, a transposon-based vector is injected into the lumen of the oviduct, and more preferably, the lumen of the magnum or the infundibulum of the oviduct. The transposon-based vectors may additionally or alternatively be placed in an artery supplying the reproductive organ. Administering the vectors to the artery supplying the ovary results in transfection of follicles and oocytes in the ovary to create a germline transgenic animal. Alternatively, supplying the vectors through an artery leading to the oviduct would preferably transfect the tubular gland and epithelial cells. Such transfected cells could manufacture a desired protein or peptide for deposition in the egg white. In one embodiment, a transposon-based vector is administered into the lumen of the magnum or the infundibulum of the oviduct and to an artery supplying the oviduct. Indirect administration to the oviduct epithelium may occur through the cloaca. Direct administration into the mammary gland comprises introduction into the duct system of the mammary gland.
Administration of transposon-based vectors may occur in arteries supplying the ovary and or through direct intrathecal administration into the ovary through injection.
The transposon-based vectors may be administered in a single administration, multiple administrations, continuously, or intermittently. The transposon-based vectors may be administered by injection, via a catheter, an osmotic mini-pump or any other method. In some embodiments, the transposon-based vector is administered to an animal in multiple administrations, each administration containing the vector and a different transfecting reagent.
The transposon-based vectors may be administered to the animal at any point during the lifetime of the animal, however, it is preferable that the vectors are administered prior to the animal reaching sexual maturity. The transposon-based vectors are preferably administered to a chicken between approximately 14 and 16 weeks of age and to a quail between approximately 5 and 10 weeks of age, more preferably 5 and 8 weeks of age, and most preferably between 5 and 6 weeks of age, when standard poultry rearing practices are used. The vectors may be administered at earlier ages when exogenous hormones are used to induce early sexual maturation in the bird. In some embodiments, the transposon-based vector is administered to an animal following an increase in proliferation of the oviduct epithelial cells and/or the tubular gland cells. Such an increase in proliferation normally follows an influx of reproductive hormones in the area of the oviduct. When the animal is an avian, the transposon-based vector is administered following an increase in proliferation of the oviduct epithelial cells and before the avian begins to produce egg white constituents.
In a preferred embodiment, the animal is an egg-laying animal, and more preferably, an avian. In one embodiment, between approximately 1 and 150 μg, 1 and 100 μg, 1 and 50 μg, preferably between 1 and 20 μg, and more preferably between 5 and 10 μg of transposon-based vector DNA is administered to the oviduct of a bird. Optimal ranges depend upon the type of bird and the bird's stage of sexual maturity. In a chicken, it is preferred that between approximately 1 and 100 μg, or 5 and 50 μg are administered. In a quail, it is preferred that between approximately 5 and 10 μg are administered. Intraoviduct administration of the transposon-based vectors of the present invention result in incorporation of the gene of interest into the cells of the oviduct as evidenced by a PCR positive signal in the oviduct tissue. In other embodiments, the transposon-based vector is administered to an artery that supplies the oviduct. These methods of administration may also be combined with any methods for facilitating transfection, including without limitation, electroporation, gene guns, injection of naked DNA, and use of dimethyl sulfoxide (DMSO).
According to the present invention, the transposon-based vector is administered in conjunction with an acceptable carrier and/or transfection reagent. Acceptable carriers include, but are not limited to, water, saline, Hanks Balanced Salt Solution (HBSS), Tris-EDTA (TE) and lyotropic liquid crystals. Transfection reagents commonly known to one of ordinary skill in the art that may be employed include, but are not limited to, the following: cationic lipid transfection reagents, cationic lipid mixtures, polyamine reagents, liposomes and combinations thereof; SUPERFECT®, Cytofectene, BioPORTER®, GenePORTER®, NeuroPORTER®, and perfectin from Gene Therapy Systems; lipofectamine, cellfectin, DMRIE-C oligofectamine, TROJENE® and PLUS reagent from InVitrogen; Xtreme gene, fugene, DOSPER and DOTAP from Roche; Lipotaxi and Genejammer from Strategene; and Escort from SIGMA. In one embodiment, the transfection reagent is SUPERFECT®. The ratio of DNA to transfection reagent may vary based upon the method of administration. In one embodiment, the transposon-based vector is administered to the oviduct and the ratio of DNA to transfection reagent can be from 1:1.5 to 1:15, preferably 1:2 to 1:5, all expressed as wt/vol. Transfection may also be accomplished using other means known to one of ordinary skill in the art, including without limitation electroporation, gene guns, injection of naked DNA, and use of dimethyl sulfoxide (DMSO).
Depending upon the cell or tissue type targeted for transfection, the form of the transposon-based vector may be important. Plasmids harvested from bacteria are generally closed circular supercoiled molecules, and this is the preferred state of a vector for gene delivery because of the ease of preparation. In some instances, transposase expression and insertion may be more efficient in a relaxed, closed circular configuration or in a linear configuration. In still other instances, a purified transposase protein may be co-injected with a transposon-based vector containing the gene of interest for more immediate insertion. This could be accomplished by using a transfection reagent complexed with both the purified transposase protein and the transposon-based vector.
Testing for and Breeding Animals Carrying the Transgene
Following administration of a transposon-based vector to an animal, DNA is extracted from the animal to confirm integration of the gene of interest. Advantages provided by the present invention include the high rates of integration, or incorporation, and transcription of the gene of interest when administered to a bird via an intraoviduct or intraovarian route (including intraarterial administrations to arteries leading to the oviduct or ovary). Example 6 below describes isolation of a proinsulin/ENT TAG protein from a transgenic hen following ammonium sulfate precipitation and ion exchange chromatography. FIG. 5 demonstrates successful administration of a transposon-based vector to a hen, successful integration of the gene of interest, successful production of a protein encoded by the gene of interest, and successful deposition of the protein in egg white produced by the transgenic hen.
Actual frequencies of integration may be estimated both by comparative strength of the PCR signal, and by histological evaluation of the tissues by quantitative PCR. Another method for estimating the rate of transgene insertion is the so-called primed in situ hybridization technique (PRINS). This method determines not only which cells carry a transgene of interest, but also into which chromosome the gene has inserted, and even what portion of the chromosome. Briefly, labeled primers are annealed to chromosome spreads (affixed to glass slides) through one round of PCR, and the slides are then developed through normal in situ hybridization procedures. This technique combines the best features of in situ PCR and fluorescence in situ hybridization (FISH) to provide distinct chromosome location and copy number of the gene in question.
Breeding experiments are also conducted to determine if germline transmission of the transgene has occurred. In a general bird breeding experiment performed according to the present invention, each male bird was exposed to 2-3 different adult female birds for 3-4 days each. This procedure was continued with different females for a total period of 6-12 weeks. Eggs are collected daily for up to 14 days after the last exposure to the transgenic male, and each egg is incubated in a standard incubator. The resulting embryos are examined for transgene presence at day 3 or 4 using PCR. It is to be understood that the above procedure can be modified to suit animals other than birds and that selective breeding techniques may be performed to amplify gene copy numbers and protein output.
Production of Desired Proteins or Peptides in Egg White
In one embodiment, the transposon-based vectors of the present invention may be administered to a bird for production of desired proteins or peptides in the egg white. These transposon-based vectors preferably contain one or more of an ovalbumin promoter, an ovomucoid promoter, an ovalbumin signal sequence and an ovomucoid signal sequence. Oviduct-specific ovalbumin promoters are described in B. O'Malley et al., 1987. EMBO J., vol. 6, pp. 2305-12; A. Qiu et al., 1994. Proc. Nat. Acad. Sci. (USA), vol. 91, pp. 4451-4455; D. Monroe et al., 2000. Biochim. Biophys. Acta, 1517 (1):27-32; H. Park et al., 2000. Biochem., 39:8537-8545; and T. Muramatsu et al., 1996. Poult. Avian Biol. Rev., 6:107-123. Examples oftransposon-based vectors designed for production of a desired protein in an egg white are shown in FIGS. 2 and 3.
Production of Desired Proteins or Peptides in Egg Yolk
The present invention is particularly advantageous for production of recombinant peptides and proteins of low solubility in the egg yolk. Such proteins include, but are not limited to, membrane-associated or membrane-bound proteins, lipophilic compounds; attachment factors, receptors, and components of second messenger transduction machinery. Low solubility peptides and proteins are particularly challenging to produce using conventional recombinant protein production techniques (cell and tissue cultures) because they aggregate in water-based, hydrophilic environments. Such aggregation necessitates denaturation and re-folding of the recombinantly-produced proteins, which may deleteriously affect their structure and function. Moreover, even highly soluble recombinant peptides and proteins may precipitate and require denaturation and renaturation when produced in sufficiently high amounts in recombinant protein production systems. The present invention provides an advantageous resolution of the problem of protein and peptide solubility during production of large amounts of recombinant proteins.
In one embodiment of the present invention wherein germ line transfection is obtained via intraovarian administration of the transposon-based vector, deposition of a desired protein into the egg yolk is accomplished in offspring by attaching a sequence encoding a protein capable of binding to the yolk vitellogenin receptor to a gene of interest that encodes a desired protein. This transposon-based vector can be used for the receptor-mediated uptake of the desired protein by the oocytes. In a preferred embodiment, the sequence ensuring the binding to the vitellogenin receptor is a targeting sequence of a vitellogenin protein. The invention encompasses various vitellogenin proteins and their targeting sequences. In a preferred embodiment, a chicken vitellogenin protein targeting sequence is used, however, due to the high degree of conservation among vitellogenin protein sequences and known cross-species reactivity of vitellogenin targeting sequences with their egg-yolk receptors, other vitellogenin targeting sequences can be substituted. One example of a construct for use in the transposon-based vectors of the present invention and for deposition of an insulin protein in an egg yolk is a transposon-based vector containing a vitellogenin promoter, a vitellogenin targeting sequence, a TAG sequence, a pro-insulin sequence and a synthetic polyA sequence. The present invention includes, but is not limited to, vitellogenin targeting sequences residing in the N-terminal domain of vitellogenin, particularly in lipovitellin I. In one embodiment, the vitellogenin targeting sequence contains the polynucleotide sequence of SEQ ID NO:22. In a preferred embodiment, the transposon-based vector contains a transposase gene operably-linked to a constitutive promoter and a gene of interest operably-linked to a liver-specific promoter and a vitellogenin targeting sequence.
Isolation and Purification of Desired Protein or Peptide
For large-scale production of protein, an animal breeding stock that is homozygous for the transgene is preferred. Such homozygous individuals are obtained and identified through, for example, standard animal breeding procedures or PCR protocols.
Once expressed, peptides, polypeptides and proteins can be purified according to standard procedures known to one of ordinary skill in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, high performance liquid chromatography, immunoprecipitation and the like. Substantially pure compositions of about 50 to 99% homogeneity are preferred, and 80 to 95% or greater homogeneity are most preferred for use as therapeutic agents.
In one embodiment of the present invention, the animal in which the desired protein is produced is an egg-laying animal. In a preferred embodiment of the present invention, the animal is an avian and a desired peptide, polypeptide or protein is isolated from an egg white. Egg white containing the exogenous protein or peptide is separated from the yolk and other egg constituents on an industrial scale by any of a variety of methods known in the egg industry. See, e.g., W. Stadelman et al. (Eds.), Egg Science & Technology, Haworth Press, Binghamton, N.Y. (1995). Isolation of the exogenous peptide or protein from the other egg white constituents is accomplished by any of a number of polypeptide isolation and purification methods well known to one of ordinary skill in the art. These techniques include, for example, chromatographic methods such as gel permeation, ion exchange, affinity separation, metal chelation, HPLC, and the like, either alone or in combination. Another means that may be used for isolation or purification, either in lieu of or in addition to chromatographic separation methods, includes electrophoresis. Successful isolation and purification is confirmed by standard analytic techniques, including HPLC, mass spectroscopy, and spectrophotometry. These separation methods are often facilitated if the first step in the separation is the removal of the endogenous ovalbumin fraction of egg white, as doing so will reduce the total protein content to be further purified by about 50%.
To facilitate or enable purification of a desired protein or peptide, transposon-based vectors may include one or more additional epitopes or domains. Such epitopes or domains include DNA sequences encoding enzymatic or chemical cleavage sites including, but not limited to, an enterokinase cleavage site; the glutathione binding domain from glutathione S-transferase; polylysine; hexa-histidine or other cationic amino acids; thioredoxin; hemagglutinin antigen; maltose binding protein; a fragment of gp41 from HIV; and other purification epitopes or domains commonly known to one of skill in the art.
In one representative embodiment, purification of desired proteins from egg white utilizes the antigenicity of the ovalbumin carrier protein and particular attributes of a TAG linker sequence that spans ovalbumin and the desired protein. The TAG sequence is particularly useful in this process because it contains 1) a highly antigenic epitope, a fragment of gp41 from HIV, allowing for stringent affinity purification, and, 2) a recognition site for the protease enterokinase immediately juxtaposed to the desired protein. In a preferred embodiment, the TAG sequence comprises approximately 50 amino acids. A representative TAG sequence is provided below.







(SEQ ID NO: 35)


Pro Ala Asp Asp Ala Pro Ala Asp Asp Ala Pro Ala


 


Asp Asp Ala Pro Ala Asp Asp Ala Pro Ala Asp Asp


 


Ala Pro Ala Asp Asp Ala Thr Thr Cys Ile Leu Lys


 


Gly Ser Cys Gly Trp Ile Gly Leu Leu Asp Asp Asp


 


Asp Lys






The underlined sequences were taken from the hairpin loop domain of HIV gp-41 (SEQ ID NO:33). Sequences in italics represent the cleavage site for enterokinase (SEQ ID NO:34). The spacer sequence upstream of the loop domain was made from repeats of (Pro Ala Asp Asp Ala) (SEQ ID NO:31) to provide free rotation and promote surface availability of the hairpin loop from the ovalbumin carrier protein.

Isolation and purification of a desired protein is performed as follows:

    
    
        1. Enrichment of the egg white protein fraction containing ovalbumin and the transgenic ovalbumin-TAG-desired protein.
        2. Size exclusion chromatography to isolate only those proteins within a narrow range of molecular weights (a further enrichment of step 1).
        3. Ovalbumin affinity chromatography. Highly specific antibodies to ovalbumin will eliminate virtually all extraneous egg white proteins except ovalbumin and the transgenic ovalbumin-TAG-desired protein.
        4. gp41 affinity chromatography using anti-gp41 antibodies. Stringent application of this step will result in virtually pure transgenic ovalbumin-TAG-desired protein.
        5. Cleavage of the transgene product can be accomplished in at least one of two ways:
        
            a. The transgenic ovalbumin-TAG-desired protein is left attached to the gp41 affinity resin (beads) from step 4 and the protease enterokinase is added. This liberates the transgene target protein from the gp41 affinity resin while the ovalbumin-TAG sequence is retained. Separation by centrifugation (in a batch process) or flow through (in a column purification), leaves the desired protein together with enterokinase in solution. Enterokinase is recovered and reused.
            b. Alternatively, enterokinase is immobilized on resin (beads) by the addition of poly-lysine moieties to a non-catalytic area of the protease. The transgenic ovalbumin-TAG-desired protein eluted from the affinity column of step 4 is then applied to the protease resin. Protease action cleaves the ovalbumin-TAG sequence from the desired protein and leaves both entities in solution. The immobilized enterokinase resin is recharged and reused.
            c. The choice of these alternatives is made depending upon the size and chemical composition of the transgene target protein.
        
        
        6. A final separation of either of these two (5a or 5b) protein mixtures is made using size exclusion, or enterokinase affinity chromatography. This step allows for desalting, buffer exchange and/or polishing, as needed.
    
    


Cleavage of the transgene product (ovalbumin-TAG-desired protein) by enterokinase, then, results in two products: ovalbumin-TAG and the desired protein. More specific methods for isolation using the TAG label is provided in the Examples. Some desired proteins may require additions or modifications of the above-described approach as known to one of ordinary skill in the art. The method is scaleable from the laboratory bench to pilot and production facility largely because the techniques applied are well documented in each of these settings.
In another representative embodiment, egg whites containing a protein of interest were pooled and separated, in any order, from the yolks and other egg constituents by methods known to one skilled in the art. A variety of such methods is described in manuals known in the art, such as Egg Science & Technology, W. Stadelman, et al. (Eds.), Haworth Press, Binghamton, N.Y. (1995).
One non-limiting example of a method for isolating a desired peptide, polypeptide or protein from an egg white is as follows. It is to be understood that this method may be employed to isolate any desired peptide, polypeptide or protein from the eggs of transgenic animals of the present invention. This present example involved transgenes that used a portion of or the entire ovalbumin protein, or specific ovalbumin epitopes, as a carrier, linked to the protein of interest via the specified TAG sequence, or another affinity/cleavage sequence. The TAG sequence contains the hairpin loop epitope from HIV I followed by an enterokinase cleavage site.
First, the viscosity of the egg white was lowered by subjecting the egg white to low shear forces of 3140 cps (Tung et al., 1969). The resulting pourable solution was then filtered to remove chalazae. An ammonium sulfate precipitation was then used to enrich the fraction of transgenic protein (see, for example, Practical Protein Chemistry A Handbook A. Darbre (Ed.), John Wiley & Sons Ltd., 1986). Other methods of crude fractionation known in the art are also used as needed. The supernatant of this separation was then fractionated using size-exclusion chromatography, further enriching the transgenic fusion protein fraction and eliminating the ammonium sulfate from the material. The fusion protein was isolated by anti-ovalbumin affinity chromatography (batch or column) using methods known to one skilled in the art. This step may capture native ovalbumin in addition to an ovalbumin-transgene fusion protein. After elution from the anti-ovalbumin affinity resin, the transgenic protein was specifically isolated using anti-gp41 affinity chromatography (batch or column) using methods known to one skilled in the art.
Cleavage of the transgene product from the carrier and the TAG sequences was accomplished in one of at least two ways:
1) The transgenic ovalbumin-TAG-transgene target protein was left attached to the gp41 affinity resin and the protease enterokinase was added. Cleavage of the transgene by enterokinase liberated the transgene target protein from the gp41 affinity resin while the ovalbumin-TAG sequence was retained. Separation by centrifugation (in a batch process) or flow through (in a column purification), kept the transgene target protein together with enterokinase in solution. Enterokinase was recovered and reused.
2) Alternatively, enterokinase was immobilized on resin (beads) by the addition of poly-lysine moieties to a non-catalytic area of the protease. The transgenic ovalbumin-TAG-transgene target protein was eluted from the gp41 affinity chromatography resin and then applied to the protease resin. Protease action cleaved the ovalbumin-TAG sequence from the transgene target protein and left both entities in solution. The immobilized enterokinase resin was recharged and reused. The choice between these alternatives is made on a case-by case basis, depending upon the size and chemical composition of the transgene target protein.
A final separation of either of these two (process 1 or 2) protein mixtures was made using size exclusion chromatography, or enterokinase affinity chromatography. This step also allows for desalting, concentrating, buffer exchange and/or polishing, as needed.
It is believed that a typical chicken egg produced by a transgenic animal of the present invention will contain at least 0.001 mg, from about 0.001 to 1.0 mg, or from about 0.001 to 100.0 mg of exogenous protein, peptide or polypeptide, in addition to the normal constituents of egg white (or possibly replacing a small fraction of the latter). In some embodiments, a chicken egg will contain between 50 and 75 mg of exogenous protein.
One of skill in the art will recognize that after biological expression or purification, the desired proteins, fragments thereof and peptides may possess a conformation substantially different than the native conformations of the proteins, fragments thereof and peptides. In this case, it is often necessary to denature and reduce protein and then to cause the protein to re-fold into the preferred conformation. Methods of reducing and denaturing proteins and inducing re-folding are well known to those of skill in the art.
Production of Protein or Peptide in Milk
In addition to methods of producing eggs containing transgenic proteins or peptides, the present invention encompasses methods for the production of milk containing transgenic proteins or peptides. These methods include the administration of a transposon-based vector described above to a mammal through the duct system. In one embodiment, the transposon-based vector contains a transposase operably-linked to a constitutive promoter and a gene of interest operably-linked to mammary specific promoter. Genes of interest can include, but are not limited to antiviral and antibacterial proteins and immunoglobulins. In other embodiments, a transposon-based vector is administered to the ovary of an animal and germline transformation is obtained. In these embodiments, offspring of the transfected animal express a gene of interest in the mammary gland under the control of a mammary gland-specific promoter.
The following examples will serve to further illustrate the present invention without, at the same time, however, constituting any limitation thereof. On the contrary, it is to be clearly understood that resort may be had to various embodiments, modifications and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the invention.
EXAMPLE 1
IntraOviduct Administration of Transposon-Based Vectors
Quail or chicken were selected for administration of the transposon-based vectors of the present invention. Feathers were removed from the area where surgery was performed and the area was cleansed and sterilized by rinsing it with ethanol (alcohol) and 0.5% chlorhexidine. Using the scalpel, a dorsolateral incision was made through the skin over the ovary approximately 2 cm in length. Using blunt scissors, a second incision was made through the muscle between the last two ribs to expose the oviduct beneath. A small animal retractor was used to spread the last two ribs, exposing the oviduct beneath. The oviduct was further exposed using retractors to pull the intestines to one side.
A delivery solution containing a transposon-based vector and SUPERFECT® was prepared fresh immediately before surgery. Specific ratios of vector and SUPERFECT® that were used in each experiment are provided in the Examples below. The delivery solution was warmed to room temperature prior to injection into the bird. Approximately 250-500 μl of the delivery solution was injected into the lumen of the magnum of the oviduct using a 1 cc syringe with a 27 gauge needle attached. The wound was closed and antibiotic cream liberally applied to the area surrounding the wound.
EXAMPLE 2
Preparation of Transposon-Based Vector pTnMod
A vector was designed for inserting a desired coding sequence into the genome of eukaryotic cells, given below as SEQ ID NO:3. The vector of SEQ ID NO:3, termed pTnMod, was constructed and its sequence verified.
This vector employed a cytomegalovirus (CMV) promoter. A modified Kozak sequence (ACCATG) (SEQ ID NO:1) was added to the promoter. The nucleotide in the wobble position in nucleotide triplet codons encoding the first 10 amino acids of transposase was changed to an adenine (A) or thymine (T), which did not alter the amino acid encoded by this codon. Two stop codons were added and a synthetic polyA was used to provide a strong termination sequence. This vector uses a promoter designed to be active soon after entering the cell (without any induction) to increase the likelihood of stable integration. The additional stop codons and synthetic polyA insures proper termination without read through to potential genes downstream.
The first step in constructing this vector was to modify the transposase to have the desired changes. Modifications to the transposase were accomplished with the primers High Efficiency forward primer (Hef) Altered transposase (ATS)-Hef 5′ ATCTCGAGACCATGTGTGAACTTGATATTTTACATGATTCTCTTTACC 3′ (SEQ ID NO:36) and Altered transposase-High efficiency reverse primer (Her) 5′ GATTGATCATTATCATAATTTCCCCAAAGCGTAACC 3′ (SEQ ID NO:37, a reverse complement primer). In the 5′ forward primer ATS-Hef, the sequence CTCGAG (SEQ ID NO:38) is the recognition site for the restriction enzyme Xho I, which permits directional cloning of the amplified gene. The sequence ACCATG (SEQ ID NO:1) contains the Kozak sequence and start codon for the transposase and the underlined bases represent changes in the wobble position to an A or T of codons for the first 10 amino acids (without changing the amino acid coded by the codon). Primer ATS-Her (SEQ ID NO:37) contains an additional stop codon TAA in addition to native stop codon TGA and adds a Bcl I restriction site, TGATCA (SEQ ID NO:39), to allow directional cloning. These primers were used in a PCR reaction with pTnLac (p defines plasmid, tn defines transposon, and lac defines the beta fragment of the lactose gene, which contains a multiple cloning site) as the template for the transposase and a FailSafeTM PCR System (which includes enzyme, buffers, dNTP's, MgCl2 and PCR Enhancer; Epicentre Technologies, Madison, Wis.). Amplified PCR product was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and visualized on an ultraviolet transilluminator. A band corresponding to the expected size was excised from the gel and purified from the agarose using a Zymo Clean Gel Recovery Kit (Zymo Research, Orange, Calif.). Purified DNA was digested with restriction enzymes Xho 1 (5′) and Bcl I (3′) (New England Biolabs, Beverly, Mass.) according to the manufacturer's protocol. Digested DNA was purified from restriction enzymes using a Zymo DNA Clean and Concentrator kit (Zymo Research).
Plasmid gwhiz (Gene Therapy Systems, San Diego, Calif.) was digested with restriction enzymes Sal I and BamH I (New England Biolabs), which are compatible with Xho I and Bcl I, but destroy the restriction sites. Digested gwhiz was separated on an agarose gel, the desired band excised and purified as described above. Cutting the vector in this manner facilitated directional cloning of the modified transposase (mATS) between the CMV promoter and synthetic polyA.
To insert the mATS between the CMV promoter and synthetic polyA in gWhiz, a Stratagene T4 Ligase Kit (Stratagene, Inc. La Jolla, Calif.) was used and the ligation set up according to the manufacturer's protocol. Ligated product was transformed into E. coli Top10 competent cells (Invitrogen Life Technologies, Carlsbad, Calif.) using chemical transformation according to Invitrogen's protocol. Transformed bacteria were incubated in 1 ml of SOC (GIBCO BRL, CAT#15544-042) medium for 1 hour at 37° C. before being spread to LB (Luria-Bertani media (broth or agar)) plates supplemented with 100 μg/ml ampicillin (LB/amp plates). These plates were incubated overnight at 37° C. and resulting colonies picked to LB/amp broth for overnight growth at 37° C. Plasmid DNA was isolated using a modified alkaline lysis protocol (Sambrook et al., 1989), electrophoresed on a 1% agarose gel, and visualized on a U.V. transilluminator after ethidium bromide staining. Colonies producing a plasmid of the expected size (approximately 6.4 kbp) were cultured in at least 250 ml of LB/amp broth and plasmid DNA harvested using a Qiagen Maxi-Prep Kit (column purification) according to the manufacturer's protocol (Qiagen, Inc., Chatsworth, Calif.). Column purified DNA was used as template for sequencing to verify the changes made in the transposase were the desired changes and no further changes or mutations occurred due to PCR amplification. For sequencing, Perkin-Elmer's Big Dye Sequencing Kit was used. All samples were sent to the Gene Probes and Expression Laboratory (LSU School of Veterinary Medicine) for sequencing on a Perkin-Elmer Model 377 Automated Sequencer.
Once a clone was identified that contained the desired mATS in the correct orientation, primers CMVf-NgoM IV (5′ TTGCCGGCATCAGATTGGCTAT (SEQ ID NO:40); underlined bases denote a NgoM IV recognition site) and Syn-polyA-BstE II (5′ AGAGGTCACCGGGTCAATTCTTCAGCACCTGGTA (SEQ ID NO:41); underlined bases denote a BstE II recognition site) were used to PCR amplify the entire CMV promoter, mATS, and synthetic polyA for cloning upstream of the transposon in pTnLac. The PCR was conducted with FailSafeTM as described above, purified using the Zymo Clean and Concentrator kit, the ends digested with NgoM IV and BstE II (New England Biolabs), purified with the Zymo kit again and cloned upstream of the transposon in pTnLac as described below.
Plasmid pTnLac was digested with NgoM IV and BstE II to remove the ptac promoter and transposase and the fragments separated on an agarose gel. The band corresponding to the vector and transposon was excised, purified from the agarose, and dephosphorylated with calf intestinal alkaline phosphatase (New England Biolabs) to prevent self-annealing. The enzyme was removed from the vector using a Zymo DNA Clean and Concentrator-5. The purified vector and CMVp/mATS/polyA were ligated together using a Stratagene T4 Ligase Kit and transformed into E. coli as described above.
Colonies resulting from this transformation were screened (mini-preps) as describe above and clones that were the correct size were verified by DNA sequence analysis as described above. The vector was given the name pTnMod (SEQ ID NO:3) and includes the following components:
Base pairs 1-130 are a remainder of F1(−) on from pBluescriptII sk(−) (Stratagene), corresponding to base pairs 1-130 of pBluescriptII sk(−).
Base pairs 131-132 are a residue from ligation of restriction enzyme sites used in constructing the vector.
Base pairs 133-1777 are the CMV promoter/enhancer taken from vector pGWiz (Gene Therapy Systems), corresponding to bp 229-1873 of pGWiz. The CMV promoter was modified by the addition of an ACC sequence upstream of ATG.
Base pairs 1778-1779 are a residue from ligation of restriction enzyme sites used in constructing the vector.
Base pairs 1780-1785 are the Kozak sequence of SEQ ID NO: 1, and base pairs 1783-2987 are the coding sequence for the transposase, modified from Tn10 (GenBank accession J01829) by optimizing codons for stability of the transposase mRNA and for the expression of protein. More specifically, in each of the codons for the first ten amino acids of the transposase, G or C was changed to A or T when such a substitution would not alter the amino acid that was encoded.
Base pairs 2988-2993 are two engineered stop codons.
Base pair 2994 is a residue from ligation of restriction enzyme sites used in constructing the vector.
Base pairs 2995-3410 are a synthetic polyA sequence taken from the pGWiz vector (Gene Therapy Systems), corresponding to bp 1922-2337 of 10 pGWiz.
Base pairs 3415-3718 are non-coding DNA that is residual from vector pNK2859.
Base pairs 3719-3761 are non-coding λ DNA that is residual from pNK2859.
Base pairs 3762-3831 are the 70 bp of the left insertion sequence recognized by the transposon Tn10.
Base pairs 3832-3837 are a residue from ligation of restriction enzyme sites used in constructing the vector.
Base pairs 3838-4527 are the multiple cloning site from pBluescriptII sk(20), corresponding to bp 924-235 of pBluescriptII sk(−). This multiple cloning site may be used to insert any coding sequence of interest into the vector.
Base pairs 4528-4532 are a residue from ligation of restriction enzyme sites used in constructing the vector.
Base pairs 4533-4602 are the 70 bp of the right insertion sequence recognized by the transposon Tn10.
Base pairs 4603-4644 are non-coding λ DNA that is residual from pNK2859.
Base pairs 4645-5488 are non-coding DNA that is residual from pNK2859.
Base pairs 5489-7689 are from the pBluescriptII sk(−) base vector—(Stratagene, Inc.), corresponding to bp 761-2961 of pBluescriptII sk(−).
Completing pTnMod is a pBlueScript backbone that contains a colE I origin of replication and an antibiotic resistance marker (ampicillin).
It should be noted that all non-coding DNA sequences described above can be replaced with any other non-coding DNA sequence(s). Missing nucleotide sequences in the above construct represent restriction site remnants.
All plasmid DNA was isolated by standard procedures. Briefly, Escherichia coli containing the plasmid was grown in 500 mL aliquots of LB broth (supplemented with an appropriate antibiotic) at 37° C. overnight with shaking. Plasmid DNA was recovered from the bacteria using a Qiagen Maxi-Prep kit (Qiagen, Inc., Chatsworth, Calif.) according to the manufacturer's protocol. Plasmid DNA was resuspended in 500 μL of PCR-grade water and stored at −20° C. until used.
EXAMPLE 3
Transposon-Based Vector pTnMCS
Another transposon-based vector was designed for inserting a desired coding sequence into the genome of eukaryotic cells. This vector was termed pTnMCS and its constituents are provided below. The sequence of the pTnMCS vector is provided in SEQ ID NO:2. The pTnMCS vector contains an avian optimized polyA sequence operably-linked to the transposase gene. The avian optimized polyA sequence contains approximately 40 nucleotides that precede the A nucleotide string.
Bp 1-130 Remainder of F1 (−) ori of pBluescriptII sk(−) (Stratagene) bp1-130
Bp 133-1777 CMV promoter/enhancer taken from vector pGWIZ (Gene Therapy Systems) bp 229-1873
Bp 1783-2991 Transposase, from Tn10 (GenBank accession #J01829) bp 108-1316
Bp 2992-3344 Non coding DNA from vector pNK2859
Bp 3345-3387 Lambda DNA from pNK2859
Bp 3388-3457 70 bp of IS10 left insertion sequence from Tn10
Bp 3464-3670 Multiple cloning site from pBluescriptII sk(−), thru the XmaI site bp 924-718
Bp 3671-3715 Multiple cloning site from pBluescriptII sk(−), from the XmaI site thru the XhoI site. These base pairs are usually lost when cloning into pTnMCS bp 717-673
Bp 3716-4153 Multiple cloning site from pBluescriptII sk(−), from the XhoI site bp 672-235
Bp 4159-4228 70 bp of IS10 right insertion sequence from Tn10
Bp 4229-4270 Lambda DNA from pNK2859
Bp 4271-5114 Non-coding DNA from pNK2859
Bp 5115-7315 pBluescript sk (−) base vector (Stratagene, Inc.) bp 761-2961.
EXAMPLE 4
Preparation of Transposon-Based Vector pTnMod(Oval/ENT TAG/ProIns/PA)—Chicken
A vector was designed to insert a human proinsulin coding sequence under the control of a chicken ovalbumin promoter, and a ovalbumin gene including an ovalbumin signal sequence, into the genome of a bird given below as SEQ ID NO:42.
Base pairs 1-130 are a remainder of F1(−) ori of pBluescriptII sk(−) (Stratagene) corresponding to base pairs 1-130 of pBluescriptII sk(−).
Base pairs 133-1777 are a CMV promoter/enhancer taken from vector pGWiz (Gene Therapy Systems) corresponding to base pairs 229-1873 of pGWiz.
Base pairs 1780-1785 are the Kozak sequence of SEQ ID NO: 1, and base pairs 1783-2987 are the coding sequence for the a transposase, modified from Tn10 (GenBank accession number J01829).
Base pairs 2988-2993 are two engineered stop codons.
Base pairs 2995-3410 are a synthetic polyA from pGWiz (Gene Therapy Systems) corresponding to base pairs 1922-2337 of pGWiz.
Base pairs 3415-37.18 are non coding DNA that is residual from vector pNK2859.
Base pairs 3719-3761 are λ DNA that is residual from pNK2859.
Base pairs 3762-3831 are the 70 base pairs of the left insertion sequence (IS10) recognized by the transposon Tn10.
Base pairs 3838-4044 are a multiple cloning site from pBlueScriptII sk(−) corresponding to base pairs 924-718 of pBluescriptII sk(−).
Base pairs 4050-4951 are a chicken ovalbumin promoter (including SDRE) that corresponds to base pairs 431-1332 of the chicken ovalbumin promoter in GenBank Accession Number J00895 M24999.
Base pairs 4958-6115 are a chicken ovalbumin signal sequence and ovalbumin gene that correspond to base pairs 66-1223 of GenBank Accession Number V00383.1. (The STOP codon being omitted).
Base pairs 6122-6271 are a TAG sequence containing a gp41 hairpin loop from HIV I, an enterokinase cleavage site and a spacer (synthetic).
Base pairs 6272-6531 are a proinsulin gene.
Base pairs 6539-6891 are a synthetic polyadenylation sequence from pGWiz (Gene Therapy Systems) corresponding to base pairs 1920-2272 of pGWiz.
Base pairs 6897-7329 are a multiple cloning site from pBlueScriptII sk(−) corresponding to base pairs 667-235 of pBluescriptII sk(−).
Base pairs 7335-7404 are the 70 base pairs of the right insertion sequence (IS10) recognized by the transposon Tn10.
Base pairs 7405-7446 are λ DNA that is residual from pNK2859.
Base pairs 7447-8311 are non coding DNA that is residual from pNK2859.
Base pairs 8312-10512 are pBlueScript sk(−) base vector (Stratagene, Inc.) corresponding to base pairs 761-2961 of pBluescriptII sk(−).
It should be noted that all non-coding DNA sequences described above can be replaced with any other non-coding DNA sequence(s). Missing nucleotide sequences in the above construct represent restriction site remnants.
EXAMPLE 5
Transposon-Based Vector pTnMOD (CMV-CHOVg-ent-ProInsulin-synPA)
A vector was designed to insert a proinsulin coding sequence under the control of a quail ovalbumin promoter, and a ovalbumin gene including an ovalbumin signal sequence, into the genome of a bird given below as SEQ ID NO:43.
Bp 1-4045 from vector pTnMod, bp 1-4045
Bp 4051-5695 CMV promoter/enhancer taken from vector pGWIZ (Gene therapy systems), bp 230-1864
Bp 5702-6855 Chicken ovalbumin gene taken from GenBank accession # V00383, bp 66-1219
Bp 6862-7011 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site
Bp 7012-7272 Human Proinsulin taken from GenBank accession # NM000207, bp 117-377
Bp 7273-7317 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and pGWIZ (Gene Therapy Systems)
Bp 7318-7670 Synthetic polyA from the cloning vector pGWIZ (Gene Therapy Systems), bp 1920-2271
Bp 7672-11271 from cloning vector pTnMCS, bp 3716-7315
EXAMPLE 6
Transfection of Japanese Quail Using a Transposon-Based Vector Containing a Proinsulin Gene Via Oviduct Injections
Two experiments were conducted in Japanese quail using transposon-based vectors containing either Oval promoter/Oval gene/GP41 Enterokinase TAG/Proinsulin/Poly A (SEQ ID NO:42) or CMV promoter/Oval gene/GP41 Enterokinase TAG/Proinsulin/Poly A (SEQ ID NO:43).
In the first experiment, the Oval promoter/Oval gene/GP41 Enterokinase TAG/Proinsulin/Poly A containing construct was injected into the lumen of the oviduct of sexually mature quail; three hens received 5 μg at a 1:3 SUPERFECT® ratio and three received 10 μg at a 1:3 SUPERFECT® ratio. As of the writing of the present application, at least one bird that received above-mentioned construct was producing human proinsulin in egg white (other birds remain to be tested). This experiment indicates that 1) the DNA has been stable for at least 3 months; 2) protein levels are comparable to those observed with a constitutive promoter such as the CMV promoter; and 3) sexually mature birds can be injected and results obtained without the need for cell culture. It is estimated that each quail egg contains approximately 1.4 mg/ml of the proinsulin protein. It is also estimated that each transgenic chicken egg contains 50-75 mg of protein encoded by the gene of interest.
In the second experiment, the transposon-based vector containing CMV promoter/Oval gene/GP41 Enterokinase TAG/Proinsulin/Poly A was injected into the lumen of the oviduct of sexually immature Japanese quail. A total of 9 birds were injected. Of the 8 survivors, 3 produced human proinsulin in the white of their eggs for over 6 weeks. An ELISA assay described in detail below was developed to detect GP41 in the fusion peptide (Oval gene/GP41 Enterokinase TAG/Proinsulin) since the GP41 peptide sequence is unique and not found as part of normal egg white protein. In all ELISA assays, the same birds produced positive results and all controls worked as expected.
ELISA Procedure: Individual egg white samples were diluted in sodium carbonate buffer, pH 9.6, and added to individual wells of 96 well microtiter ELISA plates at a total volume of 0.1 ml. These plates were then allowed to coat overnight at 4° C. Prior to ELISA development, the plates were allowed warm to room temperature. Upon decanting the coating solutions and blotting away any excess, non-specific binding of antibodies was blocked by adding a solution of phosphate buffered saline (PBS), 1% (w/v) BSA, and 0.05% (v/v) Tween 20 and allowing it to incubate with shaking for a minimum of 45 minutes. This blocking solution was subsequently decanted and replaced with a solution of the primary antibody (Goat Anti-GP41 TAG) diluted in fresh PBS/BSA/Tween 20. After a two hour period of incubation with the primary antibody, each plate was washed with a solution of PBS and 0.05% Tween 20 in an automated plate washer to remove unbound antibody. Next, the secondary antibody, Rabbit anti-Goat Alkaline Phosphatase-conjugated, was diluted in PBS/BSA/Tween 20 and allowed to incubate 1 hour. The plates were then subjected to a second wash with PBS/Tween 20. Antigen was detected using a solution of p-Nitrophenyl Phosphate in Diethanolamine Substrate Buffer for Alkaline Phosphatase and measuring the absorbance at 30 minutes and 1 hour.
Additionally, a proinsulin fusion protein produced using a construct described above was isolated from egg white using ammonium sulfate precipitation and ion exchange chromatography. A pooled fraction of the isolated fusion protein was run on an SDS-PAGE gel shown in FIG. 5, lanes 4 and 6. Lanes 1 and 10 of the gel contain molecular weight standards, lanes 2 and 8 contain non-transgenic chicken egg white, whereas lanes 3, 5, 7 and 9 are blank.
EXAMPLE 7
Isolation of Human Proinsulin Using Anti-Tag Column Chromotography
A HiTrap NHS-activated 1 mL column (Amersham) was charged with a 30 amino acid peptide that contained the gp-41 epitope containing gp-41's native disulfide bond that stabilizes the formation of the gp-41 hairpin loop. The 30 amino acid gp41 peptide is provided as SEQ ID NO:32. Approximately 10 mg of the peptide was dissolved in coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3 and the ligand was circulated on the column for 2 hours at room temperature at 0.5 mL/minute. Excess active groups were then deactivated using 6 column volumes of 0.5 M ethanolamine, 0.5 M NaCl, pH 8.3 and the column was washed alternately with 6 column volumes of acetate buffer (0.1 M acetate, 0.5 M NaCl, pH 4.0) and ethanolamine (above). The column was neutralized using 1×PBS. The column was then washed with buffers to be used in affinity purification: 75 mM Tris, pH 8.0 and elution buffer, 100 mM glycine-HCl, 0.5 M NaCl, pH 2.7. Finally, the column was equilibrated in 75 mM Tris buffer, pH 8.0.
Antibodies to gp-41 were raised in goats by inoculation with the gp-41 peptide described above. More specifically, goats were inoculated, given a booster injection of the gp-41 peptide and blood samples were obtained by veinupuncture. Serum was harvested by centrifugation. Approximately 30 mL of goat serum was filtered to 0.45 uM and passed over a TAG column at a rate of 0.5 mL/min. The column was washed with 75 mM Tris, pH 8.0 until absorbance at 280 nm reached a baseline. Three column volumes (3 mL) of elution buffer (100 mM glycine, 0.5 M NaCl, pH 2.7) was applied, followed by 75 mM Tris buffer, pH 8.0, all at a rate of 0.5 mL/min. One milliliter fractions were collected. Fractions were collected into 200 uL 1 M Tris, pH 9.0 to neutralize acidic factions as rapidly as possible. A large peak eluted from the column, coincident with the application the elution buffer. Fractions were pooled. Analysis by SDS-PAGE showed a high molecular weight species that separated into two fragments under reducing condition, in keeping with the heavy and light chain structure of IgG.
Pooled antibody fractions were used to charge two 1 mL HiTrap NHS-activated columns, attached in series. Coupling was carried out in the same manner as that used for charging the TAG column.
Isolation of Ovalbumin-TAG-Proinsulin from Egg White
Egg white from quail and chickens treated by intra-oviduct injection of the CMV-ovalbumin-TAG-proinsulin construct were pooled. Viscosity was lowered by subjecting the allantoid fluid to successively finer pore sizes using negative pressure filtration, finishing with a 0.22 μM pore size. Through the process, egg white was diluted approximately 1:16. The clarified sample was loaded on the Anti-TAG column and eluted in the same manner as described for the purification of the anti-TAG antibodies. A peak of absorbance at 280 nm, coincident with the application of the elution buffer, indicated that protein had been specifically eluted from the Anti-TAG column. Fractions containing the eluted peak were pooled for analysis.
The pooled fractions from the Anti-TAG affinity column were characterized by SDS-PAGE and western blot analysis. SDS-PAGE of the pooled fractions revealed a 60 kDal molecular weight band not present in control egg white fluid, consistent with the predicted molecular weight of the transgenic protein. Although some contaminating bands were observed, the 60 kDal species was greatly enriched compared to the other proteins. An aliquot of the pooled fractions was cleaved overnight at room temperature with the protease, enterokinase. SDS-PAGE analysis of the cleavage product, revealed a band not present in the uncut material that co-migrated with a commercial human proinsulin positive control. Western blot analysis showed specific binding to the 60 kDal species under non-reducing condition (which preserved the hairpin epitope of gp-41 by retaining the disulfide bond). Western analysis of the low molecular weight species that appeared upon cleavage with an anti-human proinsulin antibody, conclusively identified the cleaved fragment as human proinsulin.
EXAMPLE 8
Purification Procedures for Insulin
I. ELISA Data for Egg Characterization/Identification
An ELISA was employed for the initial screening of eggs and, thereby, identification of hens producing positive eggs. With further modifications this procedure was used for the initial quantification of recombinant protein amounts. These procedures were aided by the successful purification of an initial stock of the recombinant proinsulin (RPI). This stock of protein is used in the development of a double antibody assay that increases the sensitivity and reduces the background in the assay. Subsequent identification of hens producing positive eggs obviate the need to screen each egg collected. Only periodic checks are needed to determine if production levels are consistent.
II. Egg White (EW) or Albumin Preparation
A. Clarification—Ovomucin Precipitation
Eggs from hens positively identified as producing RPI are pooled for RPI purification. The initial purification step involved diluting the pool 1:1 with 100 mM Tris-HCl, pH 8 for a final concentration of 50 mM Tris-HCl. The pH of this solution was then adjusted to 6 and ovomucin was allowed to precipitate at 4° C. for a minimum of 3 hrs (preferably overnight) with constant stirring. The precipitated ovomucin was then pelleted and removed by centrifugation at 2400×g. After collection of the RPI containing supernatant, the pH of this solution was readjusted to 8.
B. Filtration
To prepare the egg white for loading onto the column and, thereby, minimize the potential for clogging the columns during loading, the egg white solution was filtered to at least 0.45 um.
Initially, the ovomucin precipitated egg white solution was subjected to successive filtration steps with the pore size of the filtration membrane decreasing at each step. This procedure involved time and dilution of the egg white solution to reach 0.45 um filtration.
Amersham's hollow-fiber ultrafiltration apparatus was used to produced a column-ready solution filtered down to <0.2 um with an undiluted starting solution. This approach minimized the time and the solution dilution needed to prepare the egg white solution for column loading.
III. Purification
A. Affinity Chromatography—
Using antibody with specificity to a synthetic peptide modeled after the enterokinase recognition site, initial purification schemes involved developing a one-step column purification procedure for the RPI.
Goats immunized with the synthetic Ent peptide were employed to produce anti-Ent Tag antiserum which was used in the egg screening ELISAs followed by antibody purification. The purified goat Anti-Ent Tag antibodies were covalently bound to the matrix of HiTrap NHS-activated HP columns (Amersham) and subsequently used to specifically bind and purify the RPI.
An initial attempt was made to direct the first purification step against the ovalbumin portion of the recombinant protein using an antibody specific for the ovalbumin portion. The present purification scheme employed a combination of classical techniques such as ammonium sulfate precipitation, ion exchange, and gel filtration chromatography.
After the initial ovomucin precipitation, the egg white solution was subjected to protein precipitation using a 40% ammonium sulfate fractionation. The precipitated protein was subsequently collected via centrifugation and resuspended in 50 mM Tris-HCl, pH 8. The resuspended protein solution was dialyzed to remove residual (NH4)2SO4 or subjected to gel filtration to remove the (NH4)2SO4 and partially isolate the RPI from the remaining egg white protein. The RPI was further isolated via anion exchange chromatography using a 0 to 0.5M NaCl gradient in 50 mM Tris-HCl, pH 8. Two possible elution profiles were observed. One at approximately 25% of the 0.5 M NaCl gradient without (NH4)2SO4 precipitation. The second was observed at less than 16% gradient (approximately 7%) following 40% (NH4)2SO4 precipitation and a longer gradient. Fractions containing RPI were identified by SDS-PAGE analysis and pooled.
Three gel filtration columns, differing by column size and fractionation range, were employed in RPI purification and/or desalting: Superdex 75 10/300 GL, Hiload 26/60 Superdex 75, and Hiload 26/60 Superdex 200. Using these individual columns at different steps in the purification scheme increased the efficiency of the process. Fractions containing RPI were identified by SDS-PAGE analysis and pooled.
Cleavage of the RPI Enterokinase recognition site was accomplished using purified enterokinase from Sigma. Enterokinase, 0.004 Unit/μl per reaction, was applied to the pooled and, if necessary, concentrated protein solution. The digestion reaction was incubated at room temperature (up to 30° C. in a rolling hybridization oven) for a minimum of 16 h and in some cases up to 48 hrs of incubation. The digestion efficiency was followed using 16.5% Tris-Tricine SDS-PAGE peptide gels. All gel staining utilized Simply Blue Coomassie Staining Solutions. Free Proinsulin was observed on gels after digestion.
A subsequent gel filtration separation was employed to obtain purified Proinsulin, and to remove the remaining Ovalbumin portion of the RPI and residual native EW proteins. Select steps in the purification process were analyzed using the 2-dimensional Beckman Coulter ProteomeLab PF2D Protein Fractionation System.
EXAMPLE 9
Optimization of Intra-Oviduct and Intra-Ovarian Arterial Injections
Overall transfection rates of oviduct cells in a flock of chicken or quail hens are enhanced by synchronizing the development of the oviduct and ovary within the flock. When the development of the oviducts and ovaries are uniform across a group of hens and when the stage of oviduct and ovarian development can be determined or predicted, timing of injections is optimized to transfect the greatest number of cells. Accordingly, oviduct development is synchronized as described below to ensure that a large and uniform proportion of oviduct secretory cells are transfected with the gene of interest.
Hens are treated with estradiol to stimulate oviduct maturation as described in Oka and Schimke (T. Oka and R T Schimke, J. Cell Biol., 41, 816 (1969)), Palmiter, Christensen and Schimke (J. Biol. Chem. 245(4):833-845, 1970). Specifically, repeated daily injections of 1 mg estradiol benzoate are performed sometime before the onset of sexual maturation, a period ranging from 1-14 weeks of age. After a stimulation period sufficient to maximize development of the oviduct, hormone treatment is withdrawn thereby causing regression in oviduct secretory cell size but not cell number. At an optimum time after hormone withdrawal, the lumens of the oviducts of treated hens are injected with the transposon-based vector. Hens are subjected to additional estrogen stimulation after an optimized time during which the transposon-based vector is taken up into oviduct secretory cells. Re-stimulation by estrogen activates transposon expression, causing the integration of the gene of interest into the host genome. Estrogen stimulation is then withdrawn and hens continue normal sexual development. If a developmentally regulated promoter such as the ovalbumin promoter is used, expression of the transposon-based vector initiates in the oviduct at the time of sexual maturation. Intra-ovarian artery injection during this window allows for high and uniform transfection efficiencies of ovarian follicles to produce germ-line transfections and possibly oviduct expression.
Other means are also used to synchronize the development, or regression, of the oviduct and ovary to allow high and uniform transfection efficiencies. Alterations of lighting and/or feed regimens, for example, cause hens to ‘molt’ during which time the oviduct and ovary regress. Molting is used to synchronize hens for transfection, and may be used in conjunction with other hormonal methods to control regression and/or development of the oviduct and ovary.
EXAMPLE 10
Preparation of Transposon-Based Vector pTnMod(Oval/ENT TAG/ProIns/PA)—Quail
A vector is designed for inserting a proinsulin gene under the control of a quail ovalbumin promoter, and a ovalbumin gene including an ovalbumin signal sequence, into the genome of a bird given below as SEQ ID NO:44.
Base pairs 1-130 are a remainder of F1(−) ori of pBluescriptII sk(−) (Stratagene) corresponding to base pairs 1-130 of pBluescriptII sk(−).
Base pairs 133-1777 are a CMV promoter/enhancer taken from vector pGWiz (Gene Therapy Systems) corresponding to base pairs 229-1873 of pGWiz.
Base pairs 1780-1785 are the Kozak sequence of SEQ ID NO: 1, and base pairs 1783-2987 are the coding sequence for the a transposase, modified from Tn10 (GenBank accession number J01829).
Base pairs 2988-2993 are an engineered stop codon.
Base pairs 2995-3410 are a synthetic polyA from pGWiz (Gene Therapy Systems) corresponding to base pairs 1922-2337 of pGWiz.
Base pairs 3415-3718 are non coding DNA that is residual from vector pNK2859.
Base pairs 3719-3761 are λ DNA that is residual from pNK2859.
Base pairs 3762-3831 are the 70 base pairs of the left insertion sequence (IS10) recognized by the transposon Tn10.
Base pairs 3838-4044 are a multiple cloning site from pBlueScriptII sk(−) corresponding to base pairs 924-718 of pBluescriptII sk(−).
Base pairs 4050-4938 are the Japanese quail ovalbumin promoter (including SDRE, steroid-dependent response element). The Japanese quail ovalbumin promoter was isolated by its high degree of homology to the chicken ovalbumin promoter (GenBank accession number J00895 M24999, base pairs 431-1332). Some deletions were noted in the quail sequence, as compared to the chicken sequence.
Base pairs 4945-6092 are a quail ovalbumin signal sequence and ovalbumin gene that corresponds to base pairs 54-1201 of GenBank accession number X53964.1. (The STOP codon being omitted).
Base pairs 6093-6246 are a TAG sequence containing a gp41 hairpin loop from HIV I an enterokinase cleavage site and a spacer (synthetic).
Base pairs 6247-6507 are a proinsulin gene.
Base pairs 6514-6866 are a synthetic polyadenylation sequence from pGWiz (Gene Therapy Systems) corresponding to base pairs 1920-2272 of pGWiz.
Base pairs 6867-7303 are a multiple cloning site from pBlueScriptII sk(−) corresponding to base pairs 667-235 of pBluescriptII sk(−).
Base pairs 7304-7379 are the 70 base pairs of the right insertion sequence (IS10) recognized by the transposon Tn10.
Base pairs 7380-7421 are λ DNA that is residual from pNK2859.
Base pairs 7422-8286 are non coding DNA that is residual from pNK2859.
Base pairs 8287-10487 are pBlueScript sk(−) base vector (Stratagene, Inc.) corresponding to base pairs 761-2961 of pBluescriptII sk(−).
It should be noted that all non-coding DNA sequences described above can be replaced with any other non-coding DNA sequence(s). Missing nucleotide sequences in the above construct represent restriction site remnants.
EXAMPLE 11
Preparation of Transposon-Based Vector pTnMod(Oval/ENT TAG/p146/PA)—Chicken
A vector was designed for inserting a p146 gene under the control of a chicken ovalbumin promoter, and a ovalbumin gene including an ovalbumin signal sequence, into the genome of a bird. The vector sequence is provided below as SEQ ID NO:45.
Base pairs 1-130 are a remainder of F1(−) ori of pBluescriptII sk(−) (Stratagene) corresponding to base pairs 1-130 of pBluescriptII sk(−).
Base pairs 133-1777 are a CMV promoter/enhancer taken from vector pGWiz (Gene Therapy Systems) corresponding to base pairs 229-1873 of pGWiz.
Base pairs 1780-1785 are the Kozak sequence of SEQ ID NO: 1, and base pairs 1783-2987 are the coding sequence for the a transposase, modified from Tn10 (GenBank accession number J01829).
Base pairs 2988-2993 are an engineered stop codon.
Base pairs 2995-3410 are a synthetic polyA from pGWiz (Gene Therapy Systems) corresponding to base pairs 1922-2337 of pGWiz.
Base pairs 3415-3718 are non coding DNA that is residual from vector pNK2859.
Base pairs 3719-3761 are λ DNA that is residual from pNK2859.
Base pairs 3762-3831 are the 70 base pairs of the left insertion sequence (IS10) recognized by the transposon Tn10.
Base pairs 3838-4044 are a multiple cloning site from pBlueScriptII sk(−) corresponding to base pairs 924-718 of pBluescriptII sk(−).
Base pairs 4050-4951 are a chicken ovalbumin promoter (including SDRE, steroid-dependent response element) that corresponds to base pairs 431-1332 of the chicken ovalbumin promoter in GenBank Accession Number J00895 M24999.
Base pairs 4958-6115 are a chicken ovalbumin signal sequence and Ovalbumin gene that correspond to base pairs 66-1223 of GenBank Accession Number V00383.1 (The STOP codon being omitted).
Base pairs 6122-6271 are a TAG sequence containing a gp41 hairpin loop from HIV I, an enterokinase cleavage site and a spacer (synthetic).
Base pairs 6272-6316 are a p146 sequence (synthetic) with 2 added stop codons.
Base pairs 6324-6676 are a synthetic polyadenylation sequence from pGWiz (Gene Therapy Systems) corresponding to base pairs 1920-2272 of pGWiz.
Base pairs 6682-7114 are a multiple cloning site from pBlueScriptII sk(−) corresponding to base pairs 667-235 of pBluescriptII sk(−).
Base pairs 7120-7189 are the 70 base pairs of the right insertion sequence (IS10) recognized by the transposon Tn10.
Base pairs 7190-7231 are λ DNA that is residual from pNK2859.
Base pairs 7232-8096 are non coding DNA that is residual from pNK2859.
Base pairs 8097-10297 are pBlueScript sk(−) base vector (Stratagene, Inc.) corresponding to base pairs 761-2961 of pBluescriptII sk(−).
It should be noted that all non-coding DNA sequences described above can be replaced with any other non-coding DNA sequence(s). Missing nucleotide sequences in the above construct represent restriction site remnants.
EXAMPLE 12
Preparation of Transposon-Based Vector pTnMod(Oval/ENT TAG/p146/PA)—Quail
A vector was designed for inserting a p146 gene under the control of a quail ovalbumin promoter, and a ovalbumin gene including an ovalbumin signal sequence, into the genome of a bird. The vector sequence is given below as SEQ ID NO:46.
Base pairs 1-130 are a remainder of F1(−) ori of pBluescriptII sk(−) (Stratagene) corresponding to base pairs 1-130 of pBluescriptII sk(−).
Base pairs 133-1777 are a CMV promoter/enhancer taken from vector pGWiz (Gene Therapy Systems) corresponding to base pairs 229-1873 of pGWiz.
Base pairs 1780-1785 are the Kozak sequence of SEQ ID NO: 1, and base pairs 1783-2987 are the coding sequence for the a transposase, modified from Tn10 (GenBank accession number J01829).
Base pairs 2988-2993 are an engineered stop codon.
Base pairs 2995-3410 are a synthetic polyA from pGWiz (Gene Therapy Systems) corresponding to base pairs 1922-2337 of pGWiz.
Base pairs 3415-3718 are non coding DNA that is residual from vector pNK2859.
Base pairs 3719-3761 are λ DNA that is residual from pNK2859.
Base pairs 3762-3831 are the 70 base pairs of the left insertion sequence (IS10) recognized by the transposon Tn10.
Base pairs 3838-4044 are a multiple cloning site from pBlueScriptII sk(−) corresponding to base pairs 924-718 of pBluescriptII sk(−).
Base pairs 4050-4938 are the Japanese quail ovalbumin promoter (including SDRE, steroid-dependent response element). The Japanese quail ovalbumin promoter was isolated by its high degree of homology to the chicken ovalbumin promoter (GenBank accession number J00895 M24999, base pairs 431-1332).
Bp 4945-6092 are a quail ovalbumin signal sequence and ovalbumin gene that corresponds to base pairs 54-1201 of GenBank accession number X53964.1. (The STOP codon being omitted).
Base pairs 6097-6246 are a TAG sequence containing a gp41 hairpin loop from HIV I, an enterokinase cleavage site and a spacer (synthetic).
Base pairs 6247-6291 are a p146 sequence (synthetic) with 2 added stop codons.
Base pairs 6299-6651 are a synthetic polyadenylation sequence from pGWiz (Gene Therapy Systems) corresponding to base pairs 1920-2272 of pGWiz.
Base pairs 6657-7089 are a multiple cloning site from pBlueScriptII sk(−) corresponding to base pairs 667-235 of pBluescriptII sk(−).
Base pairs 7095-7164 are the 70 base pairs of the right insertion sequence (IS10) recognized by the transposon Tn10.
Base pairs 7165-7206 are λ DNA that is residual from pNK2859.
Base pairs 7207-8071 are non coding DNA that is residual from pNK2859.
Base pairs 8072-10272 are pBlueScript sk(−) base vector (Stratagene, Inc.) corresponding to base pairs 761-2961 of pBluescriptII sk(−).
It should be noted that all non-coding DNA sequences described above can be replaced with any other non-coding DNA sequence(s). Missing nucleotide sequences in the above construct represent restriction site remnants.
EXAMPLE 13
Additional Transposon-Based Vectors for Administration to an Animal
The following example provides a description of various transposon-based vectors of the present invention and several constructs that have been made for insertion into the transposon-based vectors of the present invention. These examples are not meant to be limiting in any way. The constructs for insertion into a transposon-based vector are provided in a cloning vector pTnMCS or pTnMod, both described above.
pTnMCS (CMV-CHOVg-ent-ProInsulin-synPA) (SEQ ID NO:47)
Bp 1-3670 from vector PTnMCS, bp 1-3670
Bp 3676-5320 CMV promoter/enhancer taken from vector pGWIZ (Gene Therapy Systems), bp 230-1864
Bp 5327-6480 Chicken ovalbumin gene taken from GenBank accession # V00383, bp 66-1219
Bp 6487-6636 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site
Bp 6637-6897 Human Proinsulin taken from GenBank accession # NM000207, bp 117-377
Bp 6898-6942 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and pGWIZ (Gene Therapy Systems)
Bp 6943-7295 Synthetic polyA from the cloning vector pGWIZ (Gene Therapy Systems), bp 1920-2271
Bp 7296-10895 from cloning vector pTnMCS, bp 3716-7315
pTnMCS (CMV-prepro-ent-ProInsulin-synPA)
Bp 1-3670 from vector PTnMCS, bp 1-3670
Bp 3676-5320 CMV promoter/enhancer taken from vector pGWIZ (Gene Therapy Systems), bp 230-1864
Bp 5326-5496 Capsite/prepro taken from GenBank accession # X07404, bp 563-733
Bp 5504-5652 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site
Bp 5653-5913 Human Proinsulin taken from GenBank accession # NM000207, bp 117-377
Bp 5914-5958 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and pGWIZ (Gene Therapy Systems)
Bp 5959-6310 Synthetic polyA from the cloning vector pGWIZ (Gene Therapy Systems), bp 1920-2271
Bp 6313-9912 from cloning vector pTnMCS, bp 3716-7315
pTnMCS(Chicken OVep+OVg′+ENT+proins+syn polyA)
Bp 1-3670 from vector pTnMCS, bp 1-3670
Bp 3676-4350 Chicken Ovalbumin enhancer taken from GenBank accession #S82527.1 bp 1-675
Bp 4357-5692 Chicken Ovalbumin promoter taken from GenBank accession # J00895M24999 bp 1-1336
Bp 5699-6917 Chicken Ovalbumin gene from GenBank Accession # V00383.1 bp 2-1220. (This sequence includes the 5′UTR, containing putative cap site, bp 5699-5762.)
Bp 6924-7073 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site
Bp 7074-7334 Human proinsulin GenBank Accession # NM000207 bp 117-377
Bp 7335-7379 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and gWIZ (Gene Therapy Systems)
Bp 7380-7731 Synthetic polyA from the cloning vector gWIZ (Gene Therapy Systems) bp 1920-2271
Bp 7733-11332 from vector pTnMCS, bp 3716-7315
pTnMCS(Chicken OVep+prepro+ENT+proins+syn polyA)
Bp 1-3670 from cloning vector pTnMCS, bp 1-3670
Bp 3676-4350 Chicken Ovalbumin enhancer taken from GenBank accession # S82527.1 bp 1-675
Bp 4357-5692 Chicken Ovalbumin promoter taken from GenBank accession # J00895-M24999 bp 1-1336
Bp 5699-5869 Cecropin cap site and prepro, Genbank accession # X07404 bp 563-733
Bp 5876-6025 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site
Bp 6026-6286 Human proinsulin GenBank Accession # NM000207 bp 117-377
Bp 6287-6331 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and gWIZ (Gene Therapy Systems)
Bp 6332-6683 Synthetic polyA from the cloning vector gWIZ (Gene Therapy Systems) bp 1920-2271
Bp 6685-10284 from cloning vector pTnMCS, bp 3716-7315
pTnMCS(Quail OVep+OVg′+ENT+proins+syn polyA)
Bp 1-3670 from cloning vector pTnMCS, bp 1-3670
Bp 3676-4333 Quail Ovalbumin enhancer: 658 bp sequence, amplified in-house from quail genomic DNA, roughly equivalent to the far-upstream chicken ovalbumin enhancer, GenBank accession # S82527.1, bp 1-675. (There are multiple base pair substitutions and deletions in the quail sequence, relative to chicken, so the number of bases does not correspond exactly.)

Bp 4340-5705 Quail Ovalbumin promoter: 1366 bp sequence, amplified in-house from quail genomic DNA, roughly corresponding to chicken ovalbumin promoter, GenBank accession # J00895-M24999 bp 1-1336. (There are multiple base pair substitutions and deletions between the quail and chicken sequences, so the number of bases does not correspond exactly.)

Bp 5712-6910 Quail Ovalbumin gene, EMBL accession # X53964, bp 1-1199. (This sequence includes the 5′UTR, containing putative cap site bp 5712-5764.)

Bp 6917-7066 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site

Bp 7067-7327 Human proinsulin GenBank Accession # NM000207 bp 117-377

Bp 7328-7372 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and gWIZ (Gene Therapy Systems)

Bp 7373-7724 Synthetic polyA from the cloning vector gWIZ (Gene Therapy Systems) bp 1920-2271

Bp 7726-11325 from cloning vector pTnMCS, bp 3716-7315

pTnMCS(Quail OVep+prepro+ENT+proins+syn polyA)

Bp 1-3670 from cloning vector pTnMCS, bp 1-3670

Bp 3676-4333 Quail Ovalbumin enhancer: 658 bp sequence, amplified from quail genomic DNA, roughly equivalent to the far-upstream chicken ovalbumin enhancer, GenBank accession #S82527.1, bp 1-675. (There are multiple base pair substitutions and deletions in the quail sequence, relative to chicken, so the number of bases does not correspond exactly.)

Bp 4340-5705 Quail Ovalbumin promoter: 1366 bp sequence, amplified from quail genomic DNA, roughly corresponding to chicken ovalbumin promoter, GenBank accession # J00895-M24999 bp 1-1336. (There are multiple base pair substitutions and deletions between the quail and chicken sequences, so the number of bases does not correspond exactly.)

Bp 5712-5882 Cecropin cap site and prepro, Genbank accession # X07404 bp 563-733

Bp 5889-6038 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site

Bp 6039-6299 Human proinsulin GenBank Accession # NM000207 bp 117-377

Bp 6300-6344 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and gWIZ (Gene Therapy Systems)

Bp 6345-6696 Synthetic polyA from the cloning vector gWIZ (Gene Therapy Systems) bp 1920-2271

Bp 6698-10297 from cloning vector pTnMCS, bp 3716-7315.

pTnMOD (CMV-prepro-ent-proins-synPA)

Bp 1-4045 from vector pTnMOD, bp 1-4045

Bp 4051-5695 CMV promoter/enhancer taken from vector pGWIZ (Gene therapy systems), bp 230-1864

Bp 5701-5871 Capsite/prepro taken from GenBank accession # X07404, bp 563-733

Bp 5879-6027 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site

Bp 6028-6288 Human Proinsulin taken from GenBank accession # NM000207, bp 117-377

Bp 6289-6333 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and pGWIZ (Gene Therapy Systems)

Bp 6334-6685 Synthetic polyA from the cloning vector pGWIZ (Gene Therapy Systems), bp 1920-2271

Bp 6687-10286 from cloning vector pTnMOD, bp 3716-7315

pTnMOD(Chicken OVep+OVg′+ENT+proins+syn polyA)

Bp 1-4045 from cloning vector pTnMod, bp 1-4045

Bp 4051-4725 Chicken Ovalbumin enhancer taken from GenBank accession # S82527.1 bp 1-675

Bp 4732-6067 Chicken Ovalbumin promoter taken from GenBank accession # J00895-M24999 bp 1-1336

Bp 6074-7292 Chicken Ovalbumin gene from GenBank Accession # V00383.1 bp 2-1220. (This sequence includes the 5′UTR, containing putative cap site bp 6074-6137.)

Bp 7299-7448 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site

Bp 7449-7709 Human proinsulin GenBank Accession # NM000207 bp 117-377

Bp 7710-7754 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and gWIZ (Gene Therapy Systems)

Bp 7755-8106 Synthetic polyA from the cloning vector gWIZ (Gene Therapy Systems) bp 1920-2271

Bp 8108-11707 from cloning vector pTnMod, bp 3716-7315

pTnMOD(Chicken OVep+prepro+ENT+proins+syn polyA)

Bp 1-4045 from cloning vector pTnMOD, bp 1-4045

Bp 4051-4725 Chicken Ovalbumin enhancer taken from GenBank accession # S82527.1 bp 1-675

Bp 4732-6067 Chicken Ovalbumin promoter taken from GenBank accession # J00895-M24999 bp 1-1336

Bp 6074-6244 Cecropin cap site and prepro, Genbank accession # X07404 bp 563-733

Bp 6251-6400 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site

Bp 6401-6661 Human proinsulin GenBank Accession # NM000207 bp 117-377

Bp 6662-6706 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and gWIZ (Gene Therapy Systems)

Bp 6707-7058 Synthetic polyA from the cloning vector gWIZ (Gene Therapy Systems) bp 1920-2271

Bp 7060-10659 from cloning vector pTnMOD, bp 3716-7315

pTnMOD(Quail OVep+OVg′+ENT+proins+syn polyA)

Bp 1-4045 from cloning vector pTnMOD, bp 1-4045

Bp 4051-4708 Quail Ovalbumin enhancer: 658 bp sequence, amplified in-house from quail genomic DNA, roughly equivalent to the far-upstream chicken ovalbumin enhancer, GenBank accession # S82527.1, bp 1-675. (There are multiple base pair substitutions and deletions in the quail sequence, relative to chicken, so the number of bases does not correspond exactly.)

Bp 4715-6080 Quail Ovalbumin promoter: 1366 bp sequence, amplified in-house from quail genomic DNA, roughly corresponding to chicken ovalbumin promoter, GenBank accession # J00895-M24999 bp 1-1336. (There are multiple base pair substitutions and deletions between the quail and chicken sequences, so the number of bases does not correspond exactly.)

Bp 6087-7285 Quail Ovalbumin gene, EMBL accession # X53964, bp 1-1199. (This sequence includes the 5′UTR, containing putative cap site by 6087-6139.)

Bp 7292-7441 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site

Bp 7442-7702 Human proinsulin GenBank Accession # NM000207 bp 117-377

Bp 7703-7747 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and gWIZ (Gene Therapy Systems)

Bp 7748-8099 Synthetic polyA from the cloning vector gWIZ (Gene Therapy Systems) bp 1920-2271

Bp 8101-11700 from cloning vector pTnMOD, bp 3716-7315

pTnMOD(Quail OVep+prepro+ENT+proins+syn polyA

Bp 1-4045 from cloning vector pTnMOD, bp 1-4045

Bp 4051-4708 Quail Ovalbumin enhancer: 658 bp sequence, amplified in-house from quail genomic DNA, roughly equivalent to the far-upstream chicken ovalbumin enhancer, GenBank accession #S82527.1, bp 1-675. (There are multiple base pair substitutions and deletions in the quail sequence, relative to chicken, so the number of bases does not correspond exactly.)

Bp 4715-6080 Quail Ovalbumin promoter: 1366 bp sequence, amplified in-house from quail genomic DNA, roughly corresponding to chicken ovalbumin promoter, GenBank accession # J00895-M24999 bp 1-1336. (There are multiple base pair substitutions and deletions between the quail and chicken sequences, so the number of bases does not correspond exactly.)

Bp 6087-6257 Cecropin cap site and Prepro, Genbank accession # X07404 bp 563-733

Bp 6264-6413 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site

Bp 6414-6674 Human proinsulin GenBank Accession # NM000207 bp 117-377

Bp 6675-6719 Spacer DNA, derived as an artifact from the cloning vectors pTOPO Blunt II (Invitrogen) and gWIZ (Gene Therapy Systems)

Bp 6720-7071 Synthetic polyA from the cloning vector gWIZ (Gene Therapy Systems) bp 1920-2271

Bp 7073-10672 from cloning vector pTnMOD pTnMCS, bp 3716-7315

pTnMOD (CMV-prepro-ent-hGH-CPA)

Bp 1-4045 from vector PTnMOD, bp 1-4045

Bp 4051-5694 CMV promoter/enhancer taken from vector pGWIZ (Gene therapy systems), bp 230-1873

Bp 5701-5871 Capsite/Prepro taken from GenBank accession # X07404, bp 563-733

Bp 5878-6012 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site

Bp 6013-6666 Human growth hormone taken from GenBank accession # V00519, bp 1-654

Bp 6673-7080 Conalbumin polyA taken from GenBank accession # Y00407, bp 10651-11058

Bp 7082-10681 from cloning vector pTnMOD, bp 4091-7690

pTnMCS(CHOVep-prepro-ent-hGH-CPA)

Bp 1-3670 from vector PTnMCS, bp 1-3670

Bp 3676-4350 Chicken Ovalbumin enhancer taken from GenBank accession # S82527.1, bp 1-675

Bp 4357-5692 Chicken Ovalbumin promoter taken from GenBank accession # J00899-M24999, bp 1-1336

Bp 5699-5869 Capsite/Prepro taken from GenBank accession # X07404, bp 563-733

Bp 5876-6010 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site

Bp 6011-6664 Human growth hormone taken from GenBank accession # V00519, bp 1-654

Bp 6671-7078 Conalbumin polyA taken from GenBank accession # Y00407, bp 10651-11058

Bp 7080-10679 from cloning vector pTnMCS, bp 3716-7315

pTnMCS (CMV-prepro-ent-hGH-CPA)

Bp 1-3670 from vector PTnMCS, bp 1-3670

Bp 3676-5319 CMV promoter/enhancer taken from vector pGWIZ (Gene therapy systems), bp 230-1873

Bp 5326-5496 Capsite/Prepro taken from GenBank accession # X07404, bp 563-733

Bp 5503-5637 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site

Bp 5638-6291 Human growth hormone taken from GenBank accession # V00519, bp 1-654

Bp 6298-6705 Conalbumin polyA taken from GenBank accession # Y00407, bp 10651-11058

Bp 6707-10306 from cloning vector pTnMCS, bp 3716-7315

pTnMOD (CHOVep-prepro-ent-hGH-CPA)

Bp 1-4045 from vector PTnMOD, bp 1-4045

Bp 4051-4725 Chicken Ovalbumin enhancer taken from GenBank accession # S82527.1, bp 1-675

Bp 4732-6067 Chicken Ovalbumin promoter taken from GenBank accession # J00899-M24999, bp 1-1336

Bp 60746244 Capsite/Prepro taken from GenBank accession # X07404, bp 563-733

Bp 6251-6385 Synthetic spacer sequence and hairpin loop of HIV gp41 with an added enterokinase cleavage site

Bp 6386-7039 Human growth hormone taken from GenBank accession # V00519, bp 1-654

Bp 7046-7453 Conalbumin polyA taken from GenBank accession # Y00407, bp 10651-11058

Bp 7455-11054 from cloning vector pTnMOD, bp 4091-7690

PTnMod(CMV/Transposase/ChickOvep/prepro/ProteinA/ConpolyA

BP 1-130 remainder of F1 (−) ori of pBluescriptII sk(−) (Stragagene) bp 1-130.

BP 133-1777 CMV promoter/enhancer taken from vector pGWIZ (Gene Therapy Systems) bp 229-1873.

BP 1780-2987 Transposase, modified from Tn10 (GenBank #J01829).

BP 2988-2993 Engineered DOUBLE stop codon.

BP 2994-3343 non coding DNA from vector pNK2859.

BP 3344-3386 Lambda DNA from pNK2859.

BP 3387-3456 70 bp of IS10 left from Tn10.

BP 3457-3674 multiple cloning site from pBluescriptII sk(−) bp 924-707.

BP 3675-5691 Chicken Ovalbumin enhancer plus promoter from a Topo Clone 10 maxi 040303 (5′ XmaI, 3′ BamHI)

BP 5698-5865 prepro with Cap site amplified from cecropin of pMON200 GenBank # X07404 (5′BamHI, 3′KpnI)

BP 5872-7338 Protein A gene from GenBank# J01786, mature peptide bp 292-1755 (5′KpnI, 3′SacII)

BP 7345-7752 ConPolyA from Chicken conalbumin polyA from GenBank # Y00407 bp 10651-11058. (5′SacII, 3′XhoI)

BP 7753-8195 multiple cloning site from pBluescriptII sk(−) bp 677-235.

BP 8196-8265 70 bp of IS 10 left from Tn10.

BP 8266-8307 Lamda DNA from pNK2859

BP 8308-9151 noncoding DNA from pNK2859

BP 9152-11352 pBluescriptII sk(−) base vector (Stratagene, INC.) bp 761-2961

All patents, publications and abstracts cited above are incorporated herein by reference in their entirety. It should be understood that the foregoing relates only to preferred embodiments of the present invention and that numerous modifications or alterations may be made therein without departing from the spirit and the scope of the present invention as defined in the following claims.
APPENDIX A








SEQ ID NO: 1 (modified Kozak sequence)



ACCATG


 


SEQ ID NO: 2 (pTnMCS)


   1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga


 


  61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg


 


 121 ccacgttcgc cggcatcaga ttggctattg gccattgcat acgttgtatc catatcataa


 


 181 tatgtacatt tatattggct catgtccaac attaccgcca tgttgacatt gattattgac


 


 241 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg


 


 301 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt


 


 361 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca


 


 421 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc


 


 481 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta


 


 541 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac


 


 601 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg


 


 661 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg


 


 721 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt


 


 781 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg


 


 841 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccg


 


 901 ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta ccgcctatag


 


 961 actctatagg cacacccctt tggctcttat gcatgctata ctgtttttgg cttggggcct


 


1021 atacaccccc gcttccttat gctataggtg atggtatagc ttagcctata ggtgtgggtt


 


1081 attgaccatt attgaccact cccctattgg tgacgatact ttccattact aatccataac


 


1141 atggctcttt gccacaacta tctctattgg ctatatgcca atactctgtc cttcagagac


 


1201 tgacacggac tctgtatttt tacaggatgg ggtcccattt attatttaca aattcacata


 


1261 tacaacaacg ccgtcccccg tgcccgcagt ttttattaaa catagcgtgg gatctccacg


 


1321 cgaatctcgg gtacgtgttc cggacatggg ctcttctccg gtagcggcgg agcttccaca


 


1381 tccgagccct ggtcccatgc ctccagcggc tcatggtcgc tcggcagctc cttgctccta


 


1441 acagtggagg ccagacttag gcacagcaca atgcccacca ccaccagtgt gccgcacaag


 


1501 gccgtggcgg tagggtatgt gtctgaaaat gagcgtggag attgggctcg cacggctgac


 


1561 gcagatggaa gacttaaggc agcggcagaa gaagatgcag gcagctgagt tgttgtattc


 


1621 tgataagagt cagaggtaac tcccgttgcg gtgctgttaa cggtggaggg cagtgtagtc


 


1681 tgagcagtac tcgttgctgc cgcgcgcgcc accagacata atagctgaca gactaacaga


 


1741 ctgttccttt ccatgggtct tttctgcagt caccgtcgga ccatgtgcga actcgatatt


 


1801 ttacacgact ctctttacca attctgcccc gaattacact taaaacgact caacagctta


 


1861 acgttggctt gccacgcatt acttgactgt aaaactctca ctcttaccga acttggccgt


 


1921 aacctgccaa ccaaagcgag aacaaaacat aacatcaaac gaatcgaccg attgttaggt


 


1981 aatcgtcacc tccacaaaga gcgactcgct gtataccgtt ggcatgctag ctttatctgt


 


2041 tcgggcaata cgatgcccat tgtacttgtt gactggtctg atattcgtga gcaaaaacga


 


2101 cttatggtat tgcgagcttc agtcgcacta cacggtcgtt ctgttactct ttatgagaaa


 


2161 gcgttcccgc tttcagagca atgttcaaag aaagctcatg accaatttct agccgacctt


 


2221 gcgagcattc taccgagtaa caccacaccg ctcattgtca gtgatgctgg ctttaaagtg


 


2281 ccatggtata aatccgttga gaagctgggt tggtactggt taagtcgagt aagaggaaaa


 


2341 gtacaatatg cagacctagg agcggaaaac tggaaaccta tcagcaactt acatgatatg


 


2401 tcatctagtc actcaaagac tttaggctat aagaggctga ctaaaagcaa tccaatctca


 


2461 tgccaaattc tattgtataa atctcgctct aaaggccgaa aaaatcagcg ctcgacacgg


 


2521 actcattgtc accacccgtc acctaaaatc tactcagcgt cggcaaagga gccatgggtt


 


2581 ctagcaacta acttacctgt tgaaattcga acacccaaac aacttgttaa tatctattcg


 


2641 aagcgaatgc agattgaaga aaccttccga gacttgaaaa gtcctgccta cggactaggc


 


2701 ctacgccata gccgaacgag cagctcagag cgttttgata tcatgctgct aatcgccctg


 


2761 atgcttcaac taacatgttg gcttgcgggc gttcatgctc agaaacaagg ttgggacaag


 


2821 cacttccagg ctaacacagt cagaaatcga aacgtactct caacagttcg cttaggcatg


 


2881 gaagttttgc ggcattctgg ctacacaata acaagggaag acttactcgt ggctgcaacc


 


2941 ctactagctc aaaatttatt cacacatggt tacgctttgg ggaaattatg aggggatcgc


 


3001 tctagagcga tccgggatct cgggaaaagc gttggtgacc aaaggtgcct tttatcatca


 


3061 ctttaaaaat aaaaaacaat tactcagtgc ctgttataag cagcaattaa ttatgattga


 


3121 tgcctacatc acaacaaaaa ctgatttaac aaatggttgg tctgccttag aaagtatatt


 


3181 tgaacattat cttgattata ttattgataa taataaaaac cttatcccta tccaagaagt


 


3241 gatgcctatc attggttgga atgaacttga aaaaaattag ccttgaatac attactggta


 


3301 aggtaaacgc cattgtcagc aaattgatcc aagagaacca acttaaagct ttcctgacgg


 


3361 aatgttaatt ctcgttgacc ctgagcactg atgaatcccc taatgatttt ggtaaaaatc


 


3421 attaagttaa ggtggataca catcttgtca tatgatcccg gtaatgtgag ttagctcact


 


3481 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg


 


3541 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt


 


3601 aaccctcact aaagggaaca aaagctggag ctccaccgcg gtggcggccg ctctagaact


 


3661 agtggatccc ccgggctgca ggaattcgat atcaagctta tcgataccgc tgacctcgag


 


3721 ggggggcccg gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt


 


3781 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc


 


3841 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca


 


3901 acagttgcgc agcctgaatg gcgaatggaa attgtaagcg ttaatatttt gttaaaattc


 


3961 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc


 


4021 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag


 


4081 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc


 


4141 gatggcccac tactccggga tcatatgaca agatgtgtat ccaccttaac ttaatgattt


 


4201 ttaccaaaat cattagggga ttcatcagtg ctcagggtca acgagaatta acattccgtc


 


4261 aggaaagctt atgatgatga tgtgcttaaa aacttactca atggctggtt atgcatatcg


 


4321 caatacatgc gaaaaaccta aaagagcttg ccgataaaaa aggccaattt attgctattt


 


4381 accgcggctt tttattgagc ttgaaagata aataaaatag ataggtttta tttgaagcta


 


4441 aatcttcttt atcgtaaaaa atgccctctt gggttatcaa gagggtcatt atatttcgcg


 


4501 gaataacatc atttggtgac gaaataacta agcacttgtc tcctgtttac tcccctgagc


 


4561 ttgaggggtt aacatgaagg tcatcgatag caggataata atacagtaaa acgctaaacc


 


4621 aataatccaa atccagccat cccaaattgg tagtgaatga ttataaataa cagcaaacag


 


4681 taatgggcca ataacaccgg ttgcattggt aaggctcacc aataatccct gtaaagcacc


 


4741 ttgctgatga ctctttgttt ggatagacat cactccctgt aatgcaggta aagcgatccc


 


4801 accaccagcc aataaaatta aaacagggaa aactaaccaa ccttcagata taaacgctaa


 


4861 aaaggcaaat gcactactat ctgcaataaa tccgagcagt actgccgttt tttcgcccat


 


4921 ttagtggcta ttcttcctgc cacaaaggct tggaatactg agtgtaaaag accaagaccc


 


4981 gtaatgaaaa gccaaccatc atgctattca tcatcacgat ttctgtaata gcaccacacc


 


5041 gtgctggatt ggctatcaat gcgctgaaat aataatcaac aaatggcatc gttaaataag


 


5101 tgatgtatac cgatcagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa


 


5161 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata


 


5221 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta


 


5281 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa


 


5341 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg


 


5401 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag


 


5461 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa


 


5521 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc


 


5581 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca


 


5641 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg


 


5701 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct


 


5761 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt


 


5821 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag


 


5881 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc


 


5941 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac


 


6001 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga


 


6061 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc


 


6121 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg


 


6181 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca


 


6241 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt


 


6301 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca


 


6361 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg


 


6421 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca


 


6481 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt


 


6541 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt


 


6601 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca


 


6661 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca


 


6721 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga


 


6781 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact


 


6841 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga


 


6901 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg


 


6961 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc


 


7021 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga


 


7081 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat


 


7141 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt


 


7201 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt


 


7261 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccac


 


SEQ ID NO: 3 (pTnMod)


CTGACGCGCC CTGTAGCGGC GCATTAAGCG CGGCGGGTGT GGTGGTTACG   50


 


CGCAGCGTGA CCGCTACACT TGCCAGCGCC CTAGCGCCCG CTCCTTTCGC  100


 


TTTCTTCCCT TCCTTTCTCG CCACGTTCGC CGGCATCAGA TTGGCTATTG  150


 


GCCATTGCAT ACGTTGTATC CATATCATAA TATGTACATT TATATTGGCT  200


 


CATGTCCAAC ATTACCGCCA TGTTGACATT GATTATTGAC TAGTTATTAA  250


 


TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG  300


 


CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC  350


 


CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA  400


 


GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA  450


 


CTTGGCAGTA CATCAAGTGT ATCATATGCC AAGTACGCCC CCTATTGACG  500


 


TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA CATGACCTTA  550


 


TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC  600


 


CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG  650


 


ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG  700


 


TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC  750


 


CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG GTCTATATAA  800


 


GCAGAGCTCG TTTAGTGAAC CGTCAGATCG CCTGGAGACG CCATCCACGC  850


 


TGTTTTGACC TCCATAGAAG ACACCGGGAC CGATCCAGCC TCCGCGGCCG  900


 


GGAACGGTGC ATTGGAACGC GGATTCCCCG TGCCAAGAGT GACGTAAGTA  950


 


CCGCCTATAG ACTCTATAGG CACACCCCTT TGGCTCTTAT GCATGCTATA 1000


 


CTGTTTTTGG CTTGGGGCCT ATACACCCCC GCTTCCTTAT GCTATAGGTG 1050


 


ATGGTATAGC TTAGCCTATA GGTGTGGGTT ATTGACCATT ATTGACCACT 1100


 


CCCCTATTGG TGACGATACT TTCCATTACT AATCCATAAC ATGGCTCTTT 1150


 


GCCACAACTA TCTCTATTGG CTATATGCCA ATACTCTGTC CTTCAGAGAC 1200


 


TGACACGGAC TCTGTATTTT TACAGGATGG GGTCCCATTT ATTATTTACA 1250


 


AATTCACATA TACAACAACG CCGTCCCCCG TGCCCGCAGT TTTTATTAAA 1300


 


CATAGCGTGG GATCTCCACG CGAATCTCGG GTACGTGTTC CGGACATGGG 1350


 


CTCTTCTCCG GTAGCGGCGG AGCTTCCACA TCCGAGCCCT GGTCCCATGC 1400


 


CTCCAGCGGC TCATGGTCGC TCGGCAGCTC CTTGCTCCTA ACAGTGGAGG 1450


 


CCAGACTTAG GCACAGCACA ATGCCCACCA CCACCAGTGT GCCGCACAAG 1500


 


GCCGTGGCGG TAGGGTATGT GTCTGAAAAT GAGCGTGGAG ATTGGGCTCG 1550


 


CACGGCTGAC GCAGATGGAA GACTTAAGGC AGCGGCAGAA GAAGATGCAG 1600


 


GCAGCTGAGT TGTTGTATTC TGATAAGAGT CAGAGGTAAC TCCCGTTGCG 1650


 


GTGCTGTTAA CGGTGGAGGG CAGTGTAGTC TGAGCAGTAC TCGTTGCTGC 1700


 


CGCGCGCGCC ACCAGACATA ATAGCTGACA GACTAACAGA CTGTTCCTTT 1750


 


CCATGGGTCT TTTCTGCAGT CACCGTCGGA CCATGTGTGA ACTTGATATT 1800


 


TTACATGATT CTCTTTACCA ATTCTGCCCC GAATTACACT TAAAACGACT 1850


 


CAACAGCTTA ACGTTGGCTT GCCACGCATT ACTTGACTGT AAAACTCTCA 1900


 


CTCTTACCGA ACTTGGCCGT AACCTGCCAA CCAAAGCGAG AACAAAACAT 1950


 


AACATCAAAC GAATCGACCG ATTGTTAGGT AATCGTCACC TCCACAAAGA 2000


 


GCGACTCGCT GTATACCGTT GGCATGCTAG CTTTATCTGT TCGGGAATAC 2050


 


GATGCCCATT GTACTTGTTG ACTGGTCTGA TATTCGTGAG CAAAAACGAC 2100


 


TTATGGTATT GCGAGCTTCA GTCGCACTAC ACGGTCGTTC TGTTACTCTT 2150


 


TATGAGAAAG CGTTCCCGCT TTCAGAGCAA TGTTCAAAGA AAGCTCATGA 2200


 


CCAATTTCTA GCCGACCTTG CGAGCATTCT ACCGAGTAAC ACCACACCGC 2250


 


TCATTGTCAG TGATGCTGGC TTTAAAGTGC CATGGTATAA ATCCGTTGAG 2300


 


AAGCTGGGTT GGTACTGGTT AAGTCGAGTA AGAGGAAAAG TACAATATGC 2350


 


AGACCTAGGA GCGGAAAACT GGAAACCTAT CAGCAACTTA CATGATATGT 2400


 


CATCTAGTCA CTCAAAGACT TTAGGCTATA AGAGGCTGAC TAAAAGCAAT 2450


 


CCAATCTCAT GCCAAATTCT ATTGTATAAA TCTCGCTCTA AAGGCCGAAA 2500


 


AAATCAGCGC TCGACACGGA CTCATTGTCA CCACCCGTCA CCTAAAATCT 2550


 


ACTCAGCGTC GGCAAAGGAG CCATGGGTTC TAGCAACTAA CTTACCTGTT 2600


 


GAAATTCGAA CACCCAAACA ACTTGTTAAT ATCTATTCGA AGCGAATGCA 2650


 


GATTGAAGAA ACCTTCCGAG ACTTGAAAAG TCCTGCCTAC GGACTAGGCC 2700


 


TACGCCATAG CCGAACGAGC AGCTCAGAGC GTTTTGATAT CATGCTGCTA 2750


 


ATCGCCCTGA TGCTTCAACT AACATGTTGG CTTGCGGGCG TTCATGCTCA 2800


 


GAAACAAGGT TGGGACAAGC ACTTCCAGGC TAACACAGTC AGAAATCGAA 2850


 


ACGTACTCTC AACAGTTCGC TTAGGCATGG AAGTTTTGCG GCATTCTGGC 2900


 


TACACAATAA CAAGGGAAGA CTTACTCGTG GCTGCAACCC TACTAGCTCA 2950


 


AAATTTATTC ACACATGGTT ACGCTTTGGG GAAATTATGA TAATGATCCA 3000


 


GATCACTTCT GGCTAATAAA AGATCAGAGC TCTAGAGATC TGTGTGTTGG 3050


 


TTTTTTGTGG ATCTGCTGTG CCTTCTAGTT GCCAGCCATC TGTTGTTTGC 3100


 


CCCTCCCCCG TGCCTTCCTT GACCCTGGAA GGTGCCACTC CCACTGTCCT 3150


 


TTCCTAATAA AATGAGGAAA TTGCATCGCA TTGTCTGAGT AGGTGTCATT 3200


 


CTATTCTGGG GGGTGGGGTG GGGCAGCACA GCAAGGGGGA GGATTGGGAA 3250


 


GACAATAGCA GGCATGCTGG GGATGCGGTG GGCTCTATGG GTACCTCTCT 3300


 


CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTCGGTAC CTCTCTCTCT 3350


 


CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CGGTACCAGG TGCTGAAGAA 3400


 


TTGACCCGGT GACCAAAGGT GCCTTTTATC ATCACTTTAA AAATAAAAAA 3450


 


CAATTACTCA GTGCCTGTTA TAAGCAGCAA TTAATTATGA TTGATGCCTA 3500


 


CATCACAACA AAAACTGATT TAACAAATGG TTGGTCTGCC TTAGAAAGTA 3550


 


TATTTGAACA TTATCTTGAT TATATTATTG ATAATAATAA AAACCTTATC 3600


 


CCTATCCAAG AAGTGATGCC TATCATTGGT TGGAATGAAC TTGAAAAAAA 3650


 


TTAGCCTTGA ATACATTACT GGTAAGGTAA ACGCCATTGT CAGCAAATTG 3700


 


ATCCAAGAGA ACCAACTTAA AGCTTTCCTG ACGGAATGTT AATTCTCGTT 3750


 


GACCCTGAGC ACTGATGAAT CCCCTAATGA TTTTGGTAAA AATCATTAAG 3800


 


TTAAGGTGGA TACACATCTT GTCATATGAT CCCGGTAATG TGAGTTAGCT 3850


 


CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT 3900


 


TGTGTGGAAT TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC 3950


 


CATGATTACG CCAAGCGCGC AATTAACCCT CACTAAAGGG AACAAAAGCT 4000


 


GGAGCTCCAC CGCGGTGGCG GCCGCTCTAG AACTAGTGGA TCCCCCGGGC 4050


 


TGCAGGAATT CGATATCAAG CTTATCGATA CCGCTGACCT CGAGGGGGGG 4100


 


CCCGGTACCC AATTCCCCCT ATAGTGAGTC GTATTACGCG CGCTCACTGG 4150


 


CCGTCGTTTT ACAACGTCGT GACTGGGAAA ACCCTGGCGT TACCCAACTT 4200


 


AATCGCCTTG CAGCACATCC CCCTTTCGCC AGCTGGCGTA ATAGCGAAGA 4250


 


GGCCCGCACC GATCGCCCTT CCCAACAGTT GCGCAGCCTG AATGGCGAAT 4300


 


GGAAATTGTA AGCGTTAATA TTTTGTTAAA ATTCGCGTTA AATTTTTGTT 4350


 


AAATCAGCTC ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT 4400


 


AAATCAAAAG AATAGACCGA GATAGGGTTG AGTGTTGTTC CAGTTTGGAA 4450


 


CAAGAGTCCA CTATTAAAGA ACGTGGACTC CAACGTCAAA GGGCGAAAAA 4500


 


CCGTCTATCA GGGCGATGGC CCACTACTCC GGGATCATAT GACAAGATGT 4550


 


GTATCCACCT TAACTTAATG ATTTTTACCA AAATCATTAG GGGATTCATC 4600


 


AGTGCTCAGG GTCAACGAGA ATTAACATTC CGTCAGGAAA GCTTATGATG 4650


 


ATGATGTGCT TAAAAACTTA CTCAATGGCT GGTTATGCAT ATCGCAATAC 4700


 


ATGCGAAAAA CCTAAAAGAG CTTGCCGATA AAAAAGGCCA ATTTATTGCT 4750


 


ATTTACCGCG GCTTTTTATT GAGCTTGAAA GATAAATAAA ATAGATAGGT 4800


 


TTTATTTGAA GCTAAATCTT CTTTATCGTA AAAAATGCCC TCTTGGGTTA 4850


 


TCAAGAGGGT CATTATATTT CGCGGAATAA CATCATTTGG TGACGAAATA 4900


 


ACTAAGCACT TGTCTCCTGT TTACTCCCCT GAGCTTGAGG GGTTAACATG 4950


 


AAGGTCATCG ATAGCAGGAT AATAATACAG TAAAACGCTA AACCAATAAT 5000


 


CCAAATCCAG CCATCCCAAA TTGGTAGTGA ATGATTATAA ATAACAGCAA 5050


 


ACAGTAATGG GCCAATAACA CCGGTTGCAT TGGTAAGGCT CACCAATAAT 5100


 


CCCTGTAAAG CACCTTGCTG ATGACTCTTT GTTTGGATAG ACATCACTCC 5150


 


CTGTAATGCA GGTAAAGCGA TCCCACCACC AGCCAATAAA ATTAAAACAG 5200


 


GGAAAACTAA CCAACCTTCA GATATAAACG CTAAAAAGGC AAATGCACTA 5250


 


CTATCTGCAA TAAATCCGAG CAGTACTGCC GTTTTTTCGC CCATTTAGTG 5300


 


GCTATTCTTC CTGCCACAAA GGCTTGGAAT ACTGAGTGTA AAAGACCAAG 5350


 


ACCCGTAATG AAAAGCCAAC CATCATGCTA TTCATCATCA CGATTTCTGT 5400


 


AATAGCACCA CACCGTGCTG GATTGGCTAT CAATGCGCTG AAATAATAAT 5450


 


CAACAAATGG CATCGTTAAA TAAGTGATGT ATACCGATCA GCTTTTGTTC 5500


 


CCTTTAGTGA GGGTTAATTG CGCGCTTGGC GTAATCATGG TCATAGCTGT 5550


 


TTCCTGTGTG AAATTGTTAT CCGCTCACAA TTCCACACAA CATACGAGCC 5600


 


GGAAGCATAA AGTGTAAAGC CTGGGGTGCC TAATGAGTGA GCTAACTCAC 5650


 


ATTAATTGCG TTGCGCTCAC TGCCCGCTTT CCAGTCGGGA AACCTGTCGT 5700


 


GCCAGCTGCA TTAATGAATC GGCCAACGCG CGGGGAGAGG CGGTTTGCGT 5750


 


ATTGGGCGCT CTTCCGCTTC CTCGCTCACT GACTCGCTGC GCTCGGTCGT 5800


 


TCGGCTGCGG CGAGCGGTAT CAGCTCACTC AAAGGCGGTA ATACGGTTAT 5850


 


CCACAGAATC AGGGGATAAC GCAGGAAAGA ACATGTGAGC AAAAGGCCAG 5900


 


CAAAAGGCCA GGAACCGTAA AAAGGCCGCG TTGCTGGCGT TTTTCCATAG 5950


 


GCTCCGCCCC CCTGACGAGC ATCACAAAAA TCGACGCTCA AGTCAGAGGT 6000


 


GGCGAAACCC GACAGGACTA TAAAGATACC AGGCGTTTCC CCCTGGAAGC 6050


 


TCCCTCGTGC GCTCTCCTGT TCCGACCCTG CCGCTTACCG GATACCTGTC 6100


 


CGCCTTTCTC CCTTCGGGAA GCGTGGCGCT TTCTCATAGC TCACGCTGTA 6150


 


GGTATCTCAG TTCGGTGTAG GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC 6200


 


GAACCCCCCG TTCAGCCCGA CCGCTGCGCC TTATCCGGTA ACTATCGTCT 6250


 


TGAGTCCAAC CCGGTAAGAC ACGACTTATC GCCACTGGCA GCAGCCACTG 6300


 


GTAACAGGAT TAGCAGAGCG AGGTATGTAG GCGGTGCTAC AGAGTTCTTG 6350


 


AAGTGGTGGC CTAACTACGG CTACACTAGA AGGACAGTAT TTGGTATCTG 6400


 


CGCTCTGCTG AAGCCAGTTA CCTTCGGAAA AAGAGTTGGT AGCTCTTGAT 6450


 


CCGGCAAACA AACCACCGCT GGTAGCGGTG GTTTTTTTGT TTGCAAGCAG 6500


 


CAGATTACGC GCAGAAAAAA AGGATCTCAA GAAGATCCTT TGATCTTTTC 6550


 


TACGGGGTCT GACGCTCAGT GGAACGAAAA CTCACGTTAA GGGATTTTGG 6600


 


TCATGAGATT ATCAAAAAGG ATCTTCACCT AGATCCTTTT AAATTAAAAA 6650


 


TGAAGTTTTA AATCAATCTA AAGTATATAT GAGTAAACTT GGTCTGACAG 6700


 


TTACCAATGC TTAATCAGTG AGGCACCTAT CTCAGCGATC TGTCTATTTC 6750


 


GTTCATCCAT AGTTGCCTGA CTCCCCGTCG TGTAGATAAC TACGATACGG 6800


 


GAGGGCTTAC CATCTGGCCC CAGTGCTGCA ATGATACCGC GAGACCCACG 6850


 


CTCACCGGCT CCAGATTTAT CAGCAATAAA CCAGCCAGCC GGAAGGGCCG 6900


 


AGCGCAGAAG TGGTCCTGCA ACTTTATCCG CCTCCATCCA GTCTATTAAT 6950


 


TGTTGCCGGG AAGCTAGAGT AAGTAGTTCG CCAGTTAATA GTTTGCGCAA 7000


 


CGTTGTTGCC ATTGCTACAG GCATCGTGGT GTCACGCTCG TCGTTTGGTA 7050


 


TGGCTTCATT CAGCTCCGGT TCCCAACGAT CAAGGCGAGT TACATGATCC 7100


 


CCCATGTTGT GCAAAAAAGC GGTTAGCTCC TTCGGTCCTC CGATCGTTGT 7150


 


CAGAAGTAAG TTGGCCGCAG TGTTATCACT CATGGTTATG GCAGCACTGC 7200


 


ATAATTCTCT TACTGTCATG CCATCCGTAA GATGCTTTTC TGTGACTGGT 7250


 


GAGTACTCAA CCAAGTCATT CTGAGAATAG TGTATGCGGC GACCGAGTTG 7300


 


CTCTTGCCCG GCGTCAATAC GGGATAATAC CGCGCCACAT AGCAGAACTT 7350


 


TAAAAGTGCT CATCATTGGA AAACGTTCTT CGGGGCGAAA ACTCTCAAGG 7400


 


ATCTTACCGC TGTTGAGATC CAGTTCGATG TAACCCACTC GTGCACCCAA 7450


 


CTGATCTTCA GCATCTTTTA CTTTCACCAG CGTTTCTGGG TGAGCAAAAA 7500


 


CAGGAAGGCA AAATGCCGCA AAAAAGGGAA TAAGGGCGAC ACGGAAATGT 7550


 


TGAATACTCA TACTCTTCCT TTTTCAATAT TATTGAAGCA TTTATCAGGG 7600


 


TTATTGTCTC ATGAGCGGAT ACATATTTGA ATGTATTTAG AAAAATAAAC 7650


 


AAATAGGGGT TCCGCGCACA TTTCCCCGAA AAGTGCCAC             7689


 


SEQ ID NO: 4 (a Kozak sequence)


ACCATGG


 


SEQ ID NO: 52 (a Kozak sequence)


ACCATGT


 


SEQ ID NO: 5 (a Kozak sequence)


AAGATGT


 


SEQ ID NO: 6 (a Kozak sequence)


ACGATGA


 


SEQ ID NO: 7 (a Kozak sequence)


AAGATGG


 


SEQ ID NO: 8 (a Kozak sequence)


GACATGA


 


SEQ ID NO: 9 (a Kozak sequence)


ACCATGA


 


SEQ ID NO: 11 (conalbumin polyA)


tctgccattg ctgcttcctc tgcccttcct cgtcactctg aatgtggctt cttcgctact


 


gccacagcaa gaaataaaat ctcaacatct aaatgggttt cctgaggttt ttcaagagtc


 


gttaagcaca ttccttcccc agcacccctt gctgcaggcc agtgccaggc accaacttgg


 


ctactgctgc ccatgagaga aatccagttc aatattttcc aaagcaaaat ggattacata


 


tgccctagat cctgattaac aggcgtttgt attatctagt gctttcgctt cacccagatt


 


atcccattgc ctccc


 


SEQ ID NO: 12 (synthetic polyA)


GGCGCCTGGATCCAGATCACTTCTGGCTAATAAAAGATCAGAGCTCTAGAGATCTGTGTGTTGGTTTTT


 


TGTGGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACC


 


CTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGG


 


TGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGCACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGG


 


CATGCTGGGGATGCGGTGGGCTCTATGGGTACCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTC


 


TCTCGGTACCTCTCTC


 


SEQ ID NO: 13 (avian optimized polyA)


ggggatcgc tctagagcga tccgggatct cgggaaaagc gttggtgacc aaaggtgcct


 


tttatcatca ctttaaaaat aaaaaacaat tactcagtgc ctgttataag cagcaattaa


 


ttatgattga tgcctacatc acaacaaaaa ctgatttaac aaatggttgg tctgccttag


 


aaagtatatt tgaacattat cttgattata ttattgataa taataaaaac cttatcccta


 


tccaagaagt gatgcctatc attggttgga atgaacttga aaaaaattag ccttgaatac


 


attactggta aggtaaacgc cattgtcagc aaattgatcc aagagaacca a


 


SEQ ID NO: 14 (vitellogenin promoter)


TGAATGTGTT CTTGTGTTAT CAATATAAAT CACAGTTAGT GATGAAGTTG GCTGCAAGCC


 


TGCATCAGTT CAGCTACTTG GCTGCATTTT GTATTTGGTT CTGTAGGAAA TGCAAAAGGT


 


TCTAGGCTGA CCTGCACTTC TATCCCTCTT GCCTTACTGC TGAGAATCTC TGCAGGTTTT


 


AATTGTTCAC ATTTTGCTCC CATTTACTTT GGAAGATAAA ATATTTACAG AATGCTTATG


 


AAACCTTTGT TCATTTAAAA ATATTCCTGG TCAGCGTGAC CGGAGCTGAA AGAACACATT


 


GATCCCGTGA TTTCAATAAA TACATATGTT CCATATATTG TTTCTCAGTA GCCTCTTAAA


 


TCATGTGCGT TGGTGCACAT ATGAATACAT GAATAGCAAA GGTTTATCTG GATTACGCTC


 


TGGCCTGCAG GAATGGCCAT AAACCAAAGC TGAGGGAAGA GGGAGAGTAT AGTCAATGTA


 


GATTATACTG ATTGCTGATT GGGTTATTAT CAGCTAGATA ACAACTTGGG TCAGGTGCCA


 


GGTCAACATA ACCTGGGCAA AACCAGTCTC ATCTGTGGCA GGACCATGTA CCAGCAGCCA


 


GCCGTGACCC AATCTAGGAA AGCAAGTAGC ACATCAATTT TAAATTTATT GTAAATGCCG


 


TAGTAGAAGT GTTTTACTGT GATACATTGA AACTTCTGGT CAATCAGAAA AAGGTTTTTT


 


ATCAGAGATG CCAAGGTATT ATTTGATTTT CTTTATTCGC CGTGAAGAGA ATTTATGATT


 


GCAAAAAGAG GAGTGTTTAC ATAAACTGAT AAAAAACTTG AGGAATTCAG CAGAAAACAG


 


CCACGTGTTC CTGAACATTC TTCCATAAAA GTCTCACCAT GCCTGGCAGA GCCCTATTCA


 


CCTTCGCT


 


SEQ ID NO: 15 (fragment of ovalbumin promoter - chicken)


GAGGTCAGAAT GGTTTCTTTA CTGTTTGTCA ATTCTATTAT TTCAATACAG


 


AACAATAGCT TCTATAACTG AAATATATTT GCTATTGTAT ATTATGATTG


 


TCCCTCGAAC CATGAACACT CCTCCAGCTG AATTTCACAA TTCCTCTGTC


 


ATCTGCCAGG CCATTAAGTT ATTCATGGAA GATCTTTGAG GAACACTGCA


 


AGTTCATATC ATAAACACAT TTGAAATTGA GTATTGTTTT GCATTGTATG


 


GAGCTATGTT TTGCTGTATC CTCAGAAAAA AAGTTTGTTA TAAAGCATTC


 


ACACCCATAA AAAGATAGAT TTAAATATTC CAGCTATAGG AAAGAAAGTG


 


CGTCTGCTCT TCACTCTAGT CTCAGTTGGC TCCTTCACAT GCATGCTTCT


 


TTATTTCTCC TATTTTGTCA AGAAAATAAT AGGTCACGTC TTGTTCTCAC


 


TTATGTCCTG CCTAGCATGG CTCAGATGCA CGTTGTAGAT ACAAGAAGGA


 


TCAAATGAAA CAGACTTCTG GTCTGTTACT ACAACCATAG TAATAAGCAC


 


ACTAACTAAT AATTGCTAAT TATGTTTTCC ATCTCTAAGG TTCCCACATT


 


TTTCTGTTTT CTTAAAGATC CCATTATCTG GTTGTAACTG AAGCTCAATG


 


GAACATGAGC AATATTTCCC AGTCTTCTCT CCCATCCAAC AGTCCTGATG


 


GATTAGCAGA ACAGGCAGAA AACACATTGT TACCCAGAAT TAAAAACTAA


 


TATTTGCTCT CCATTCAATC CAAAATGGAC CTATTGAAAC TAAAATCTAA


 


CCCAATCCCA TTAAATGATT TCTATGGCGT CAAAGGTCAA ACTTCTGAAG


 


GGAACCTGTG GGTGGGTCAC AATTCAGGCT ATATATTCCC CAGGGCTCAG


 


SEQ ID NO: 16 (chicken ovalbumin ehancer)


ccgggctgca gaaaaatgcc aggtggacta tgaactcaca tccaaaggag


 


cttgacctga tacctgattt tcttcaaact ggggaaacaa cacaatccca caaaacagct


 


cagagagaaa ccatcactga tggctacagc accaaggtat gcaatggcaa tccattcgac


 


attcatctgt gacctgagca aaatgattta tctctccatg aatggttgct tctttccctc


 


atgaaaaggc aatttccaca ctcacaatat gcaacaaaga caaacagaga acaattaatg


 


tgctccttcc taatgtcaaa attgtagtgg caaagaggag aacaaaatct caagttctga


 


gtaggtttta gtgattggat aagaggcttt gacctgtgag ctcacctgga cttcatatcc


 


ttttggataa aaagtgcttt tataactttc aggtctccga gtctttattc atgagactgt


 


tggtttaggg acagacccac aatgaaatgc ctggcatagg aaagggcagc agagccttag


 


ctgacctttt cttgggacaa gcattgtcaa acaatgtgtg acaaaactat ttgtactgct


 


ttgcacagct gtgctgggca gggcaatcca ttgccaccta tcccaggtaa ccttccaact


 


gcaagaagat tgttgcttac tctctctaga


 


SEQ ID NO: 17 (5′ untranslated region)


GTGGATCAACATACAGCTAGAAAGCTGTATTGCCTTTAGCACTCAAGCTCAAAAGACAACTCAGAGTTC


 


ACC


 


SEQ ID NO: 18 (putative cap site)


ACATACAGCTAG AAAGCTGTAT TGCCTTTAGC ACTCAAGCTC AAAAGACAAC TCAGAGTTCA


 


SEQ ID NO: 19 (Chicken Ovalbumin Signal Sequence)


ATG GGCTCCATCG GCGCAGCAAG CATGGAATTT TGTTTTGATG TATTCAAGGA GCTCAAAGTC


 


CACCATGCCA ATGAGAACAT CTTCTACTGC CCCATTGCCA TCATGTCAGC TCTAGCCATG


 


GTATACCTGG GTGCAAAAGA CAGCACCAGG ACACAGATAA ATAAGGTTGT TCGCTTTGAT


 


AAACTTCCAG GATTCGGAGA CAGTATTGAA GCTCAGTGTG GCACATCTGT AAACGTTCAC


 


TCTTCACTTA GAGACATCCT CAACCAAATC ACCAAACCAA ATGATGTTTA TTCGTTCAGC


 


CTTGCCAGTA GACTTTATGC TGAAGAGAGA TACCCAATCC TGCCAGAATA CTTGCAGTGT


 


GTGAAGGAAC TGTATAGAGG AGGCTTGGAA CCTATCAACT TTCAAACAGC TGCAGATCAA


 


GCCAGAGAGC TCATCAATTC CTGGGTAGAA AGTCAGACAA ATGGAATTAT CAGAAATGTC


 


CTTCAGCCAA GCTCCGTGGA TTCTCAAACT GCAATGGTTC TGGTTAATGC CATTGTCTTC


 


AAAGGACTGT GGGAGAAAAC ATTTAAGGAT GAAGACACAC AAGCAATGCC TTTCAGAGTG


 


ACTGAGCAAG AAAGCAAACC TGTGCAGATG ATGTACCAGA TTGGTTTATT TAGAGTGGCA


 


TCAATGGCTT CTGAGAAAAT GAAGATCCTG GAGCTTCCAT TTGCCAGTGG GACAATGAGC


 


ATGTTGGTGC TGTTGCCTGA TGAAGTCTCA GGCCTTGAGC AGCTTGAGAG TATAATCAAC


 


TTTGAAAAAC TGACTGAATG GACCAGTTCT AATGTTATGG AAGAGAGGAA GATCAAAGTG


 


TACTTACCTC GCATGAAGAT GGAGGAAAAA TACAACCTCA CATCTGTCTT AATGGCTATG


 


GGCATTACTG ACGTGTTTAG CTCTTCAGCC AATCTGTCTG GCATCTCCTC AGCAGAGAGC


 


CTGAAGATAT CTCAAGCTGT CCATGCAGCA CATGCAGAAA TCAATGAAGC AGGCAGAGAG


 


GTGGTAGGGT CAGCAGAGGC TGGAGTGGAT GCTGCAAGCG TCTCTGAAGA ATTTAGGGCT


 


GACCATCCAT TCCTCTTCTG TATCAAGCAC ATCGCAACCA ACGCCGTTCT CTTCTTTGGC


 


AGATGTGTTT CCCCT


 


SEQ ID NO: 20 (Chicken Ovalbumin Signal Sequence - shortened 50 bp)


ATG GGCTCCATCG GCGCAGCAAG CATGGAATTT TGTTTTGATG TATTCAAGGA


 


SEQ ID NO: 21 (Chicken Ovalbumin Signal Sequence - shortened 100 bp)


ATG GGCTCCATCG GCGCAGCAAG CATGGAATTT TGTTTTGATG TATTCAAGGA GCTCAAAGTC


 


CACCATGCCA ATGAGAACAT CTTCTACTGC CCCATTGCCA


 


SEQ ID NO: 22 (vitellogenin targeting sequence)


ATGAGGGGGATCATACTGGCATTAGTGCTCACCCTTGTAGGCAGCCAGAAGTTTGACATTGGT


 


SEQ ID NO: 23 (pro-insulin sequence)


TTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTACCTAGTGTGCGGGGAACGAGGC


 


TTCTTCTACACACCCAAGACCCGCCGGGAGGCAGAGGACCTGCAGGTGGGGCAGGTGGAGCTGGGCGGG


 


GGCCCTGGTGCAGGCAGCCTGCAGCCCTTGGCCCTGGAGGGGTCCCTGCAGAAGCGTGGCATTGTGGAA


 


CAATGCTGTACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTCTGCAACTAG


 


SEQ ID NO: 24 (p146 protein)


KYKKALKKLAKLL


 


SEQ ID NO: 25 (p146 coding sequence)


AAATACAAAAAAGCACTGAAAAAACTGGCAAAACTGCTG


 


SEQ ID NO: 26 (spacer)


(GPGG)x


 


SEQ ID NO: 27 (spacer)


GPGGGPGGGPGG


 


SEQ ID NO: 28 (spacer)


GGGGSGGGGSGGGGS


 


SEQ ID NO: 29 (spacer)


GGGGSGGGGSGGGGSGGGGS


 


SEQ ID NO: 30 (repeat domain in TAG spacer sequence)


Pro Ala Asp Asp Ala


 


SEQ ID NO: 31 (TAG spacer sequence)


Pro Ala Asp Asp Ala Pro Ala Asp Asp Ala Pro Ala Asp Asp Ala Pro Ala Asp Asp


 


Ala Pro Ala Asp Asp Ala Pro Ala Asp Asp


 


SEQ ID NO: 32 (gp41 epitope)


Ala Thr Thr Cys Ile Leu Lys Gly Ser Cys Gly Trp Ile Gly Leu Leu


 


SEQ ID NO: 33 (polynucleotide sequence encoding gp41 epitope)


Pro Ala Asp Asp Ala Pro Ala Asp Asp Ala Thr Thr Cys Ile Leu Lys Gly


 


Ser Cys Gly Trp Ile Gly Leu Leu Asp Asp Asp Asp Lys


 


SEQ ID NO: 34 (enterokinase cleavage site)


DDDDK


 


SEQ ID NO: 35 (TAG sequence)


Pro Ala Asp Asp Ala Pro Ala Asp Asp Ala Pro Ala Asp Asp Ala Pro Ala Asp Asp


 


Ala Pro Ala Asp Asp Ala Pro Ala Asp Asp Ala Thr Thr Cys Ile Leu Lys Gly Ser


 


Cys Gly Trp Ile Gly Leu Leu Asp Asp Asp Asp Lys


 


SEQ ID NO: 36 (altered transposase Hef forward primer)


ATCTCGAGACCATGTGTGAACTTGATATTTTACATGATTCTCTTTACC


 


SEQ ID NO: 37 (altered transposase Her reverse primer)


GATTGATCATTATCATAATTTCCCCAAAGCGTAACC


 


SEQ ID NO: 38 (Xho I restriction site)


CTCGAG


 


SEQ ID NO: 39 (Bcl I restriction site)


TGATCA


 


SEQ ID NO: 40 (CMVf-NgoM IV primer)


TTGCCGGCATCAGATTGGCTAT


 


SEQ ID NO: 41 (Syn-polyAr-BstE II primer)


AGAGGTCACCGGGTCAATTCTTCAGCACCTGGTA


 


SEQ ID NO: 42 (pTnMod(Oval/ENT tag/Proins/PA) - Chicken)


CTGACGCGCC CTGTAGCGGC GCATTAAGCG CGGCGGGTGT GGTGGTTACG    50


 


CGCAGCGTGA CCGCTACACT TGCCAGCGCC CTAGCGCCCG CTCCTTTCGC   100


 


TTTCTTCCCT TCCTTTCTCG CCACGTTCGC CGGCATCAGA TTGGCTATTG   150


 


GCCATTGCAT ACGTTGTATC CATATCATAA TATGTACATT TATATTGGCT   200


 


CATGTCCAAC ATTACCGCCA TGTTGACATT GATTATTGAC TAGTTATTAA   250


 


TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG   300


 


CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC   350


 


CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA   400


 


GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA   450


 


CTTGGCAGTA CATCAAGTGT ATCATATGCC AAGTACGCCC CCTATTGACG   500


 


TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA CATGACCTTA   550


 


TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC   600


 


CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG   650


 


ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG   700


 


TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC   750


 


CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG GTCTATATAA   800


 


GCAGAGCTCG TTTAGTGAAC CGTCAGATCG CCTGGAGACG CCATCCACGC   850


 


TGTTTTGACC TCCATAGAAG ACACCGGGAC CGATCCAGCC TCCGCGGCCG   900


 


GGAACGGTGC ATTGGAACGC GGATTCCCCG TGCCAAGAGT GACGTAAGTA   950


 


CCGCCTATAG ACTCTATAGG CACACCCCTT TGGCTCTTAT GCATGCTATA  1000


 


CTGTTTTTGG CTTGGGGCCT ATACACCCCC GCTTCCTTAT GCTATAGGTG  1050


 


ATGGTATAGC TTAGCCTATA GGTGTGGGTT ATTGACCATT ATTGACCACT  1100


 


CCCCTATTGG TGACGATACT TTCCATTACT AATCCATAAC ATGGCTCTTT  1150


 


GCCACAACTA TCTCTATTGG CTATATGCCA ATACTCTGTC CTTCAGAGAC  1200


 


TGACACGGAC TCTGTATTTT TACAGGATGG GGTCCCATTT ATTATTTACA  1250


 


AATTCACATA TACAACAACG CCGTCCCCCG TGCCCGCAGT TTTTATTAAA  1300


 


CATAGCGTGG GATCTCCACG CGAATCTCGG GTACGTGTTC CGGACATGGG  1350


 


CTCTTCTCCG GTAGCGGCGG AGCTTCCACA TCCGAGCCCT GGTCCCATGC  1400


 


CTCCAGCGGC TCATGGTCGC TCGGCAGCTC CTTGCTCCTA ACAGTGGAGG  1450


 


CCAGACTTAG GCACAGCACA ATGCCCACCA CCACCAGTGT GCCGCACAAG  1500


 


GCCGTGGCGG TAGGGTATGT GTCTGAAAAT GAGCGTGGAG ATTGGGCTCG  1550


 


CACGGCTGAC GCAGATGGAA GACTTAAGGC AGCGGCAGAA GAAGATGCAG  1600


 


GCAGCTGAGT TGTTGTATTC TGATAAGAGT CAGAGGTAAC TCCCGTTGCG  1650


 


GTGCTGTTAA CGGTGGAGGG CAGTGTAGTC TGAGCAGTAC TCGTTGCTGC  1700


 


CGCGCGCGCC ACCAGACATA ATAGCTGACA GACTAACAGA CTGTTCCTTT  1750


 


CCATGGGTCT TTTCTGCAGT CACCGTCGGA CCATGTGTGA ACTTGATATT  1800


 


TTACATGATT CTCTTTACCA ATTCTGCCCC GAATTACACT TAAAACGACT  1850


 


CAACAGCTTA ACGTTGGCTT GCCACGCATT ACTTGACTGT AAAACTCTCA  1900


 


CTCTTACCGA ACTTGGCCGT AACCTGCCAA CCAAAGCGAG AACAAAACAT  1950


 


AACATCAAAC GAATCGACCG ATTGTTAGGT AATCGTCACC TCCACAAAGA  2000


 


GCGACTCGCT GTATACCGTT GGCATGCTAG CTTTATCTGT TCGGGAATAC  2050


 


GATGCCCATT GTACTTGTTG ACTGGTCTGA TATTCGTGAG CAAAAACGAC  2100


 


TTATGGTATT GCGAGCTTCA GTCGCACTAC ACGGTCGTTC TGTTACTCTT  2150


 


TATGAGAAAG CGTTCCCGCT TTCAGAGCAA TGTTCAAAGA AAGCTCATGA  2200


 


CCAATTTCTA GCCGACCTTG CGAGCATTCT ACCGAGTAAC ACCACACCGC  2250


 


TCATTGTCAG TGATGCTGGC TTTAAAGTGC CATGGTATAA ATCCGTTGAG  2300


 


AAGCTGGGTT GGTACTGGTT AAGTCGAGTA AGAGGAAAAG TACAATATGC  2350


 


AGACCTAGGA GCGGAAAACT GGAAACCTAT CAGCAACTTA CATGATATGT  2400


 


CATCTAGTCA CTCAAAGACT TTAGGCTATA AGAGGCTGAC TAAAAGCAAT  2450


 


CCAATCTCAT GCCAAATTCT ATTGTATAAA TCTCGCTCTA AAGGCCGAAA  2500


 


AAATCAGCGC TCGACACGGA CTCATTGTCA CCACCCGTCA CCTAAAATCT  2550


 


ACTCAGCGTC GGCAAAGGAG CCATGGGTTC TAGCAACTAA CTTACCTGTT  2600


 


GAAATTCGAA CACCCAAACA ACTTGTTAAT ATCTATTCGA AGCGAATGCA  2650


 


GATTGAAGAA ACCTTCCGAG ACTTGAAAAG TCCTGCCTAC GGACTAGGCC  2700


 


TACGCCATAG CCGAACGAGC AGCTCAGAGC GTTTTGATAT CATGCTGCTA  2750


 


ATCGCCCTGA TGCTTCAACT AACATGTTGG CTTGCGGGCG TTCATGCTCA  2800


 


GAAACAAGGT TGGGACAAGC ACTTCCAGGC TAACACAGTC AGAAATCGAA  2850


 


ACGTACTCTC AACAGTTCGC TTAGGCATGG AAGTTTTGCG GCATTCTGGC  2900


 


TACACAATAA CAAGGGAAGA CTTACTCGTG GCTGCAACCC TACTAGCTCA  2950


 


AAATTTATTC ACACATGGTT ACGCTTTGGG GAAATTATGA TAATGATCCA  3000


 


GATCACTTCT GGCTAATAAA AGATCAGAGC TCTAGAGATC TGTGTGTTGG  3050


 


TTTTTTGTGG ATCTGCTGTG CCTTCTAGTT GCCAGCCATC TGTTGTTTGC  3100


 


CCCTCCCCCG TGCCTTCCTT GACCCTGGAA GGTGCCACTC CCACTGTCCT  3150


 


TTCCTAATAA AATGAGGAAA TTGCATCGCA TTGTCTGAGT AGGTGTCATT  3200


 


CTATTCTGGG GGGTGGGGTG GGGCAGCACA GCAAGGGGGA GGATTGGGAA  3250


 


GACAATAGCA GGCATGCTGG GGATGCGGTG GGCTCTATGG GTACCTCTCT  3300


 


CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTCGGTAC CTCTCTCTCT  3350


 


CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CGGTACCAGG TGCTGAAGAA  3400


 


TTGACCCGGT GACCAAAGGT GCCTTTTATC ATCACTTTAA AAATAAAAAA  3450


 


CAATTACTCA GTGCCTGTTA TAAGCAGCAA TTAATTATGA TTGATGCCTA  3500


 


CATCACAACA AAAACTGATT TAACAAATGG TTGGTCTGCC TTAGAAAGTA  3550


 


TATTTGAACA TTATCTTGAT TATATTATTG ATAATAATAA AAACCTTATC  3600


 


CCTATCCAAG AAGTGATGCC TATCATTGGT TGGAATGAAC TTGAAAAAAA  3650


 


TTAGCCTTGA ATACATTACT GGTAAGGTAA ACGCCATTGT CAGCAAATTG  3700


 


ATCCAAGAGA ACCAACTTAA AGCTTTCCTG ACGGAATGTT AATTCTCGTT  3750


 


GACCCTGAGC ACTGATGAAT CCCCTAATGA TTTTGGTAAA AATCATTAAG  3800


 


TTAAGGTGGA TACACATCTT GTCATATGAT CCCGGTAATG TGAGTTAGCT  3850


 


CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT  3900


 


TGTGTGGAAT TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC  3950


 


CATGATTACG CCAAGCGCGC AATTAACCCT CACTAAAGGG AACAAAAGCT  4000


 


GGAGCTCCAC CGCGGTGGCG GCCGCTCTAG AACTAGTGGA TCCCCCGGGG  4050


 


AGGTCAGAAT GGTTTCTTTA CTGTTTGTCA ATTCTATTAT TTCAATACAG  4100


 


AACAATAGCT TCTATAACTG AAATATATTT GCTATTGTAT ATTATGATTG  4150


 


TCCCTCGAAC CATGAACACT CCTCCAGCTG AATTTCACAA TTCCTCTGTC  4200


 


ATCTGCCAGG CCATTAAGTT ATTCATGGAA GATCTTTGAG GAACACTGCA  4250


 


AGTTCATATC ATAAACACAT TTGAAATTGA GTATTGTTTT GCATTGTATG  4300


 


GAGCTATGTT TTGCTGTATC CTCAGAAAAA AAGTTTGTTA TAAAGCATTC  4350


 


ACACCCATAA AAAGATAGAT TTAAATATTC CAGCTATAGG AAAGAAAGTG  4400


 


CGTCTGCTCT TCACTCTAGT CTCAGTTGGC TCCTTCACAT GCATGCTTCT  4450


 


TTATTTCTCC TATTTTGTCA AGAAAATAAT AGGTCACGTC TTGTTCTCAC  4500


 


TTATGTCCTG CCTAGCATGG CTCAGATGCA CGTTGTAGAT ACAAGAAGGA  4550


 


TCAAATGAAA CAGACTTCTG GTCTGTTACT ACAACCATAG TAATAAGCAC  4600


 


ACTAACTAAT AATTGCTAAT TATGTTTTCC ATCTCTAAGG TTCCCACATT  4650


 


TTTCTGTTTT CTTAAAGATC CCATTATCTG GTTGTAACTG AAGCTCAATG  4700


 


GAACATGAGC AATATTTCCC AGTCTTCTCT CCCATCCAAC AGTCCTGATG  4750


 


GATTAGCAGA ACAGGCAGAA AACACATTGT TACCCAGAAT TAAAAACTAA  4800


 


TATTTGCTCT CCATTCAATC CAAAATGGAC CTATTGAAAC TAAAATCTAA  4850


 


CCCAATCCCA TTAAATGATT TCTATGGCGT CAAAGGTCAA ACTTCTGAAG  4900


 


GGAACCTGTG GGTGGGTCAC AATTCAGGCT ATATATTCCC CAGGGCTCAG  4950


 


CGGATCCATG GGCTCCATCG GCGCAGCAAG CATGGAATTT TGTTTTGATG  5000


 


TATTCAAGGA GCTCAAAGTC CACCATGCCA ATGAGAACAT CTTCTACTGC  5050


 


CCCATTGCCA TCATGTCAGC TCTAGCCATG GTATACCTGG GTGCAAAAGA  5100


 


CAGCACCAGG ACACAGATAA ATAAGGTTGT TCGCTTTGAT AAACTTCCAG  5150


 


GATTCGGAGA CAGTATTGAA GCTCAGTGTG GCACATCTGT AAACGTTCAC  5200


 


TCTTCACTTA GAGACATCCT CAACCAAATC ACCAAACCAA ATGATGTTTA  5250


 


TTCGTTCAGC CTTGCCAGTA GACTTTATGC TGAAGAGAGA TACCCAATCC  5300


 


TGCCAGAATA CTTGCAGTGT GTGAAGGAAC TGTATAGAGG AGGCTTGGAA  5350


 


CCTATCAACT TTCAAACAGC TGCAGATCAA GCCAGAGAGC TCATCAATTC  5400


 


CTGGGTAGAA AGTCAGACAA ATGGAATTAT CAGAAATGTC CTTCAGCCAA  5450


 


GCTCCGTGGA TTCTCAAACT GCAATGGTTC TGGTTAATGC CATTGTCTTC  5500


 


AAAGGACTGT GGGAGAAAAC ATTTAAGGAT GAAGACACAC AAGCAATGCC  5550


 


TTTCAGAGTG ACTGAGCAAG AAAGCAAACC TGTGCAGATG ATGTACCAGA  5600


 


TTGGTTTATT TAGAGTGGCA TCAATGGCTT CTGAGAAAAT GAAGATCCTG  5650


 


GAGCTTCCAT TTGCCAGTGG GACAATGAGC ATGTTGGTGC TGTTGCCTGA  5700


 


TGAAGTCTCA GGCCTTGAGC AGCTTGAGAG TATAATCAAC TTTGAAAAAC  5750


 


TGACTGAATG GACCAGTTCT AATGTTATGG AAGAGAGGAA GATCAAAGTG  5800


 


TACTTACCTC GCATGAAGAT GGAGGAAAAA TACAACCTCA CATCTGTCTT  5850


 


AATGGCTATG GGCATTACTG ACGTGTTTAG CTCTTCAGCC AATCTGTCTG  5900


 


GCATCTCCTC AGCAGAGAGC CTGAAGATAT CTCAAGCTGT CCATGCAGCA  5950


 


CATGCAGAAA TCAATGAAGC AGGCAGAGAG GTGGTAGGGT CAGCAGAGGC  6000


 


TGGAGTGGAT GCTGCAAGCG TCTCTGAAGA ATTTAGGGCT GACCATCCAT  6050


 


TCCTCTTCTG TATCAAGCAC ATCGCAACCA ACGCCGTTCT CTTCTTTGGC  6100


 


AGATGTGTTT CCCCTCCGCG GCCAGCAGAT GACGCACCAG CAGATGACGC  6150


 


ACCAGCAGAT GACGCACCAG CAGATGACGC ACCAGCAGAT GACGCACCAG  6200


 


CAGATGACGC AACAACATGT ATCCTGAAAG GCTCTTGTGG CTGGATCGGC  6250


 


CTGCTGGATG ACGATGACAA ATTTGTGAAC CAACACCTGT GCGGCTCACA  6300


 


CCTGGTGGAA GCTCTCTACC TAGTGTGCGG GGAACGAGGC TTCTTCTACA  6350


 


CACCCAAGAC CCGCCGGGAG GCAGAGGACC TGCAGGTGGG GCAGGTGGAG  6400


 


CTGGGCGGGG GCCCTGGTGC AGGCAGCCTG CAGCCCTTGG CCCTGGAGGG  6450


 


GTCCCTGCAG AAGCGTGGCA TTGTGGAACA ATGCTGTACC AGCATCTGCT  6500


 


CCCTCTACCA GCTGGAGAAC TACTGCAACT AGGGCGCCTG GATCCAGATC  6550


 


ACTTCTGGCT AATAAAAGAT CAGAGCTCTA GAGATCTGTG TGTTGGTTTT  6600


 


TTGTGGATCT GCTGTGCCTT CTAGTTGCCA GCCATCTGTT GTTTGCCCCT  6650


 


CCCCCGTGCC TTCCTTGACC CTGGAAGGTG CCACTCCCAC TGTCCTTTCC  6700


 


TAATAAAATG AGGAAATTGC ATCGCATTGT CTGAGTAGGT GTCATTCTAT  6750


 


TCTGGGGGGT GGGGTGGGGC AGCACAGCAA GGGGGAGGAT TGGGAAGACA  6800


 


ATAGCAGGCA TGCTGGGGAT GCGGTGGGCT CTATGGGTAC CTCTCTCTCT  6850


 


CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CGGTACCTCT CTCGAGGGGG  6900


 


GGCCCGGTAC CCAATTCGCC CTATAGTGAG TCGTATTACG CGCGCTCACT  6950


 


GGCCGTCGTT TTACAACGTC GTGACTGGGA AAACCCTGGC GTTACCCAAC  7000


 


TTAATCGCCT TGCAGCACAT CCCCCTTTCG CCAGCTGGCG TAATAGCGAA  7050


 


GAGGCCCGCA CCGATCGCCC TTCCCAACAG TTGCGCAGCC TGAATGGCGA  7100


 


ATGGAAATTG TAAGCGTTAA TATTTTGTTA AAATTCGCGT TAAATTTTTG  7150


 


TTAAATCAGC TCATTTTTTA ACCAATAGGC CGAAATCGGC AAAATCCCTT  7200


 


ATAAATCAAA AGAATAGACC GAGATAGGGT TGAGTGTTGT TCCAGTTTGG  7250


 


AACAAGAGTC CACTATTAAA GAACGTGGAC TCCAACGTCA AAGGGCGAAA  7300


 


AACCGTCTAT CAGGGCGATG GCCCACTACT CCGGGATCAT ATGACAAGAT  7350


 


GTGTATCCAC CTTAACTTAA TGATTTTTAC CAAAATCATT AGGGGATTCA  7400


 


TCAGTGCTCA GGGTCAACGA GAATTAACAT TCCGTCAGGA AAGCTTATGA  7450


 


TGATGATGTG CTTAAAAACT TACTCAATGG CTGGTTATGC ATATCGCAAT  7500


 


ACATGCGAAA AACCTAAAAG AGCTTGCCGA TAAAAAAGGC CAATTTATTG  7550


 


CTATTTACCG CGGCTTTTTA TTGAGCTTGA AAGATAAATA AAATAGATAG  7600


 


GTTTTATTTG AAGCTAAATC TTCTTTATCG TAAAAAATGC CCTCTTGGGT  7650


 


TATCAAGAGG GTCATTATAT TTCGCGGAAT AACATCATTT GGTGACGAAA  7700


 


TAACTAAGCA CTTGTCTCCT GTTTACTCCC CTGAGCTTGA GGGGTTAACA  7750


 


TGAAGGTCAT CGATAGCAGG ATAATAATAC AGTAAAACGC TAAACCAATA  7800


 


ATCCAAATCC AGCCATCCCA AATTGGTAGT GAATGATTAT AAATAACAGC  7850


 


AAACAGTAAT GGGCCAATAA CACCGGTTGC ATTGGTAAGG CTCACCAATA  7900


 


ATCCCTGTAA AGCACCTTGC TGATGACTCT TTGTTTGGAT AGACATCACT  7950


 


CCCTGTAATG CAGGTAAAGC GATCCCACCA CCAGCCAATA AAATTAAAAC  8000


 


AGGGAAAACT AACCAACCTT CAGATATAAA CGCTAAAAAG GCAAATGCAC  8050


 


TACTATCTGC AATAAATCCG AGCAGTACTG CCGTTTTTTC GCCCCATTTA  8100


 


GTGGCTATTC TTCCTGCCAC AAAGGCTTGG AATACTGAGT GTAAAAGACC  8150


 


AAGACCCGCT AATGAAAAGC CAACCATCAT GCTATTCCAT CCAAAACGAT  8200


 


TTTCGGTAAA TAGCACCCAC ACCGTTGCGG GAATTTGGCC TATCAATTGC  8250


 


GCTGAAAAAT AAATAATCAA CAAAATGGCA TCGTTTTAAA TAAAGTGATG  8300


 


TATACCGAAT TCAGCTTTTG TTCCCTTTAG TGAGGGTTAA TTGCGCGCTT  8350


 


GGCGTAATCA TGGTCATAGC TGTTTCCTGT GTGAAATTGT TATCCGCTCA  8400


 


CAATTCCACA CAACATACGA GCCGGAAGCA TAAAGTGTAA AGCCTGGGGT  8450


 


GCCTAATGAG TGAGCTAACT CACATTAATT GCGTTGCGCT CACTGCCCGC  8500


 


TTTCCAGTCG GGAAACCTGT CGTGCCAGCT GCATTAATGA ATCGGCCAAC  8550


 


GCGCGGGGAG AGGCGGTTTG CGTATTGGGC GCTCTTCCGC TTCCTCGCTC  8600


 


ACTGACTCGC TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA  8650


 


CTCAAAGGCG GTAATACGGT TATCCACAGA ATCAGGGGAT AACGCAGGAA  8700


 


AGAACATGTG AGCAAAAGGC CAGCAAAAGG CCAGGAACCG TAAAAAGGCC  8750


 


GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC CCCCCTGACG AGCATCACAA  8800


 


AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT  8850


 


ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC  8900


 


CTGCCGCTTA CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC  8950


 


GCTTTCTCAT AGCTCACGCT GTAGGTATCT CAGTTCGGTG TAGGTCGTTC  9000


 


GCTCCAAGCT GGGCTGTGTG CACGAACCCC CCGTTCAGCC CGACCGCTGC  9050


 


GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA GACACGACTT  9100


 


ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG  9150


 


TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT  9200


 


AGAAGGACAG TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG  9250


 


AAAAAGAGTT GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCG  9300


 


GTGGTTTTTT TGTTTGCAAG CAGCAGATTA CGCGCAGAAA AAAAGGATCT  9350


 


CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC AGTGGAACGA  9400


 


AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA  9450


 


CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA  9500


 


TATGAGTAAA CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC  9550


 


TATCTCAGCG ATCTGTCTAT TTCGTTCATC CATAGTTGCC TGACTCCCCG  9600


 


TCGTGTAGAT AACTACGATA CGGGAGGGCT TACCATCTGG CCCCAGTGCT  9650


 


GCAATGATAC CGCGAGACCC ACGCTCACCG GCTCCAGATT TATCAGCAAT  9700


 


AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT GCAACTTTAT  9750


 


CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT  9800


 


TCGCCAGTTA ATAGTTTGCG CAACGTTGTT GCCATTGCTA CAGGCATCGT  9850


 


GGTGTCACGC TCGTCGTTTG GTATGGCTTC ATTCAGCTCC GGTTCCCAAC  9900


 


GATCAAGGCG AGTTACATGA TCCCCCATGT TGTGCAAAAA AGCGGTTAGC  9950


 


TCCTTCGGTC CTCCGATCGT TGTCAGAAGT AAGTTGGCCG CAGTGTTATC 10000


 


ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC ATGCCATCCG 10050


 


TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA 10100


 


TAGTGTATGC GGCGACCGAG TTGCTCTTGC CCGGCGTCAA TACGGGATAA 10150


 


TACCGCGCCA CATAGCAGAA CTTTAAAAGT GCTCATCATT GGAAAACGTT 10200


 


CTTCGGGGCG AAAACTCTCA AGGATCTTAC CGCTGTTGAG ATCCAGTTCG 10250


 


ATGTAACCCA CTCGTGCACC CAACTGATCT TCAGCATCTT TTACTTTCAC 10300


 


CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC GCAAAAAAGG 10350


 


GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTT CCTTTTTCAA 10400


 


TATTATTGAA GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT 10450


 


TGAATGTATT TAGAAAAATA AACAAATAGG GGTTCCGCGC ACATTTCCCC 10500


 


GAAAAGTGCC AC                                          10512


 


SEQ ID NO: 43 (pTnMOD (CMV-CHOVg-ent-ProInsulin-synPA))


    1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga


 


   61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg


 


  121 ccacgttcgc cggcatcaga ttggctattg gccattgcat acgttgtatc catatcataa


 


  181 tatgtacatt tatattggct catgtccaac attaccgcca tgttgacatt gattattgac


 


  241 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg


 


  301 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt


 


  361 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca


 


  421 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc


 


  481 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta


 


  541 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac


 


  601 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg


 


  661 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg


 


  721 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt


 


  781 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg


 


  841 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccg


 


  901 ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta ccgcctatag


 


  961 actctatagg cacacccctt tggctcttat gcatgctata ctgtttttgg cttggggcct


 


 1021 atacaccccc gcttccttat gctataggtg atggtatagc ttagcctata ggtgtgggtt


 


 1081 attgaccatt attgaccact cccctattgg tgacgatact ttccattact aatccataac


 


 1141 atggctcttt gccacaacta tctctattgg ctatatgcca atactctgtc cttcagagac


 


 1201 tgacacggac tctgtatttt tacaggatgg ggtcccattt attatttaca aattcacata


 


 1261 tacaacaacg ccgtcccccg tgcccgcagt ttttattaaa catagcgtgg gatctccacg


 


 1321 cgaatctcgg gtacgtgttc cggacatggg ctcttctccg gtagcggcgg agcttccaca


 


 1381 tccgagccct ggtcccatgc ctccagcggc tcatggtcgc tcggcagctc cttgctccta


 


 1441 acagtggagg ccagacttag gcacagcaca atgcccacca ccaccagtgt gccgcacaag


 


 1501 gccgtggcgg tagggtatgt gtctgaaaat gagcgtggag attgggctcg cacggctgac


 


 1561 gcagatggaa gacttaaggc agcggcagaa gaagatgcag gcagctgagt tgttgtattc


 


 1621 tgataagagt cagaggtaac tcccgttgcg gtgctgttaa cggtggaggg cagtgtagtc


 


 1681 tgagcagtac tcgttgctgc cgcgcgcgcc accagacata atagctgaca gactaacaga


 


 1741 ctgttccttt ccatgggtct tttctgcagt caccgtcgga ccatgtgtga acttgatatt


 


 1801 ttacatgatt ctctttacca attctgcccc gaattacact taaaacgact caacagctta


 


 1861 acgttggctt gccacgcatt acttgactgt aaaactctca ctcttaccga acttggccgt


 


 1921 aacctgccaa ccaaagcgag aacaaaacat aacatcaaac gaatcgaccg attgttaggt


 


 1981 aatcgtcacc tccacaaaga gcgactcgct gtataccgtt ggcatgctag ctttatctgt


 


 2041 tcgggcaata cgatgcccat tgtacttgtt gactggtctg atattcgtga gcaaaaacga


 


 2101 cttatggtat tgcgagcttc agtcgcacta cacggtcgtt ctgttactct ttatgagaaa


 


 2161 gcgttcccgc tttcagagca atgttcaaag aaagctcatg accaatttct agccgacctt


 


 2221 gcgagcattc taccgagtaa caccacaccg ctcattgtca gtgatgctgg ctttaaagtg


 


 2281 ccatggtata aatccgttga gaagctgggt tggtactggt taagtcgagt aagaggaaaa


 


 2341 gtacaatatg cagacctagg agcggaaaac tggaaaccta tcagcaactt acatgatatg


 


 2401 tcatctagtc actcaaagac tttaggctat aagaggctga ctaaaagcaa tccaatctca


 


 2461 tgccaaattc tattgtataa atctcgctct aaaggccgaa aaaatcagcg ctcgacacgg


 


 2521 actcattgtc accacccgtc acctaaaatc tactcagcgt cggcaaagga gccatgggtt


 


 2581 ctagcaacta acttacctgt tgaaattcga acacccaaac aacttgttaa tatctattcg


 


 2641 aagcgaatgc agattgaaga aaccttccga gacttgaaaa gtcctgccta cggactaggc


 


 2701 ctacgccata gccgaacgag cagctcagag cgttttgata tcatgctgct aatcgccctg


 


 2761 atgcttcaac taacatgttg gcttgcgggc gttcatgctc agaaacaagg ttgggacaag


 


 2821 cacttccagg ctaacacagt cagaaatcga aacgtactct caacagttcg cttaggcatg


 


 2881 gaagttttgc ggcattctgg ctacacaata acaagggaag acttactcgt ggctgcaacc


 


 2941 ctactagctc aaaatttatt cacacatggt tacgctttgg ggaaattatg ataatgatcc


 


 3001 agatcacttc tggctaataa aagatcagag ctctagagat ctgtgtgttg gttttttgtg


 


 3061 gatctgctgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct


 


 3121 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc


 


 3181 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcagcac agcaaggggg


 


 3241 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg ggtacctctc


 


 3301 tctctctctc tctctctctc tctctctctc tctctcggta cctctctctc tctctctctc


 


 3361 tctctctctc tctctctctc tcggtaccag gtgctgaaga attgacccgg tgaccaaagg


 


 3421 tgccttttat catcacttta aaaataaaaa acaattactc agtgcctgtt ataagcagca


 


 3481 attaattatg attgatgcct acatcacaac aaaaactgat ttaacaaatg gttggtctgc


 


 3541 cttagaaagt atatttgaac attatcttga ttatattatt gataataata aaaaccttat


 


 3601 ccctatccaa gaagtgatgc ctatcattgg ttggaatgaa cttgaaaaaa attagccttg


 


 3661 aatacattac tggtaaggta aacgccattg tcagcaaatt gatccaagag aaccaactta


 


 3721 aagctttcct gacggaatgt taattctcgt tgaccctgag cactgatgaa tcccctaatg


 


 3781 attttggtaa aaatcattaa gttaaggtgg atacacatct tgtcatatga tcccggtaat


 


 3841 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg


 


 3901 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac


 


 3961 gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctcca ccgcggtggc


 


 4021 ggccgctcta gaactagtgg atcccccggg catcagattg gctattggcc attgcatacg


 


 4081 ttgtatccat atcataatat gtacatttat attggctcat gtccaacatt accgccatgt


 


 4141 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc


 


 4201 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc


 


 4261 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg


 


 4321 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat


 


 4381 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc


 


 4441 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta


 


 4501 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag


 


 4561 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt


 


 4621 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa


 


 4681 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt


 


 4741 cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga


 


 4801 tccagcctcc gcggccggga acggtgcatt ggaacgcgga ttccccgtgc caagagtgac


 


 4861 gtaagtaccg cctatagact ctataggcac acccctttgg ctcttatgca tgctatactg


 


 4921 tttttggctt ggggcctata cacccccgct tccttatgct ataggtgatg gtatagctta


 


 4981 gcctataggt gtgggttatt gaccattatt gaccactccc ctattggtga cgatactttc


 


 5041 cattactaat ccataacatg gctctttgcc acaactatct ctattggcta tatgccaata


 


 5101 ctctgtcctt cagagactga cacggactct gtatttttac aggatggggt cccatttatt


 


 5161 atttacaaat tcacatatac aacaacgccg tcccccgtgc ccgcagtttt tattaaacat


 


 5221 agcgtgggat ctccacgcga atctcgggta cgtgttccgg acatgggctc ttctccggta


 


 5281 gcggcggagc ttccacatcc gagccctggt cccatgcctc cagcggctca tggtcgctcg


 


 5341 gcagctcctt gctcctaaca gtggaggcca gacttaggca cagcacaatg cccaccacca


 


 5401 ccagtgtgcc gcacaaggcc gtggcggtag ggtatgtgtc tgaaaatgag cgtggagatt


 


 5461 gggctcgcac ggctgacgca gatggaagac ttaaggcagc ggcagaagaa gatgcaggca


 


 5521 gctgagttgt tgtattctga taagagtcag aggtaactcc cgttgcggtg ctgttaacgg


 


 5581 tggagggcag tgtagtctga gcagtactcg ttgctgccgc gcgcgccacc agacataata


 


 5641 gctgacagac taacagactg ttcctttcca tgggtctttt ctgcagtcac cgtcggatca


 


 5701 atgggctcca tcggtgcagc aagcatggaa ttttgttttg atgtattcaa ggagctcaaa


 


 5761 gtccaccatg ccaatgagaa catcttctac tgccccattg ccatcatgtc agctctagcc


 


 5821 atggtatacc tgggtgcaaa agacagcacc aggacacaaa taaataaggt tgttcgcttt


 


 5881 gataaacttc caggattcgg agacagtatt gaagctcagt gtggcacatc tgtaaacgtt


 


 5941 cactcttcac ttagagacat cctcaaccaa atcaccaaac caaatgatgt ttattcgttc


 


 6001 agccttgcca gtagacttta tgctgaagag agatacccaa tcctgccaga atacttgcag


 


 6061 tgtgtgaagg aactgtatag aggaggcttg gaacctatca actttcaaac agctgcagat


 


 6121 caagccagag agctcatcaa ttcctgggta gaaagtcaga caaatggaat tatcagaaat


 


 6181 gtccttcagc caagctccgt ggattctcaa actgcaatgg ttctggttaa tgccattgtc


 


 6241 ttcaaaggac tgtgggagaa agcatttaag gatgaagaca cacaagcaat gcctttcaga


 


 6301 gtgactgagc aagaaagcaa acctgtgcag atgatgtacc agattggttt atttagagtg


 


 6361 gcatcaatgg cttctgagaa aatgaagatc ctggagcttc catttgccag tgggacaatg


 


 6421 agcatgttgg tgctgttgcc tgatgaagtc tcaggccttg agcagcttga gagtataatc


 


 6481 aactttgaaa aactgactga atggaccagt tctaatgtta tggaagagag aagatcaaag


 


 6541 tgtacttacc tcgcatgaag atggaggaaa aatacaacct cacatctgtc ttaatggcta


 


 6601 tgggcattac tgacgtgttt agctcttcag ccaatctgtc tggcatctcc tcagcagaga


 


 6661 gcctgaagat atctcaagct gtccatgcag cacatgcaga aatcaatgaa gcaggcagag


 


 6721 aggtggtagg gtcagcagag gctggagtgg atgctgcaag cgtctctgaa gaatttaggg


 


 6781 ctgaccatcc attcctcttc tgtatcaagc acatcgcaac caacgccgtt ctcttctttt


 


 6841 ggcagatgtg tttcccgcgg ccagcagatg acgcaccagc agatgacgca ccagcagatg


 


 6901 acgcaccagc agatgacgca ccagcagatg acgcaacaac atgtatcctg aaaggctctt


 


 6961 gtggctggat cggcctgctg gatgacgatg acaaatttgt gaaccaacac ctgtgcggct


 


 7021 cacacctggt ggaagctctc tacctagtgt gcggggaacg aggcttcttc tacacaccca


 


 7081 agacccgccg ggaggcagag gacctgcagg tggggcaggt ggagctgggc gggggccctg


 


 7141 gtgcaggcag cctgcagccc ttggccctgg aggggtccct gcagaagcgt ggcattgtgg


 


 7201 aacaatgctg taccagcatc tgctccctct accagctgga gaactactgc aactagggcg


 


 7261 cctaaagggc gaattatcgc ggccgctcta gaccaggcgc ctggatccag atcacttctg


 


 7321 gctaataaaa gatcagagct ctagagatct gtgtgttggt tttttgtgga tctgctgtgc


 


 7381 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag


 


 7441 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta


 


 7501 ggtgtcattc tattctgggg ggtggggtgg ggcagcacag caagggggag gattgggaag


 


 7561 acaatagcag gcatgctggg gatgcggtgg gctctatggg tacctctctc tctctctctc


 


 7621 tctctctcac tctctctctc tctcggtacc tctcctcgag ggggggcccg gtacccaatt


 


 7681 cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact


 


 7741 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct


 


 7801 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg


 


 7861 gcgaatggaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat


 


 7921 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata


 


 7981 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt


 


 8041 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tactccggga


 


 8101 tcatatgaca agatgtgtat ccaccttaac ttaatgattt ttaccaaaat cattagggga


 


 8161 ttcatcagtg ctcagggtca acgagaatta acattccgtc aggaaagctt atgatgatga


 


 8221 tgtgcttaaa aacttactca atggctggtt atgcatatcg caatacatgc gaaaaaccta


 


 8281 aaagagcttg ccgataaaaa aggccaattt attgctattt accgcggctt tttattgagc


 


 8341 ttgaaagata aataaaatag ataggtttta tttgaagcta aatcttcttt atcgtaaaaa


 


 8401 atgccctctt gggttatcaa gagggtcatt atatttcgcg gaataacatc atttggtgac


 


 8461 gaaataacta agcacttgtc tcctgtttac tcccctgagc ttgaggggtt aacatgaagg


 


 8521 tcatcgatag caggataata atacagtaaa acgctaaacc aataatccaa atccagccat


 


 8581 cccaaattgg tagtgaatga ttataaataa cagcaaacag taatgggcca ataacaccgg


 


 8641 ttgcattggt aaggctcacc aataatccct gtaaagcacc ttgctgatga ctctttgttt


 


 8701 ggatagacat cactccctgt aatgcaggta aagcgatccc accaccagcc aataaaatta


 


 8761 aaacagggaa aactaaccaa ccttcagata taaacgctaa aaaggcaaat gcactactat


 


 8821 ctgcaataaa tccgagcagt actgccgttt tttcgcccat ttagtggcta ttcttcctgc


 


 8881 cacaaaggct tggaatactg agtgtaaaag accaagaccc gtaatgaaaa gccaaccatc


 


 8941 atgctattca tcatcacgat ttctgtaata gcaccacacc gtgctggatt ggctatcaat


 


 9001 gcgctgaaat aataatcaac aaatggcatc gttaaataag tgatgtatac cgatcagctt


 


 9061 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc


 


 9121 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg


 


 9181 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc


 


 9241 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg


 


 9301 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc


 


 9361 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac


 


 9421 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa


 


 9481 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca


 


 9541 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc


 


 9601 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata


 


 9661 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta


 


 9721 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca


 


 9781 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga


 


 9841 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg


 


 9901 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg


 


 9961 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg


 


10021 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag


 


10081 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa


 


10141 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat


 


10201 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc


 


10261 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc


 


10321 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc


 


10381 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc


 


10441 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc


 


10501 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt


 


10561 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc


 


10621 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa


 


10681 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt


 


10741 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg


 


10801 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc


 


10861 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa


 


10921 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt


 


10981 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt


 


11041 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag


 


11101 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta


 


11161 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat


 


11221 aggggttccg cgcacatttc cccgaaaagt gccac


 


SEQ ID NO: 44 (pTnMod (Oval/ENT tag/Proins/PA) - QUAIL)


CTGACGCGCC CTGTAGCGGC GCATTAAGCG CGGCGGGTGT GGTGGTTACG    50


 


CGCAGCGTGA CCGCTACACT TGCCAGCGCC CTAGCGCCCG CTCCTTTCGC   100


 


TTTCTTCCCT TCCTTTCTCG CCACGTTCGC CGGCATCAGA TTGGCTATTG   150


 


GCCATTGCAT ACGTTGTATC CATATCATAA TATGTACATT TATATTGGCT   200


 


CATGTCCAAC ATTACCGCCA TGTTGACATT GATTATTGAC TAGTTATTAA   250


 


TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG   300


 


CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC   350


 


CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA   400


 


GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA   450


 


CTTGGCAGTA CATCAAGTGT ATCATATGCC AAGTACGCCC CCTATTGACG   500


 


TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA CATGACCTTA   550


 


TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC   600


 


CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG   650


 


ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG   700


 


TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC   750


 


CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG GTCTATATAA   800


 


GCAGAGCTCG TTTAGTGAAC CGTCAGATCG CCTGGAGACG CCATCCACGC   850


 


TGTTTTGACC TCCATAGAAG ACACCGGGAC CGATCCAGCC TCCGCGGCCG   900


 


GGAACGGTGC ATTGGAACGC GGATTCCCCG TGCCAAGAGT GACGTAAGTA   950


 


CCGCCTATAG ACTCTATAGG CACACCCCTT TGGCTCTTAT GCATGCTATA  1000


 


CTGTTTTTGG CTTGGGGCCT ATACACCCCC GCTTCCTTAT GCTATAGGTG  1050


 


ATGGTATAGC TTAGCCTATA GGTGTGGGTT ATTGACCATT ATTGACCACT  1100


 


CCCCTATTGG TGACGATACT TTCCATTACT AATCCATAAC ATGGCTCTTT  1150


 


GCCACAACTA TCTCTATTGG CTATATGCCA ATACTCTGTC CTTCAGAGAC  1200


 


TGACACGGAC TCTGTATTTT TACAGGATGG GGTCCCATTT ATTATTTACA  1250


 


AATTCACATA TACAACAACG CCGTCCCCCG TGCCCGCAGT TTTTATTAAA  1300


 


CATAGCGTGG GATCTCCACG CGAATCTCGG GTACGTGTTC CGGACATGGG  1350


 


CTCTTCTCCG GTAGCGGCGG AGCTTCCACA TCCGAGCCCT GGTCCCATGC  1400


 


CTCCAGCGGC TCATGGTCGC TCGGCAGCTC CTTGCTCCTA ACAGTGGAGG  1450


 


CCAGACTTAG GCACAGCACA ATGCCCACCA CCACCAGTGT GCCGCACAAG  1500


 


GCCGTGGCGG TAGGGTATGT GTCTGAAAAT GAGCGTGGAG ATTGGGCTCG  1550


 


CACGGCTGAC GCAGATGGAA GACTTAAGGC AGCGGCAGAA GAAGATGCAG  1600


 


GCAGCTGAGT TGTTGTATTC TGATAAGAGT CAGAGGTAAC TCCCGTTGCG  1650


 


GTGCTGTTAA CGGTGGAGGG CAGTGTAGTC TGAGCAGTAC TCGTTGCTGC  1700


 


CGCGCGCGCC ACCAGACATA ATAGCTGACA GACTAACAGA CTGTTCCTTT  1750


 


CCATGGGTCT TTTCTGCAGT CACCGTCGGA CCATGTGTGA ACTTGATATT  1800


 


TTACATGATT CTCTTTACCA ATTCTGCCCC GAATTACACT TAAAACGACT  1850


 


CAACAGCTTA ACGTTGGCTT GCCACGCATT ACTTGACTGT AAAACTCTCA  1900


 


CTCTTACCGA ACTTGGCCGT AACCTGCCAA CCAAAGCGAG AACAAAACAT  1950


 


AACATCAAAC GAATCGACCG ATTGTTAGGT AATCGTCACC TCCACAAAGA  2000


 


GCGACTCGCT GTATACCGTT GGCATGCTAG CTTTATCTGT TCGGGAATAC  2050


 


GATGCCCATT GTACTTGTTG ACTGGTCTGA TATTCGTGAG CAAAAACGAC  2100


 


TTATGGTATT GCGAGCTTCA GTCGCACTAC ACGGTCGTTC TGTTACTCTT  2150


 


TATGAGAAAG CGTTCCCGCT TTCAGAGCAA TGTTCAAAGA AAGCTCATGA  2200


 


CCAATTTCTA GCCGACCTTG CGAGCATTCT ACCGAGTAAC ACCACACCGC  2250


 


TCATTGTCAG TGATGCTGGC TTTAAAGTGC CATGGTATAA ATCCGTTGAG  2300


 


AAGCTGGGTT GGTACTGGTT AAGTCGAGTA AGAGGAAAAG TACAATATGC  2350


 


AGACCTAGGA GCGGAAAACT GGAAACCTAT CAGCAACTTA CATGATATGT  2400


 


CATCTAGTCA CTCAAAGACT TTAGGCTATA AGAGGCTGAC TAAAAGCAAT  2450


 


CCAATCTCAT GCCAAATTCT ATTGTATAAA TCTCGCTCTA AAGGCCGAAA  2500


 


AAATCAGCGC TCGACACGGA CTCATTGTCA CCACCCGTCA CCTAAAATCT  2550


 


ACTCAGCGTC GGCAAAGGAG CCATGGGTTC TAGCAACTAA CTTACCTGTT  2600


 


GAAATTCGAA CACCCAAACA ACTTGTTAAT ATCTATTCGA AGCGAATGCA  2650


 


GATTGAAGAA ACCTTCCGAG ACTTGAAAAG TCCTGCCTAC GGACTAGGCC  2700


 


TACGCCATAG CCGAACGAGC AGCTCAGAGC GTTTTGATAT CATGCTGCTA  2750


 


ATCGCCCTGA TGCTTCAACT AACATGTTGG CTTGCGGGCG TTCATGCTCA  2800


 


GAAACAAGGT TGGGACAAGC ACTTCCAGGC TAACACAGTC AGAAATCGAA  2850


 


ACGTACTCTC AACAGTTCGC TTAGGCATGG AAGTTTTGCG GCATTCTGGC  2900


 


TACACAATAA CAAGGGAAGA CTTACTCGTG GCTGCAACCC TACTAGCTCA  2950


 


AAATTTATTC ACACATGGTT ACGCTTTGGG GAAATTATGA TAATGATCCA  3000


 


GATCACTTCT GGCTAATAAA AGATCAGAGC TCTAGAGATC TGTGTGTTGG  3050


 


TTTTTTGTGG ATCTGCTGTG CCTTCTAGTT GCCAGCCATC TGTTGTTTGC  3100


 


CCCTCCCCCG TGCCTTCCTT GACCCTGGAA GGTGCCACTC CCACTGTCCT  3150


 


TTCCTAATAA AATGAGGAAA TTGCATCGCA TTGTCTGAGT AGGTGTCATT  3200


 


CTATTCTGGG GGGTGGGGTG GGGCAGCACA GCAAGGGGGA GGATTGGGAA  3250


 


GACAATAGCA GGCATGCTGG GGATGCGGTG GGCTCTATGG GTACCTCTCT  3300


 


CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTCGGTAC CTCTCTCTCT  3350


 


CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CGGTACCAGG TGCTGAAGAA  3400


 


TTGACCCGGT GACCAAAGGT GCCTTTTATC ATCACTTTAA AAATAAAAAA  3450


 


CAATTACTCA GTGCCTGTTA TAAGCAGCAA TTAATTATGA TTGATGCCTA  3500


 


CATCACAACA AAAACTGATT TAACAAATGG TTGGTCTGCC TTAGAAAGTA  3550


 


TATTTGAACA TTATCTTGAT TATATTATTG ATAATAATAA AAACCTTATC  3600


 


CCTATCCAAG AAGTGATGCC TATCATTGGT TGGAATGAAC TTGAAAAAAA  3650


 


TTAGCCTTGA ATACATTACT GGTAAGGTAA ACGCCATTGT CAGCAAATTG  3700


 


ATCCAAGAGA ACCAACTTAA AGCTTTCCTG ACGGAATGTT AATTCTCGTT  3750


 


GACCCTGAGC ACTGATGAAT CCCCTAATGA TTTTGGTAAA AATCATTAAG  3800


 


TTAAGGTGGA TACACATCTT GTCATATGAT CCCGGTAATG TGAGTTAGCT  3850


 


CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT  3900


 


TGTGTGGAAT TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC  3950


 


CATGATTACG CCAAGCGCGC AATTAACCCT CACTAAAGGG AACAAAAGCT  4000


 


GGAGCTCCAC CGCGGTGGCG GCCGCTCTAG AACTAGTGGA TCCCCCGGGG  4050


 


AGGTCAGAAT GGTTTCTTTA CTGTTTGTCA ATTCTATTAT TTCAATACAG  4100


 


AACAAAAGCT TCTATAACTG AAATATATTT GCTATTGTAT ATTATGATTG  4150


 


TCCCTCGAAC CATGAACACT CCTCCAGCTG AATTTCACAA TTCCTCTGTC  4200


 


ATCTGCCAGG CTGGAAGATC ATGGAAGATC TCTGAGGAAC ATTGCAAGTT  4250


 


CATACCATAA ACTCATTTGG AATTGAGTAT TATTTTGCTT TGAATGGAGC  4300


 


TATGTTTTGC AGTTCCCTCA GAAGAAAAGC TTGTTATAAA GCGTCTACAC  4350


 


CCATCAAAAG ATATATTTAA ATATTCCAAC TACAGAAAGA TTTTGTCTGC  4400


 


TCTTCACTCT GATCTCAGTT GGTTTCTTCA CGTACATGCT TCTTTATTTG  4450


 


CCTATTTTGT CAAGAAAATA ATAGGTCAAG TCCTGTTCTC ACTTATCTCC  4500


 


TGCCTAGCAT GGCTTAGATG CACGTTGTAC ATTCAAGAAG GATCAAATGA  4550


 


AACAGACTTC TGGTCTGTTA CAACAACCAT AGTAATAAAC AGACTAACTA  4600


 


ATAATTGCTA ATTATGTTTT CCATCTCTAA GGTTCCCACA TTTTTCTGTT  4650


 


TTAAGATCCC ATTATCTGGT TGTAACTGAA GCTCAATGGA ACATGAACAG  4700


 


TATTTCTCAG TCTTTTCTCC AGCAATCCTG ACGGATTAGA AGAACTGGCA  4750


 


GAAAACACTT TGTTACCCAG AATTAAAAAC TAATATTTGC TCTCCCTTCA  4800


 


ATCCAAAATG GACCTATTGA AACTAAAATC TGACCCAATC CCATTAAATT  4850


 


ATTTCTATGG CGTCAAAGGT CAAACTTTTG AAGGGAACCT GTGGGTGGGT  4900


 


CCCAATTCAG GCTATATATT CCCCAGGGCT CAGCCAGTGG ATCCATGGGC  4950


 


TCCATCGGTG CAGCAAGCAT GGAATTTTGT TTTGATGTAT TCAAGGAGCT  5000


 


CAAAGTCCAC CATGCCAATG ACAACATGCT CTACTCCCCC TTTGCCATCT  5050


 


TGTCAACTCT GGCCATGGTC TTCCTAGGTG CAAAAGACAG CACCAGGACC  5100


 


CAGATAAATA AGGTTGTTCA CTTTGATAAA CTTCCAGGAT TCGGAGACAG  5150


 


TATTGAAGCT CAGTGTGGCA CATCTGTAAA TGTTCACTCT TCACTTAGAG  5200


 


ACATACTCAA CCAAATCACC AAACAAAATG ATGCTTATTC GTTCAGCCTT  5250


 


GCCAGTAGAC TTTATGCTCA AGAGACATAC ACAGTCGTGC CGGAATACTT  5300


 


GCAATGTGTG AAGGAACTGT ATAGAGGAGG CTTAGAATCC GTCAACTTTC  5350


 


AAACAGCTGC AGATCAAGCC AGAGGCCTCA TCAATGCCTG GGTAGAAAGT  5400


 


CAGACAAACG GAATTATCAG AAACATCCTT CAGCCAAGCT CCGTGGATTC  5450


 


TCAAACTGCA ATGGTCCTGG TTAATGCCAT TGCCTTCAAG GGACTGTGGG  5500


 


AGAAAGCATT TAAGGCTGAA GACACGCAAA CAATACCTTT CAGAGTGACT  5550


 


GAGCAAGAAA GCAAACCTGT GCAGATGATG TACCAGATTG GTTCATTTAA  5600


 


AGTGGCATCA ATGGCTTCTG AGAAAATGAA GATCCTGGAG CTTCCATTTG  5650


 


CCAGTGGAAC AATGAGCATG TTGGTGCTGT TGCCTGATGA TGTCTCAGGC  5700


 


CTTGAGCAGC TTGAGAGTAT AATCAGCTTT GAAAAACTGA CTGAATGGAC  5750


 


CAGTTCTAGT ATTATGGAAG AGAGGAAGGT CAAAGTGTAC TTACCTCGCA  5800


 


TGAAGATGGA GGAGAAATAC AACCTCACAT CTCTCTTAAT GGCTATGGGA  5850


 


ATTACTGACC TGTTCAGCTC TTCAGCCAAT CTGTCTGGCA TCTCCTCAGT  5900


 


AGGGAGCCTG AAGATATCTC AAGCTGTCCA TGCAGCACAT GCAGAAATCA  5950


 


ATGAAGCGGG CAGAGATGTG GTAGGCTCAG CAGAGGCTGG AGTGGATGCT  6000


 


ACTGAAGAAT TTAGGGCTGA CCATCCATTC CTCTTCTGTG TCAAGCACAT  6050


 


CGAAACCAAC GCCATTCTCC TCTTTGGCAG ATGTGTTTCT CCGCGGCCAG  6100


 


CAGATGACGC ACCAGCAGAT GACGCACCAG CAGATGACGC ACCAGCAGAT  6150


 


GACGCACCAG CAGATGACGC ACCAGCAGAT GACGCAACAA CATGTATCCT  6200


 


GAAAGGCTCT TGTGGCTGGA TCGGCCTGCT GGATGACGAT GACAAATTTG  6250


 


TGAACCAACA CCTGTGCGGC TCACACCTGG TGGAAGCTCT CTACCTAGTG  6300


 


TGCGGGGAAC GAGGCTTCTT CTACACACCC AAGACCCGCC GGGAGGCAGA  6350


 


GGACCTGCAG GTGGGGCAGG TGGAGCTGGG CGGGGGCCCT GGTGCAGGCA  6400


 


GCCTGCAGCC CTTGGCCCTG GAGGGGTCCC TGCAGAAGCG TGGCATTGTG  6450


 


GAACAATGCT GTACCAGCAT CTGCTCCCTC TACCAGCTGG AGAACTACTG  6500


 


CAACTAGGGC GCCTGGATCC AGATCACTTC TGGCTAATAA AAGATCAGAG  6550


 


CTCTAGAGAT CTGTGTGTTG GTTTTTTGTG GATCTGCTGT GCCTTCTAGT  6600


 


TGCCAGCCAT CTGTTGTTTG CCCCTCCCCC GTGCCTTCCT TGACCCTGGA  6650


 


AGGTGCCACT CCCACTGTCC TTTCCTAATA AAATGAGGAA ATTGCATCGC  6700


 


ATTGTCTGAG TAGGTGTCAT TCTATTCTGG GGGGTGGGGT GGGGCAGCAC  6750


 


AGCAAGGGGG AGGATTGGGA AGACAATAGC AGGCATGCTG GGGATGCGGT  6800


 


GGGCTCTATG GGTACCTCTC TCTCTCTCTC TCTCTCTCTC TCTCTCTCTC  6850


 


TCTCTCGGTA CCTCTCTCGA GGGGGGGCCC GGTACCCAAT TCGCCCTATA  6900


 


GTGAGTCGTA TTACGCGCGC TCACTGGCCG TCGTTTTACA ACGTCGTGAC  6950


 


TGGGAAAACC CTGGCGTTAC CCAACTTAAT CGCCTTGCAG CACATCCCCC  7000


 


TTTCGCCAGC TGGCGTAATA GCGAAGAGGC CCGCACCGAT CGCCCTTCCC  7050


 


AACAGTTGCG CAGCCTGAAT GGCGAATGGA AATTGTAAGC GTTAATATTT  7100


 


TGTTAAAATT CGCGTTAAAT TTTTGTTAAA TCAGCTCATT TTTTAACCAA  7150


 


TAGGCCGAAA TCGGCAAAAT CCCTTATAAA TCAAAAGAAT AGACCGAGAT  7200


 


AGGGTTGAGT GTTGTTCCAG TTTGGAACAA GAGTCCACTA TTAAAGAACG  7250


 


TGGACTCCAA CGTCAAAGGG CGAAAAACCG TCTATCAGGG CGATGGCCCA  7300


 


CTACTCCGGG ATCATATGAC AAGATGTGTA TCCACCTTAA CTTAATGATT  7350


 


TTTACCAAAA TCATTAGGGG ATTCATCAGT GCTCAGGGTC AACGAGAATT  7400


 


AACATTCCGT CAGGAAAGCT TATGATGATG ATGTGCTTAA AAACTTACTC  7450


 


AATGGCTGGT TATGCATATC GCAATACATG CGAAAAACCT AAAAGAGCTT  7500


 


GCCGATAAAA AAGGCCAATT TATTGCTATT TACCGCGGCT TTTTATTGAG  7550


 


CTTGAAAGAT AAATAAAATA GATAGGTTTT ATTTGAAGCT AAATCTTCTT  7600


 


TATCGTAAAA AATGCCCTCT TGGGTTATCA AGAGGGTCAT TATATTTCGC  7650


 


GGAATAACAT CATTTGGTGA CGAAATAACT AAGCACTTGT CTCCTGTTTA  7700


 


CTCCCCTGAG CTTGAGGGGT TAACATGAAG GTCATCGATA GCAGGATAAT  7750


 


AATACAGTAA AACGCTAAAC CAATAATCCA AATCCAGCCA TCCCAAATTG  7800


 


GTAGTGAATG ATTATAAATA ACAGCAAACA GTAATGGGCC AATAACACCG  7850


 


GTTGCATTGG TAAGGCTCAC CAATAATCCC TGTAAAGCAC CTTGCTGATG  7900


 


ACTCTTTGTT TGGATAGACA TCACTCCCTG TAATGCAGGT AAAGCGATCC  7950


 


CACCACCAGC CAATAAAATT AAAACAGGGA AAACTAACCA ACCTTCAGAT  8000


 


ATAAACGCTA AAAAGGCAAA TGCACTACTA TCTGCAATAA ATCCGAGCAG  8050


 


TACTGCCGTT TTTTCGCCCC ATTTAGTGGC TATTCTTCCT GCCACAAAGG  8100


 


CTTGGAATAC TGAGTGTAAA AGACCAAGAC CCGCTAATGA AAAGCCAACC  8150


 


ATCATGCTAT TCCATCCAAA ACGATTTTCG GTAAATAGCA CCCACACCGT  8200


 


TGCGGGAATT TGGCCTATCA ATTGCGCTGA AAAATAAATA ATCAACAAAA  8250


 


TGGCATCGTT TTAAATAAAG TGATGTATAC CGAATTCAGC TTTTGTTCCC  8300


 


TTTAGTGAGG GTTAATTGCG CGCTTGGCGT AATCATGGTC ATAGCTGTTT  8350


 


CCTGTGTGAA ATTGTTATCC GCTCACAATT CCACACAACA TACGAGCCGG  8400


 


AAGCATAAAG TGTAAAGCCT GGGGTGCCTA ATGAGTGAGC TAACTCACAT  8450


 


TAATTGCGTT GCGCTCACTG CCCGCTTTCC AGTCGGGAAA CCTGTCGTGC  8500


 


CAGCTGCATT AATGAATCGG CCAACGCGCG GGGAGAGGCG GTTTGCGTAT  8550


 


TGGGCGCTCT TCCGCTTCCT CGCTCACTGA CTCGCTGCGC TCGGTCGTTC  8600


 


GGCTGCGGCG AGCGGTATCA GCTCACTCAA AGGCGGTAAT ACGGTTATCC  8650


 


ACAGAATCAG GGGATAACGC AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA  8700


 


AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT TTCCATAGGC  8750


 


TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG TCAGAGGTGG  8800


 


CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC  8850


 


CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG  8900


 


CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT CTCATAGCTC ACGCTGTAGG  8950


 


TATCTCAGTT CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT GTGTGCACGA  9000


 


ACCCCCCGTT CAGCCCGACC GCTGCGCCTT ATCCGGTAAC TATCGTCTTG  9050


 


AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC AGCCACTGGT  9100


 


AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA  9150


 


GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG  9200


 


CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC  9250


 


GGCAAACAAA CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA  9300


 


GATTACGCGC AGAAAAAAAG GATCTCAAGA AGATCCTTTG ATCTTTTCTA  9350


 


CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC  9400


 


ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA ATTAAAAATG  9450


 


AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT  9500


 


ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT  9550


 


TCATCCATAG TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA  9600


 


GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA GACCCACGCT  9650


 


CACCGGCTCC AGATTTATCA GCAATAAACC AGCCAGCCGG AAGGGCCGAG  9700


 


CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT CTATTAATTG  9750


 


TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG  9800


 


TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG  9850


 


GCTTCATTCA GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC  9900


 


CATGTTGTGC AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA  9950


 


GAAGTAAGTT GGCCGCAGTG TTATCACTCA TGGTTATGGC AGCACTGCAT 10000


 


AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA 10050


 


GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT 10100


 


CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA 10150


 


AAAGTGCTCA TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT 10200


 


CTTACCGCTG TTGAGATCCA GTTCGATGTA ACCCACTCGT GCACCCAACT 10250


 


GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG AGCAAAAACA 10300


 


GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC GGAAATGTTG 10350


 


AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT 10400


 


ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA 10450


 


TAGGGGTTC CGCGCACATT TCCCCGAAAA GTGCCAC                10487


 


SEQ ID NO: 45 (pTnMod (Oval/ENT tag/P146/PA) - Chicken)


CTGACGCGCC CTGTAGCGGC GCATTAAGCG CGGCGGGTGT GGTGGTTACG    50


 


CGCAGCGTGA CCGCTACACT TGCCAGCGCC CTAGCGCCCG CTCCTTTCGC   100


 


TTTCTTCCCT TCCTTTCTCG CCACGTTCGC CGGCATCAGA TTGGCTATTG   150


 


GCCATTGCAT ACGTTGTATC CATATCATAA TATGTACATT TATATTGGCT   200


 


CATGTCCAAC ATTACCGCCA TGTTGACATT GATTATTGAC TAGTTATTAA   250


 


TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG   300


 


CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC   350


 


CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA   400


 


GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA   450


 


CTTGGCAGTA CATCAAGTGT ATCATATGCC AAGTACGCCC CCTATTGACG   500


 


TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA CATGACCTTA   550


 


TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC   600


 


CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG   650


 


ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG   700


 


TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC   750


 


CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG GTCTATATAA   800


 


GCAGAGCTCG TTTAGTGAAC CGTCAGATCG CCTGGAGACG CCATCCACGC   850


 


TGTTTTGACC TCCATAGAAG ACACCGGGAC CGATCCAGCC TCCGCGGCCG   900


 


GGAACGGTGC ATTGGAACGC GGATTCCCCG TGCCAAGAGT GACGTAAGTA   950


 


CCGCCTATAG ACTCTATAGG CACACCCCTT TGGCTCTTAT GCATGCTATA  1000


 


CTGTTTTTGG CTTGGGGCCT ATACACCCCC GCTTCCTTAT GCTATAGGTG  1050


 


ATGGTATAGC TTAGCCTATA GGTGTGGGTT ATTGACCATT ATTGACCACT  1100


 


CCCCTATTGG TGACGATACT TTCCATTACT AATCCATAAC ATGGCTCTTT  1150


 


GCCACAACTA TCTCTATTGG CTATATGCCA ATACTCTGTC CTTCAGAGAC  1200


 


TGACACGGAC TCTGTATTTT TACAGGATGG GGTCCCATTT ATTATTTACA  1250


 


AATTCACATA TACAACAACG CCGTCCCCCG TGCCCGCAGT TTTTATTAAA  1300


 


CATAGCGTGG GATCTCCACG CGAATCTCGG GTACGTGTTC CGGACATGGG  1350


 


CTCTTCTCCG GTAGCGGCGG AGCTTCCACA TCCGAGCCCT GGTCCCATGC  1400


 


CTCCAGCGGC TCATGGTCGC TCGGCAGCTC CTTGCTCCTA ACAGTGGAGG  1450


 


CCAGACTTAG GCACAGCACA ATGCCCACCA CCACCAGTGT GCCGCACAAG  1500


 


GCCGTGGCGG TAGGGTATGT GTCTGAAAAT GAGCGTGGAG ATTGGGCTCG  1550


 


CACGGCTGAC GCAGATGGAA GACTTAAGGC AGCGGCAGAA GAAGATGCAG  1600


 


GCAGCTGAGT TGTTGTATTC TGATAAGAGT CAGAGGTAAC TCCCGTTGCG  1650


 


GTGCTGTTAA CGGTGGAGGG CAGTGTAGTC TGAGCAGTAC TCGTTGCTGC  1700


 


CGCGCGCGCC ACCAGACATA ATAGCTGACA GACTAACAGA CTGTTCCTTT  1750


 


CCATGGGTCT TTTCTGCAGT CACCGTCGGA CCATGTGTGA ACTTGATATT  1800


 


TTACATGATT CTCTTTACCA ATTCTGCCCC GAATTACACT TAAAACGACT  1850


 


CAACAGCTTA ACGTTGGCTT GCCACGCATT ACTTGACTGT AAAACTCTCA  1900


 


CTCTTACCGA ACTTGGCCGT AACCTGCCAA CCAAAGCGAG AACAAAACAT  1950


 


AACATCAAAC GAATCGACCG ATTGTTAGGT AATCGTCACC TCCACAAAGA  2000


 


GCGACTCGCT GTATACCGTT GGCATGCTAG CTTTATCTGT TCGGGAATAC  2050


 


GATGCCCATT GTACTTGTTG ACTGGTCTGA TATTCGTGAG CAAAAACGAC  2100


 


TTATGGTATT GCGAGCTTCA GTCGCACTAC ACGGTCGTTC TGTTACTCTT  2150


 


TATGAGAAAG CGTTCCCGCT TTCAGAGCAA TGTTCAAAGA AAGCTCATGA  2200


 


CCAATTTCTA GCCGACCTTG CGAGCATTCT ACCGAGTAAC ACCACACCGC  2250


 


TCATTGTCAG TGATGCTGGC TTTAAAGTGC CATGGTATAA ATCCGTTGAG  2300


 


AAGCTGGGTT GGTACTGGTT AAGTCGAGTA AGAGGAAAAG TACAATATGC  2350


 


AGACCTAGGA GCGGAAAACT GGAAACCTAT CAGCAACTTA CATGATATGT  2400


 


CATCTAGTCA CTCAAAGACT TTAGGCTATA AGAGGCTGAC TAAAAGCAAT  2450


 


CCAATCTCAT GCCAAATTCT ATTGTATAAA TCTCGCTCTA AAGGCCGAAA  2500


 


AAATCAGCGC TCGACACGGA CTCATTGTCA CCACCCGTCA CCTAAAATCT  2550


 


ACTCAGCGTC GGCAAAGGAG CCATGGGTTC TAGCAACTAA CTTACCTGTT  2600


 


GAAATTCGAA CACCCAAACA ACTTGTTAAT ATCTATTCGA AGCGAATGCA  2650


 


GATTGAAGAA ACCTTCCGAG ACTTGATAAG TCCTGCCTAC GGACTAGGCC  2700


 


TACGCCATAG CCGAACGAGC AGCTCAGAGC GTTTTGATAT CATGCTGCTA  2750


 


ATCGCCCTGA TGCTTCAACT AACATGTTGG CTTGCGGGCG TTCATGCTCA  2800


 


GAAACAAGGT TGGGACAAGC ACTTCCAGGC TAACACAGTC AGAAATCGAA  2850


 


ACGTACTCTC AACAGTTCGC TTAGGCATGG AAGTTTTGCG GCATTCTGGC  2900


 


TACACAATAA CAAGGGAAGA CTTACTCGTG GCTGCAACCC TACTAGCTCA  2950


 


AAATTTATTC ACACATGGTT ACGCTTTGGG GAAATTATGA TAATGATCCA  3000


 


GATCACTTCT GGCTAATAAA AGATCAGAGC TCTAGAGATC TGTGTGTTGG  3050


 


TTTTTTGTGG ATCTGCTGTG CCTTCTAGTT GCCAGCCATC TGTTGTTTGC  3100


 


CCCTCCCCCG TGCCTTCCTT GACCCTGGAA GGTGCCACTC CCACTGTCCT  3150


 


TTCCTAATAA AATGAGGAAA TTGCATCGCA TTGTCTGAGT AGGTGTCATT  3200


 


CTATTCTGGG GGGTGGGGTG GGGCAGCACA GCAAGGGGGA GGATTGGGAA  3250


 


GACAATAGCA GGCATGCTGG GGATGCGGTG GGCTCTATGG GTACCTCTCT  3300


 


CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTCGGTAC CTCTCTCTCT  3350


 


CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CGGTACCAGG TGCTGAAGAA  3400


 


TTGACCCGGT GACCAAAGGT GCCTTTTATC ATCACTTTAA AAATAAAAAA  3450


 


CAATTACTCA GTGCCTGTTA TAAGCAGCAA TTAATTATGA TTGATGCCTA  3500


 


CATCACAACA AAAACTGATT TAACAAATGG TTGGTCTGCC TTAGAAAGTA  3550


 


TATTTGAACA TTATCTTGAT TATATTATTG ATAATAATAA AAACCTTATC  3600


 


CCTATCCAAG AAGTGATGCC TATCATTGGT TGGAATGAAC TTGAAAAAAA  3650


 


TTAGCCTTGA ATACATTACT GGTAAGGTAA ACGCCATTGT CAGCAAATTG  3700


 


ATCCAAGAGA ACCAACTTAA AGCTTTCCTG ACGGAATGTT AATTCTCGTT  3750


 


GACCCTGAGC ACTGATGAAT CCCCTAATGA TTTTGGTAAA AATCATTAAG  3800


 


TTAAGGTGGA TACACATCTT GTCATATGAT CCCGGTAATG TGAGTTAGCT  3850


 


CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT  3900


 


TGTGTGGAAT TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC  3950


 


CATGATTACG CCAAGCGCGC AATTAACCCT CACTAAAGGG AACAAAAGCT  4000


 


GGAGCTCCAC CGCGGTGGCG GCCGCTCTAG AACTAGTGGA TCCCCCGGGG  4050


 


AGGTCAGAAT GGTTTCTTTA CTGTTTGTCA ATTCTATTAT TTCAATACAG  4100


 


AACAATAGCT TCTATAACTG AAATATATTT GCTATTGTAT ATTATGATTG  4150


 


TCCCTCGAAC CATGAACACT CCTCCAGCTG AATTTCACAA TTCCTCTGTC  4200


 


ATCTGCCAGG CCATTAAGTT ATTCATGGAA GATCTTTGAG GAACACTGCA  4250


 


AGTTCATATC ATAAACACAT TTGAAATTGA GTATTGTTTT GCATTGTATG  4300


 


GAGCTATGTT TTGCTGTATC CTCAGAAAAA AAGTTTGTTA TAAAGCATTC  4350


 


ACACCCATAA AAAGATAGAT TTAAATATTC CAGCTATAGG AAAGAAAGTG  4400


 


CGTCTGCTCT TCACTCTAGT CTCAGTTGGC TCCTTCACAT GCATGCTTCT  4450


 


TTATTTCTCC TATTTTGTCA AGAAAATAAT AGGTCACGTC TTGTTCTCAC  4500


 


TTATGTCCTG CCTAGCATGG CTCAGATGCA CGTTGTAGAT ACAAGAAGGA  4550


 


TCAAATGAAA CAGACTTCTG GTCTGTTACT ACAACCATAG TAATAAGCAC  4600


 


ACTAACTAAT AATTGCTAAT TATGTTTTCC ATCTCTAAGG TTCCCACATT  4650


 


TTTCTGTTTT CTTAAAGATC CCATTATCTG GTTGTAACTG AAGCTCAATG  4700


 


GAACATGAGC AATATTTCCC AGTCTTCTCT CCCATCCAAC AGTCCTGATG  4750


 


GATTAGCAGA ACAGGCAGAA AACACATTGT TACCCAGAAT TAAAAACTAA  4800


 


TATTTGCTCT CCATTCAATC CAAAATGGAC CTATTGAAAC TAAAATCTAA  4850


 


CCCAATCCCA TTAAATGATT TCTATGGCGT CAAAGGTCAA ACTTCTGAAG  4900


 


GGAACCTGTG GGTGGGTCAC AATTCAGGCT ATATATTCCC CAGGGCTCAG  4950


 


CGGATCCATG GGCTCCATCG GCGCAGCAAG CATGGAATTT TGTTTTGATG  5000


 


TATTCAAGGA GCTCAAAGTC CACCATGCCA ATGAGAACAT CTTCTACTGC  5050


 


CCCATTGCCA TCATGTCAGC TCTAGCCATG GTATACCTGG GTGCAAAAGA  5100


 


CAGCACCAGG ACACAGATAA ATAAGGTTGT TCGCTTTGAT AAACTTCCAG  5150


 


GATTCGGAGA CAGTATTGAA GCTCAGTGTG GCACATCTGT AAACGTTCAC  5200


 


TCTTCACTTA GAGACATCCT CAACCAAATC ACCAAACCAA ATGATGTTTA  5250


 


TTCGTTCAGC CTTGCCAGTA GACTTTATGC TGAAGAGAGA TACCCAATCC  5300


 


TGCCAGAATA CTTGCAGTGT GTGAAGGAAC TGTATAGAGG AGGCTTGGAA  5350


 


CCTATCAACT TTCAAACAGC TGCAGATCAA GCCAGAGAGC TCATCAATTC  5400


 


CTGGGTAGAA AGTCAGACAA ATGGAATTAT CAGAAATGTC CTTCAGCCAA  5450


 


GCTCCGTGGA TTCTCAAACT GCAATGGTTC TGGTTAATGC CATTGTCTTC  5500


 


AAAGGACTGT GGGAGAAAAC ATTTAAGGAT GAAGACACAC AAGCAATGCC  5550


 


TTTCAGAGTG ACTGAGCAAG AAAGCAAACC TGTGCAGATG ATGTACCAGA  5600


 


TTGGTTTATT TAGAGTGGCA TCAATGGCTT CTGAGAAAAT GAAGATCCTG  5650


 


GAGCTTCCAT TTGCCAGTGG GACAATGAGC ATGTTGGTGC TGTTGCCTGA  5700


 


TGAAGTCTCA GGCCTTGAGC AGCTTGAGAG TATAATCAAC TTTGAAAAAC  5750


 


TGACTGAATG GACCAGTTCT AATGTTATGG AAGAGAGGAA GATCAAAGTG  5800


 


TACTTACCTC GCATGAAGAT GGAGGAAAAA TACAACCTCA CATCTGTCTT  5850


 


AATGGCTATG GGCATTACTG ACGTGTTTAG CTCTTCAGCC AATCTGTCTG  5900


 


GCATCTCCTC AGCAGAGAGC CTGAAGATAT CTCAAGCTGT CCATGCAGCA  5950


 


CATGCAGAAA TCAATGAAGC AGGCAGAGAG GTGGTAGGGT CAGCAGAGGC  6000


 


TGGAGTGGAT GCTGCAAGCG TCTCTGAAGA ATTTAGGGCT GACCATCCAT  6050


 


TCCTCTTCTG TATCAAGCAC ATCGCAACCA ACGCCGTTCT CTTCTTTGGC  6100


 


AGATGTGTTT CCCCTCCGCG GCCAGCAGAT GACGCACCAG CAGATGACGC  6150


 


ACCAGCAGAT GACGCACCAG CAGATGACGC ACCAGCAGAT GACGCACCAG  6200


 


CAGATGACGC AACAACATGT ATCCTGAAAG GCTCTTGTGG CTGGATCGGC  6250


 


CTGCTGGATG ACGATGACAA AAAATACAAA AAAGCACTGA AAAAACTGGC  6300


 


AAAACTGCTG TAATGAGGGC GCCTGGATCC AGATCACTTC TGGCTAATAA  6350


 


AAGATCAGAG CTCTAGAGAT CTGTGTGTTG GTTTTTTGTG GATCTGCTGT  6400


 


GCCTTCTAGT TGCCAGCCAT CTGTTGTTTG CCCCTCCCCC GTGCCTTCCT  6450


 


TGACCCTGGA AGGTGCCACT CCCACTGTCC TTTCCTAATA AAATGAGGAA  6500


 


ATTGCATCGC ATTGTCTGAG TAGGTGTCAT TCTATTCTGG GGGGTGGGGT  6550


 


GGGGCAGCAC AGCAAGGGGG AGGATTGGGA AGACAATAGC AGGCATGCTG  6600


 


GGGATGCGGT GGGCTCTATG GGTACCTCTC TCTCTCTCTC TCTCTCTCTC  6650


 


TCTCTCTCTC TCTCTCGGTA CCTCTCTCGA GGGGGGGCCC GGTACCCAAT  6700


 


TCGCCCTATA GTGAGTCGTA TTACGCGCGC TCACTGGCCG TCGTTTTACA  6750


 


ACGTCGTGAC TGGGAAAACC CTGGCGTTAC CCAACTTAAT CGCCTTGCAG  6800


 


CACATCCCCC TTTCGCCAGC TGGCGTAATA GCGAAGAGGC CCGCACCGAT  6850


 


CGCCCTTCCC AACAGTTGCG CAGCCTGAAT GGCGAATGGA AATTGTAAGC  6900


 


GTTAATATTT TGTTAAAATT CGCGTTAAAT TTTTGTTAAA TCAGCTCATT  6950


 


TTTTAACCAA TAGGCCGAAA TCGGCAAAAT CCCTTATAAA TCAAAAGAAT  7000


 


AGACCGAGAT AGGGTTGAGT GTTGTTCCAG TTTGGAACAA GAGTCCACTA  7050


 


TTAAAGAACG TGGACTCCAA CGTCAAAGGG CGAAAAACCG TCTATCAGGG  7100


 


CGATGGCCCA CTACTCCGGG ATCATATGAC AAGATGTGTA TCCACCTTAA  7150


 


CTTAATGATT TTTACCAAAA TCATTAGGGG ATTCATCAGT GCTCAGGGTC  7200


 


AACGAGAATT AACATTCCGT CAGGAAAGCT TATGATGATG ATGTGCTTAA  7250


 


AAACTTACTC AATGGCTGGT TATGCATATC GCAATACATG CGAAAAACCT  7300


 


AAAAGAGCTT GCCGATAAAA AAGGCCAATT TATTGCTATT TACCGCGGCT  7350


 


TTTTATTGAG CTTGAAAGAT AAATAAAATA GATAGGTTTT ATTTGAAGCT  7400


 


AAATCTTCTT TATCGTAAAA AATGCCCTCT TGGGTTATCA AGAGGGTCAT  7450


 


TATATTTCGC GGAATAACAT CATTTGGTGA CGAAATAACT AAGCACTTGT  7500


 


CTCCTGTTTA CTCCCCTGAG CTTGAGGGGT TAACATGAAG GTCATCGATA  7550


 


GCAGGATAAT AATACAGTAA AACGCTAAAC CAATAATCCA AATCCAGCCA  7600


 


TCCCAAATTG GTAGTGAATG ATTATAAATA ACAGCAAACA GTAATGGGCC  7650


 


AATAACACCG GTTGCATTGG TAAGGCTCAC CAATAATCCC TGTAAAGCAC  7700


 


CTTGCTGATG ACTCTTTGTT TGGATAGACA TCACTCCCTG TAATGCAGGT  7750


 


AAAGCGATCC CACCACCAGC CAATAAAATT AAAACAGGGA AAACTAACCA  7800


 


ACCTTCAGAT ATAAACGCTA AAAAGGCAAA TGCACTACTA TCTGCAATAA  7850


 


ATCCGAGCAG TACTGCCGTT TTTTCGCCCC ATTTAGTGGC TATTCTTCCT  7900


 


GCCACAAAGG CTTGGAATAC TGAGTGTAAA AGACCAAGAC CCGCTAATGA  7950


 


AAAGCCAACC ATCATGCTAT TCCATCCAAA ACGATTTTCG GTAAATAGCA  8000


 


CCCACACCGT TGCGGGAATT TGGCCTATCA ATTGCGCTGA AAAATAAATA  8050


 


ATCAACAAAA TGGCATCGTT TTAAATAAAG TGATGTATAC CGAATTCAGC  8100


 


TTTTGTTCCC TTTAGTGAGG GTTAATTGCG CGCTTGGCGT AATCATGGTC  8150


 


ATAGCTGTTT CCTGTGTGAA ATTGTTATCC GCTCACAATT CCACACAACA  8200


 


TACGAGCCGG AAGCATAAAG TGTAAAGCCT GGGGTGCCTA ATGAGTGAGC  8250


 


TAACTCACAT TAATTGCGTT GCGCTCACTG CCCGCTTTCC AGTCGGGAAA  8300


 


CCTGTCGTGC CAGCTGCATT AATGAATCGG CCAACGCGCG GGGAGAGGCG  8350


 


GTTTGCGTAT TGGGCGCTCT TCCGCTTCCT CGCTCACTGA CTCGCTGCGC  8400


 


TCGGTCGTTC GGCTGCGGCG AGCGGTATCA GCTCACTCAA AGGCGGTAAT  8450


 


ACGGTTATCC ACAGAATCAG GGGATAACGC AGGAAAGAAC ATGTGAGCAA  8500


 


AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT  8550


 


TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG  8600


 


TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC  8650


 


CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA  8700


 


TACCTGTCCG CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT CTCATAGCTC  8750


 


ACGCTGTAGG TATCTCAGTT CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT  8800


 


GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT ATCCGGTAAC  8850


 


TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC  8900


 


AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG  8950


 


AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT  9000


 


GGTATCTGCG CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG  9050


 


CTCTTGATCC GGCAAACAAA CCACCGCTGG TAGCGGTGGT TTTTTTGTTT  9100


 


GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA AGATCCTTTG  9150


 


ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG  9200


 


GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA  9250


 


ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG  9300


 


TCTGACAGTT ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG  9350


 


TCTATTTCGT TCATCCATAG TTGCCTGACT CCCCGTCGTG TAGATAACTA  9400


 


CGATACGGGA GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA  9450


 


GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC AGCCAGCCGG  9500


 


AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT  9550


 


CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT  9600


 


TTGCGCAACG TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC  9650


 


GTTTGGTATG GCTTCATTCA GCTCCGGTTC CCAACGATCA AGGCGAGTTA  9700


 


CATGATCCCC CATGTTGTGC AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG  9750


 


ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA TGGTTATGGC  9800


 


AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG  9850


 


TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA  9900


 


CCGAGTTGCT CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG  9950


 


CAGAACTTTA AAAGTGCTCA TCATTGGAAA ACGTTCTTCG GGGCGAAAAC 10000


 


TCTCAAGGAT CTTACCGCTG TTGAGATCCA GTTCGATGTA ACCCACTCGT 10050


 


GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG 10100


 


AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC 10150


 


GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT 10200


 


TATCAGGGTT ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA 10250


 


AAATAAACAA ATAGGGGTTC CGCGCACATT TCCCCGAAAA GTGCCAC    10297


 


SEQ ID NO: 46 (pTnMod (Oval/ENT tag/P146/PA) - QUAIL)


CTGACGCGCC CTGTAGCGGC GCATTAAGCG CGGCGGGTGT GGTGGTTACG    50


 


CGCAGCGTGA CCGCTACACT TGCCAGCGCC CTAGCGCCCG CTCCTTTCGC   100


 


TTTCTTCCCT TCCTTTCTCG CCACGTTCGC CGGCATCAGA TTGGCTATTG   150


 


GCCATTGCAT ACGTTGTATC CATATCATAA TATGTACATT TATATTGGCT   200


 


CATGTCCAAC ATTACCGCCA TGTTGACATT GATTATTGAC TAGTTATTAA   250


 


TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG   300


 


CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC   350


 


CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA   400


 


GGGACTTTCC ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA   450


 


CTTGGCAGTA CATCAAGTGT ATCATATGCC AAGTACGCCC CCTATTGACG   500


 


TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA CATGACCTTA   550


 


TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC   600


 


CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG   650


 


ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG   700


 


TTTTGGCACC AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC   750


 


CCCATTGACG CAAATGGGCG GTAGGCGTGT ACGGTGGGAG GTCTATATAA   800


 


GCAGAGCTCG TTTAGTGAAC CGTCAGATCG CCTGGAGACG CCATCCACGC   850


 


TGTTTTGACC TCCATAGAAG ACACCGGGAC CGATCCAGCC TCCGCGGCCG   900


 


GGAACGGTGC ATTGGAACGC GGATTCCCCG TGCCAAGAGT GACGTAAGTA   950


 


CCGCCTATAG ACTCTATAGG CACACCCCTT TGGCTCTTAT GCATGCTATA  1000


 


CTGTTTTTGG CTTGGGGCCT ATACACCCCC GCTTCCTTAT GCTATAGGTG  1050


 


ATGGTATAGC TTAGCCTATA GGTGTGGGTT ATTGACCATT ATTGACCACT  1100


 


CCCCTATTGG TGACGATACT TTCCATTACT AATCCATAAC ATGGCTCTTT  1150


 


GCCACAACTA TCTCTATTGG CTATATGCCA ATACTCTGTC CTTCAGAGAC  1200


 


TGACACGGAC TCTGTATTTT TACAGGATGG GGTCCCATTT ATTATTTACA  1250


 


AATTCACATA TACAACAACG CCGTCCCCCG TGCCCGCAGT TTTTATTAAA  1300


 


CATAGCGTGG GATCTCCACG CGAATCTCGG GTACGTGTTC CGGACATGGG  1350


 


CTCTTCTCCG GTAGCGGCGG AGCTTCCACA TCCGAGCCCT GGTCCCATGC  1400


 


CTCCAGCGGC TCATGGTCGC TCGGCAGCTC CTTGCTCCTA ACAGTGGAGG  1450


 


CCAGACTTAG GCACAGCACA ATGCCCACCA CCACCAGTGT GCCGCACAAG  1500


 


GCCGTGGCGG TAGGGTATGT GTCTGAAAAT GAGCGTGGAG ATTGGGCTCG  1550


 


CACGGCTGAC GCAGATGGAA GACTTAAGGC AGCGGCAGAA GAAGATGCAG  1600


 


GCAGCTGAGT TGTTGTATTC TGATAAGAGT CAGAGGTAAC TCCCGTTGCG  1650


 


GTGCTGTTAA CGGTGGAGGG CAGTGTAGTC TGAGCAGTAC TCGTTGCTGC  1700


 


CGCGCGCGCC ACCAGACATA ATAGCTGACA GACTAACAGA CTGTTCCTTT  1750


 


CCATGGGTCT TTTCTGCAGT CACCGTCGGA CCATGTGTGA ACTTGATATT  1800


 


TTACATGATT CTCTTTACCA ATTCTGCCCC GAATTACACT TAAAACGACT  1850


 


CAACAGCTTA ACGTTGGCTT GCCACGCATT ACTTGACTGT AAAACTCTCA  1900


 


CTCTTACCGA ACTTGGCCGT AACCTGCCAA CCAAAGCGAG AACAAAACAT  1950


 


AACATCAAAC GAATCGACCG ATTGTTAGGT AATCGTCACC TCCACAAAGA  2000


 


GCGACTCGCT GTATACCGTT GGCATGCTAG CTTTATCTGT TCGGGAATAC  2050


 


GATGCCCATT GTACTTGTTG ACTGGTCTGA TATTCGTGAG CAAAAACGAC  2100


 


TTATGGTATT GCGAGCTTCA GTCGCACTAC ACGGTCGTTC TGTTACTCTT  2150


 


TATGAGAAAG CGTTCCCGCT TTCAGAGCAA TGTTCAAAGA AAGCTCATGA  2200


 


CCAATTTCTA GCCGACCTTG CGAGCATTCT ACCGAGTAAC ACCACACCGC  2250


 


TCATTGTCAG TGATGCTGGC TTTAAAGTGC CATGGTATAA ATCCGTTGAG  2300


 


AAGCTGGGTT GGTACTGGTT AAGTCGAGTA AGAGGAAAAG TACAATATGC  2350


 


AGACCTAGGA GCGGAAAACT GGAAACCTAT CAGCAACTTA CATGATATGT  2400


 


CATCTAGTCA CTCAAAGACT TTAGGCTATA AGAGGCTGAC TAAAAGCAAT  2450


 


CCAATCTCAT GCCAAATTCT ATTGTATAAA TCTCGCTCTA AAGGCCGAAA  2500


 


AAATCAGCGC TCGACACGGA CTCATTGTCA CCACCCGTCA CCTAAAATCT  2550


 


ACTCAGCGTC GGCAAAGGAG CCATGGGTTC TAGCAACTAA CTTACCTGTT  2600


 


GAAATTCGAA CACCCAAACA ACTTGTTAAT ATCTATTCGA AGCGAATGCA  2650


 


GATTGAAGAA ACCTTCCGAG ACTTGAAAAG TCCTGCCTAC GGACTAGGCC  2700


 


TACGCCATAG CCGAACGAGC AGCTCAGAGC GTTTTGATAT CATGCTGCTA  2750


 


ATCGCCCTGA TGCTTCAACT AACATGTTGG CTTGCGGGCG TTCATGCTCA  2800


 


GAAACAAGGT TGGGACAAGC ACTTCCAGGC TAACACAGTC AGAAATCGAA  2850


 


ACGTACTCTC AACAGTTCGC TTAGGCATGG AAGTTTTGCG GCATTCTGGC  2900


 


TACACAATAA CAAGGGAAGA CTTACTCGTG GCTGCAACCC TACTAGCTCA  2950


 


AAATTTATTC ACACATGGTT ACGCTTTGGG GAAATTATGA TAATGATCCA  3000


 


GATCACTTCT GGCTAATAAA AGATCAGAGC TCTAGAGATC TGTGTGTTGG  3050


 


TTTTTTGTGG ATCTGCTGTG CCTTCTAGTT GCCAGCCATC TGTTGTTTGC  3100


 


CCCTCCCCCG TGCCTTCCTT GACCCTGGAA GGTGCCACTC CCACTGTCCT  3150


 


TTCCTAATAA AATGAGGAAA TTGCATCGCA TTGTCTGAGT AGGTGTCATT  3200


 


CTATTCTGGG GGGTGGGGTG GGGCAGCACA GCAAGGGGGA GGATTGGGAA  3250


 


GACAATAGCA GGCATGCTGG GGATGCGGTG GGCTCTATGG GTACCTCTCT  3300


 


CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTCGGTAC CTCTCTCTCT  3350


 


CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CGGTACCAGG TGCTGAAGAA  3400


 


TTGACCCGGT GACCAAAGGT GCCTTTTATC ATCACTTTAA AAATAAAAAA  3450


 


CAATTACTCA GTGCCTGTTA TAAGCAGCAA TTAATTATGA TTGATGCCTA  3500


 


CATCACAACA AAAACTGATT TAACAAATGG TTGGTCTGCC TTAGAAAGTA  3550


 


TATTTGAACA TTATCTTGAT TATATTATTG ATAATAATAA AAACCTTATC  3600


 


CCTATCCAAG AAGTGATGCC TATCATTGGT TGGAATGAAC TTGAAAAAAA  3650


 


TTAGCCTTGA ATACATTACT GGTAAGGTAA ACGCCATTGT CAGCAAATTG  3700


 


ATCCAAGAGA ACCAACTTAA AGCTTTCCTG ACGGAATGTT AATTCTCGTT  3750


 


GACCCTGAGC ACTGATGAAT CCCCTAATGA TTTTGGTAAA AATCATTAAG  3800


 


TTAAGGTGGA TACACATCTT GTCATATGAT CCCGGTAATG TGAGTTAGCT  3850


 


CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT  3900


 


TGTGTGGAAT TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC  3950


 


CATGATTACG CCAAGCGCGC AATTAACCCT CACTAAAGGG AACAAAAGCT  4000


 


GGAGCTCCAC CGCGGTGGCG GCCGCTCTAG AACTAGTGGA TCCCCCGGGG  4050


 


AGGTCAGAAT GGTTTCTTTA CTGTTTGTCA ATTCTATTAT TTCAATACAG  4100


 


AACAAAAGCT TCTATAACTG AAATATATTT GCTATTGTAT ATTATGATTG  4150


 


TCCCTCGAAC CATGAACACT CCTCCAGCTG AATTTCACAA TTCCTCTGTC  4200


 


ATCTGCCAGG CTGGAAGATC ATGGAAGATC TCTGAGGAAC ATTGCAAGTT  4250


 


CATACCATAA ACTCATTTGG AATTGAGTAT TATTTTGCTT TGAATGGAGC  4300


 


TATGTTTTGC AGTTCCCTCA GAAGAAAAGC TTGTTATAAA GCGTCTACAC  4350


 


CCATCAAAAG ATATATTTAA ATATTCCAAC TACAGAAAGA TTTTGTCTGC  4400


 


TCTTCACTCT GATCTCAGTT GGTTTCTTCA CGTACATGCT TCTTTATTTG  4450


 


CCTATTTTGT CAAGAAAATA ATAGGTCAAG TCCTGTTCTC ACTTATCTCC  4500


 


TGCCTAGCAT GGCTTAGATG CACGTTGTAC ATTCAAGAAG GATCAAATGA  4550


 


AACAGACTTC TGGTCTGTTA CAACAACCAT AGTAATAAAC AGACTAACTA  4600


 


ATAATTGCTA ATTATGTTTT CCATCTCTAA GGTTCCCACA TTTTTCTGTT  4650


 


TTAAGATCCC ATTATCTGGT TGTAACTGAA GCTCAATGGA ACATGAACAG  4700


 


TATTTCTCAG TCTTTTCTCC AGCAATCCTG ACGGATTAGA AGAACTGGCA  4750


 


GAAAACACTT TGTTACCCAG AATTAAAAAC TAATATTTGC TCTCCCTTCA  4800


 


ATCCAAAATG GACCTATTGA AACTAAAATC TGACCCAATC CCATTAAATT  4850


 


ATTTCTATGG CGTCAAAGGT CAAACTTTTG AAGGGAACCT GTGGGTGGGT  4900


 


CCCAATTCAG GCTATATATT CCCCAGGGCT CAGCCAGTGG ATCCATGGGC  4950


 


TCCATCGGTG CAGCAAGCAT GGAATTTTGT TTTGATGTAT TCAAGGAGCT  5000


 


CAAAGTCCAC CATGCCAATG ACAACATGCT CTACTCCCCC TTTGCCATCT  5050


 


TGTCAACTCT GGCCATGGTC TTCCTAGGTG CAAAAGACAG CACCAGGACC  5100


 


CAGATAAATA AGGTTGTTCA CTTTGATAAA CTTCCAGGAT TCGGAGACAG  5150


 


TATTGAAGCT CAGTGTGGCA CATCTGTAAA TGTTCACTCT TCACTTAGAG  5200


 


ACATACTCAA CCAAATCACC AAACAAAATG ATGCTTATTC GTTCAGCCTT  5250


 


GCCAGTAGAC TTTATGCTCA AGAGACATAC ACAGTCGTGC CGGAATACTT  5300


 


GCAATGTGTG AAGGAACTGT ATAGAGGAGG CTTAGAATCC GTCAACTTTC  5350


 


AAACAGCTGC AGATCAAGCC AGAGGCCTCA TCAATGCCTG GGTAGAAAGT  5400


 


CAGACAAACG GAATTATCAG AAACATCCTT CAGCCAAGCT CCGTGGATTC  5450


 


TCAAACTGCA ATGGTCCTGG TTAATGCCAT TGCCTTCAAG GGACTGTGGG  5500


 


AGAAAGCATT TAAGGCTGAA GACACGCAAA CAATACCTTT CAGAGTGACT  5550


 


GAGCAAGAAA GCAAACCTGT GCAGATGATG TACCAGATTG GTTCATTTAA  5600


 


AGTGGCATCA ATGGCTTCTG AGAAAATGAA GATCCTGGAG CTTCCATTTG  5650


 


CCAGTGGAAC AATGAGCATG TTGGTGCTGT TGCCTGATGA TGTCTCAGGC  5700


 


CTTGAGCAGC TTGAGAGTAT AATCAGCTTT GAAAAACTGA CTGAATGGAC  5750


 


CAGTTCTAGT ATTATGGAAG AGAGGAAGGT CAAAGTGTAC TTACCTCGCA  5800


 


TGAAGATGGA GGAGAAATAC AACCTCACAT CTCTCTTAAT GGCTATGGGA  5850


 


ATTACTGACC TGTTCAGCTC TTCAGCCAAT CTGTCTGGCA TCTCCTCAGT  5900


 


AGGGAGCCTG AAGATATCTC AAGCTGTCCA TGCAGCACAT GCAGAAATCA  5950


 


ATGAAGCGGG CAGAGATGTG GTAGGCTCAG CAGAGGCTGG AGTGGATGCT  6000


 


ACTGAAGAAT TTAGGGCTGA CCATCCATTC CTCTTCTGTG TCAAGCACAT  6050


 


CGAAACCAAC GCCATTCTCC TCTTTGGCAG ATGTGTTTCT CCGCGGCCAG  6100


 


CAGATGACGC ACCAGCAGAT GACGCACCAG CAGATGACGC ACCAGCAGAT  6150


 


GACGCACCAG CAGATGACGC ACCAGCAGAT GACGCAACAA CATGTATCCT  6200


 


GAAAGGCTCT TGTGGCTGGA TCGGCCTGCT GGATGACGAT GACAAAAAAT  6250


 


ACAAAAAAGC ACTGAAAAAA CTGGCAAAAC TGCTGTAATG AGGGCGCCTG  6300


 


GATCCAGATC ACTTCTGGCT AATAAAAGAT CAGAGCTCTA GAGATCTGTG  6350


 


TGTTGGTTTT TTGTGGATCT GCTGTGCCTT CTAGTTGCCA GCCATCTGTT  6400


 


GTTTGCCCCT CCCCCGTGCC TTCCTTGACC CTGGAAGGTG CCACTCCCAC  6450


 


TGTCCTTTCC TAATAAAATG AGGAAATTGC ATCGCATTGT CTGAGTAGGT  6500


 


GTCATTCTAT TCTGGGGGGT GGGGTGGGGC AGCACAGCAA GGGGGAGGAT  6550


 


TGGGAAGACA ATAGCAGGCA TGCTGGGGAT GCGGTGGGCT CTATGGGTAC  6600


 


CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CGGTACCTCT  6650


 


CTCGAGGGGG GGCCCGGTAC CCAATTCGCC CTATAGTGAG TCGTATTACG  6700


 


CGCGCTCACT GGCCGTCGTT TTACAACGTC GTGACTGGGA AAACCCTGGC  6750


 


GTTACCCAAC TTAATCGCCT TGCAGCACAT CCCCCTtTCG CCAGCTGGCG  6800


 


TAATAGCGAA GAGGCCCGCA CCGATCGCCC TTCCCAACAG TTGCGCAGCC  6850


 


TGAATGGCGA ATGGAAATTG TAAGCGTTAA TATTTTGTTA AAATTCGCGT  6900


 


TAAATTTTTG TTAAATCAGC TCATTTTTTA ACCAATAGGC CGAAATCGGC  6950


 


AAAATCCCTT ATAAATCAAA AGAATAGACC GAGATAGGGT TGAGTGTTGT  7000


 


TCCAGTTTGG AACAAGAGTC CACTATTAAA GAACGTGGAC TCCAACGTCA  7050


 


AAGGGCGAAA AACCGTCTAT CAGGGCGATG GCCCACTACT CCGGGATCAT  7100


 


ATGACAAGAT GTGTATCCAC CTTAACTTAA TGATTTTTAC CAAAATCATT  7150


 


AGGGGATTCA TCAGTGCTCA GGGTCAACGA GAATTAACAT TCCGTCAGGA  7200


 


AAGCTTATGA TGATGATGTG CTTAAAAACT TACTCAATGG CTGGTTATGC  7250


 


ATATCGCAAT ACATGCGAAA AACCTAAAAG AGCTTGCCGA TAAAAAAGGC  7300


 


CAATTTATTG CTATTTACCG CGGCTTTTTA TTGAGCTTGA AAGATAAATA  7350


 


AAATAGATAG GTTTTATTTG AAGCTAAATC TTCTTTATCG TAAAAAATGC  7400


 


CCTCTTGGGT TATCAAGAGG GTCATTATAT TTCGCGGAAT AACATCATTT  7450


 


GGTGACGAAA TAACTAAGCA CTTGTCTCCT GTTTACTCCC CTGAGCTTGA  7500


 


GGGGTTAACA TGAAGGTCAT CGATAGCAGG ATAATAATAC AGTAAAACGC  7550


 


TAAACCAATA ATCCAAATCC AGCCATCCCA AATTGGTAGT GAATGATTAT  7600


 


AAATAACAGC AAACAGTAAT GGGCCAATAA CACCGGTTGC ATTGGTAAGG  7650


 


CTCACCAATA ATCCCTGTAA AGCACCTTGC TGATGACTCT TTGTTTGGAT  7700


 


AGACATCACT CCCTGTAATG CAGGTAAAGC GATCCCACCA CCAGCCAATA  7750


 


AAATTAAAAC AGGGAAAACT AACCAACCTT CAGATATAAA CGCTAAAAAG  7800


 


GCAAATGCAC TACTATCTGC AATAAATCCG AGCAGTACTG CCGTTTTTTC  7850


 


GCCCCATTTA GTGGCTATTC TTCCTGCCAC AAAGGCTTGG AATACTGAGT  7900


 


GTAAAAGACC AAGACCCGCT AATGAAAAGC CAACCATCAT GCTATTCCAT  7950


 


CCAAAACGAT TTTCGGTAAA TAGCACCCAC ACCGTTGCGG GAATTTGGCC  8000


 


TATCAATTGC GCTGAAAAAT AAATAATCAA CAAAATGGCA TCGTTTTAAA  8050


 


TAAAGTGATG TATACCGAAT TCAGCTTTTG TTCCCTTTAG TGAGGGTTAA  8200


 


TTGCGCGCTT GGCGTAATCA TGGTCATAGC TGTTTCCTGT GTGAAATTGT  8150


 


TATCCGCTCA CAATTCCACA CAACATACGA GCCGGAAGCA TAAAGTGTAA  8200


 


AGCCTGGGGT GCCTAATGAG TGAGCTAACT CACATTAATT GCGTTGCGCT  8250


 


CACTGCCCGC TTTCCAGTCG GGAAACCTGT CGTGCCAGCT GCATTAATGA  8300


 


ATCGGCCAAC GCGCGGGGAG AGGCGGTTTG CGTATTGGGC GCTCTTCCGC  8350


 


TTCCTCGCTC ACTGACTCGC TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG  8400


 


TATCAGCTCA CTCAAAGGCG GTAATACGGT TATCCACAGA ATCAGGGGAT  8450


 


AACGCAGGAA AGAACATGTG AGCAAAAGGC CAGCAAAAGG CCAGGAACCG  8500


 


TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC CCCCCTGACG  8550


 


AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA  8600


 


CTATAAAGAT ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC  8650


 


TGTTCCGACC CTGCCGCTTA CCGGATACCT GTCCGCCTTT CTCCCTTCGG  8700


 


GAAGCGTGGC GCTTTCTCAT AGCTCACGCT GTAGGTATCT CAGTTCGGTG  8750


 


TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG CACGAACCCC CCGTTCAGCC  8800


 


CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA  8850


 


GACACGACTT ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA  8900


 


GCGAGGTATG TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA  8950


 


CGGCTACACT AGAAGGACAG TATTTGGTAT CTGCGCTCTG CTGAAGCCAG  9000


 


TTACCTTCGG AAAAAGAGTT GGTAGCTCTT GATCCGGCAA ACAAACCACC  9050


 


GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA CGCGCAGAAA  9100


 


AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC  9250


 


AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA  9200


 


AGGATCTTCA CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT  9250


 


CTAAAGTATA TATGAGTAAA CTTGGTCTGA CAGTTACCAA TGCTTAATCA  9300


 


GTGAGGCACC TATCTCAGCG ATCTGTCTAT TTCGTTCATC CATAGTTGCC  9350


 


TGACTCCCCG TCGTGTAGAT AACTACGATA CGGGAGGGCT TACCATCTGG  9400


 


CCCCAGTGCT GCAATGATAC CGCGAGACCC ACGCTCACCG GCTCCAGATT  9450


 


TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT  9500


 


GCAACTTTAT CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG  9550


 


AGTAAGTAGT TCGCCAGTTA ATAGTTTGCG CAACGTTGTT GCCATTGCTA  9600


 


CAGGCATCGT GGTGTCACGC TCGTCGTTTG GTATGGCTTC ATTCAGCTCC  9650


 


GGTTCCCAAC GATCAAGGCG AGTTACATGA TCCCCCATGT TGTGCAAAAA  9700


 


AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT AAGTTGGCCG  9750


 


CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC  9800


 


ATGCCATCCG TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC  9850


 


ATTCTGAGAA TAGTGTATGC GGCGACCGAG TTGCTCTTGC CCGGCGTCAA  9900


 


TACGGGATAA TACCGCGCCA CATAGCAGAA CTTTAAAAGT GCTCATCATT  9950


 


GGAAAACGTT CTTCGGGGCG AAAACTCTCA AGGATCTTAC CGCTGTTGAG 10000


 


ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT TCAGCATCTT 10050


 


TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC 10100


 


GCAAAAAAGG GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTT 10150


 


CCTTTTTCAA TATTATTGAA GCATTTATCA GGGTTATTGT CTCATGAGCG 10200


 


GATACATaTT TGAATGTATT TAGAAAAATA AACAAATAGG GGTTCCGCGC 10250


 


ACATTTCCCC GAAAAGTGCC AC                               10272


 


SEQ ID NO: 47 pTnMCS (CMV-CHOVg-ent-Prolnsulin-synPA)


    1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga


 


   61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg


 


  121 ccacgttcgc cggcatcaga ttggctattg gccattgcat acgttgtatc catatcataa


 


  181 tatgtacatt tatattggct catgtccaac attaccgcca tgttgacatt gattattgac


 


  241 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg


 


  301 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt


 


  361 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca


 


  421 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc


 


  481 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta


 


  541 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac


 


  601 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg


 


  661 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg


 


  721 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt


 


  781 acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg


 


  841 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccgcggccg


 


  901 ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta ccgcctatag


 


  961 actctatagg cacacccctt tggctcttat gcatgctata ctgtttttgg cttggggcct


 


 1021 atacaccccc gcttccttat gctataggtg atggtatagc ttagcctata ggtgtgggtt


 


 1081 attgaccatt attgaccact cccctattgg tgacgatact ttccattact aatccataac


 


 1141 atggctcttt gccacaacta tctctattgg ctatatgcca atactctgtc cttcagagac


 


 1201 tgacacggac tctgtatttt tacaggatgg ggtcccattt attatttaca aattcacata


 


 1261 tacaacaacg ccgtcccccg tgcccgcagt ttttattaaa catagcgtgg gatctccacg


 


 1321 cgaatctcgg gtacgtgttc cggacatggg ctcttctccg gtagcggcgg agcttccaca


 


 1381 tccgagccct ggtcccatgc ctccagcggc tcatggtcgc tcggcagctc cttgctccta


 


 1441 acagtggagg ccagacttag gcacagcaca atgcccacca ccaccagtgt gccgcacaag


 


 1501 gccgtggcgg tagggtatgt gtctgaaaat gagcgtggag attgggctcg cacggctgac


 


 1561 gcagatggaa gacttaaggc agcggcagaa gaagatgcag gcagctgagt tgttgtattc


 


 1621 tgataagagt cagaggtaac tcccgttgcg gtgctgttaa cggtggaggg cagtgtagtc


 


 1681 tgagcagtac tcgttgctgc cgcgcgcgcc accagacata atagctgaca gactaacaga


 


 1741 ctgttccttt ccatgggtct tttctgcagt caccgtcgga ccatgtgcga actcgatatt


 


 1801 ttacacgact ctctttacca attctgcccc gaattacact taaaacgact caacagctta


 


 1861 acgttggctt gccacgcatt acttgactgt aaaactctca ctcttaccga acttggccgt


 


 1921 aacctgccaa ccaaagcgag aacaaaacat aacatcaaac gaatcgaccg attgttaggt


 


 1981 aatcgtcacc tccacaaaga gcgactcgct gtataccgtt ggcatgctag ctttatctgt


 


 2041 tcgggcaata cgatgcccat tgtacttgtt gactggtctg atattcgtga gcaaaaacga


 


 2101 cttatggtat tgcgagcttc agtcgcacta cacggtcgtt ctgttactct ttatgagaaa


 


 2161 gcgttcccgc tttcagagca atgttcaaag aaagctcatg accaatttct agccgacctt


 


 2221 gcgagcattc taccgagtaa caccacaccg ctcattgtca gtgatgctgg ctttaaagtg


 


 2281 ccatggtata aatccgttga gaagctgggt tggtactggt taagtcgagt aagaggaaaa


 


 2341 gtacaatatg cagacctagg agcggaaaac tggaaaccta tcagcaactt acatgatatg


 


 2401 tcatctagtc actcaaagac tttaggctat aagaggctga ctaaaagcaa tccaatctca


 


 2461 tgccaaattc tattgtataa atctcgctct aaaggccgaa aaaatcagcg ctcgacacgg


 


 2521 actcattgtc accacccgtc acctaaaatc tactcagcgt cggcaaagga gccatgggtt


 


 2581 ctagcaacta acttacctgt tgaaattcga acacccaaac aacttgttaa tatctattcg


 


 2641 aagcgaatgc agattgaaga aaccttccga gacttgaaaa gtcctgccta cggactaggc


 


 2701 ctacgccata gccgaacgag cagctcagag cgttttgata tcatgctgct aatcgccctg


 


 2761 atgcttcaac taacatgttg gcttgcgggc gttcatgctc agaaacaagg ttgggacaag


 


 2821 cacttccagg ctaacacagt cagaaatcga aacgtactct caacagttcg cttaggcatg


 


 2881 gaagttttgc ggcattctgg ctacacaata acaagggaag acttactcgt ggctgcaacc


 


 2941 ctactagctc aaaatttatt cacacatggt tacgctttgg ggaaattatg aggggatcgc


 


 3001 tctagagcga tccgggatct cgggaaaagc gttggtgacc aaaggtgcct tttatcatca


 


 3061 ctttaaaaat aaaaaacaat tactcagtgc ctgttataag cagcaattaa ttatgattga


 


 3121 tgcctacatc acaacaaaaa ctgatttaac aaatggttgg tctgccttag aaagtatatt


 


 3181 tgaacattat cttgattata ttattgataa taataaaaac cttatcccta tccaagaagt


 


 3241 gatgcctatc attggttgga atgaacttga aaaaaattag ccttgaatac attactggta


 


 3301 aggtaaacgc cattgtcagc aaattgatcc aagagaacca acttaaagct ttcctgacgg


 


 3361 aatgttaatt ctcgttgacc ctgagcactg atgaatcccc taatgatttt ggtaaaaatc


 


 3421 attaagttaa ggtggataca catcttgtca tatgatcccg gtaatgtgag ttagctcact


 


 3481 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg


 


 3541 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt


 


 3601 aaccctcact aaagggaaca aaagctggag ctccaccgcg gtggcggccg ctctagaact


 


 3661 agtggatccc ccgggcatca gattggctat tggccattgc atacgttgta tccatatcat


 


 3721 aatatgtaca tttatattgg ctcatgtcca acattaccgc catgttgaca ttgattattg


 


 3781 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc


 


 3841 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca


 


 3901 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt


 


 3961 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg


 


 4021 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag


 


 4081 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt


 


 4141 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg


 


 4201 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa


 


 4261 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt


 


 4321 gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga


 


 4381 cgccatccac gctgttttga cctccataga agacaccggg accgatccag cctccgcggc


 


 4441 cgggaacggt gcattggaac gcggattccc cgtgccaaga gtgacgtaag taccgcctat


 


 4501 agactctata ggcacacccc tttggctctt atgcatgcta tactgttttt ggcttggggc


 


 4561 ctatacaccc ccgcttcctt atgctatagg tgatggtata gcttagccta taggtgtggg


 


 4621 ttattgacca ttattgacca ctcccctatt ggtgacgata ctttccatta ctaatccata


 


 4681 acatggctct ttgccacaac tatctctatt ggctatatgc caatactctg tccttcagag


 


 4741 actgacacgg actctgtatt tttacaggat ggggtcccat ttattattta caaattcaca


 


 4801 tatacaacaa cgccgtcccc cgtgcccgca gtttttatta aacatagcgt gggatctcca


 


 4861 cgcgaatctc gggtacgtgt tccggacatg ggctcttctc cggtagcggc ggagcttcca


 


 4921 catccgagcc ctggtcccat gcctccagcg gctcatggtc gctcggcagc tccttgctcc


 


 4981 taacagtgga ggccagactt aggcacagca caatgcccac caccaccagt gtgccgcaca


 


 5041 aggccgtggc ggtagggtat gtgtctgaaa atgagcgtgg agattgggct cgcacggctg


 


 5101 acgcagatgg aagacttaag gcagcggcag aagaagatgc aggcagctga gttgttgtat


 


 5161 tctgataaga gtcagaggta actcccgttg cggtgctgtt aacggtggag ggcagtgtag


 


 5221 tctgagcagt actcgttgct gccgcgcgcg ccaccagaca taatagctga cagactaaca


 


 5281 gactgttcct ttccatgggt cttttctgca gtcaccgtcg gatcaatggg ctccatcggt


 


 5341 gcagcaagca tggaattttg ttttgatgta ttcaaggagc tcaaagtcca ccatgccaat


 


 5401 gagaacatct tctactgccc cattgccatc atgtcagctc tagccatggt atacctgggt


 


 5461 gcaaaagaca gcaccaggac acaaataaat aaggttgttc gctttgataa acttccagga


 


 5521 ttcggagaca gtattgaagc tcagtgtggc acatctgtaa acgttcactc ttcacttaga


 


 5581 gacatcctca accaaatcac caaaccaaat gatgtttatt cgttcagcct tgccagtaga


 


 5641 ctttatgctg aagagagata cccaatcctg ccagaatact tgcagtgtgt gaaggaactg


 


 5701 tatagaggag gcttggaacc tatcaacttt caaacagctg cagatcaagc cagagagctc


 


 5761 atcaattcct gggtagaaag tcagacaaat ggaattatca gaaatgtcct tcagccaagc


 


 5821 tccgtggatt ctcaaactgc aatggttctg gttaatgcca ttgtcttcaa aggactgtgg


 


 5881 gagaaagcat ttaaggatga agacacacaa gcaatgcctt tcagagtgac tgagcaagaa


 


 5941 agcaaacctg tgcagatgat gtaccagatt ggtttattta gagtggcatc aatggcttct


 


 6001 gagaaaatga agatcctgga gcttccattt gccagtggga caatgagcat gttggtgctg


 


 6061 ttgcctgatg aagtctcagg ccttgagcag cttgagagta taatcaactt tgaaaaactg


 


 6121 actgaatgga ccagttctaa tgttatggaa gagagaagat caaagtgtac ttacctcgca


 


 6181 tgaagatgga ggaaaaatac aacctcacat ctgtcttaat ggctatgggc attactgacg


 


 6241 tgtttagctc ttcagccaat ctgtctggca tctcctcagc agagagcctg aagatatctc


 


 6301 aagctgtcca tgcagcacat gcagaaatca atgaagcagg cagagaggtg gtagggtcag


 


 6361 cagaggctgg agtggatgct gcaagcgtct ctgaagaatt tagggctgac catccattcc


 


 6421 tcttctgtat caagcacatc gcaaccaacg ccgttctctt cttttggcag atgtgtttcc


 


 6481 cgcggccagc agatgacgca ccagcagatg acgcaccagc agatgacgca ccagcagatg


 


 6541 acgcaccagc agatgacgca acaacatgta tcctgaaagg ctcttgtggc tggatcggcc


 


 6601 tgctggatga cgatgacaaa tttgtgaacc aacacctgtg cggctcacac ctggtggaag


 


 6661 ctctctacct agtgtgcggg gaacgaggct tcttctacac acccaagacc cgccgggagg


 


 6721 cagaggacct gcaggtgggg caggtggagc tgggcggggg ccctggtgca ggcagcctgc


 


 6781 agcccttggc cctggagggg tccctgcaga agcgtggcat tgtggaacaa tgctgtacca


 


 6841 gcatctgctc cctctaccag ctggagaact actgcaacta gggcgcctaa agggcgaatt


 


 6901 atcgcggccg ctctagacca ggcgcctgga tccagatcac ttctggctaa taaaagatca


 


 6961 gagctctaga gatctgtgtg ttggtttttt gtggatctgc tgtgccttct agttgccagc


 


 7021 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg


 


 7081 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc


 


 7141 tggggggtgg ggtggggcag cacagcaagg gggaggattg ggaagacaat agcaggcatg


 


 7201 ctggggatgc ggtgggctct atgggtacct ctctctctct ctctctctct ctcactctct


 


 7261 ctctctctcg gtacctctcc tcgagggggg gcccggtacc caattcgccc tatagtgagt


 


 7321 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg


 


 7381 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag


 


 7441 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggaaattgt


 


 7501 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa


 


 7561 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt


 


 7621 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa


 


 7681 agggcgaaaa accgtctatc agggcgatgg cccactactc cgggatcata tgacaagatg


 


 7741 tgtatccacc ttaacttaat gatttttacc aaaatcatta ggggattcat cagtgctcag


 


 7801 ggtcaacgag aattaacatt ccgtcaggaa agcttatgat gatgatgtgc ttaaaaactt


 


 7861 actcaatggc tggttatgca tatcgcaata catgcgaaaa acctaaaaga gcttgccgat


 


 7921 aaaaaaggcc aatttattgc tatttaccgc ggctttttat tgagcttgaa agataaataa


 


 7981 aatagatagg ttttatttga agctaaatct tctttatcgt aaaaaatgcc ctcttgggtt


 


 8041 atcaagaggg tcattatatt tcgcggaata acatcatttg gtgacgaaat aactaagcac


 


 8101 ttgtctcctg tttactcccc tgagcttgag gggttaacat gaaggtcatc gatagcagga


 


 8161 taataataca gtaaaacgct aaaccaataa tccaaatcca gccatcccaa attggtagtg


 


 8221 aatgattata aataacagca aacagtaatg ggccaataac accggttgca ttggtaaggc


 


 8281 tcaccaataa tccctgtaaa gcaccttgct gatgactctt tgtttggata gacatcactc


 


 8341 cctgtaatgc aggtaaagcg atcccaccac cagccaataa aattaaaaca gggaaaacta


 


 8401 accaaccttc agatataaac gctaaaaagg caaatgcact actatctgca ataaatccga


 


 8461 gcagtactgc cgttttttcg cccatttagt ggctattctt cctgccacaa aggcttggaa


 


 8521 tactgagtgt aaaagaccaa gacccgtaat gaaaagccaa ccatcatgct attcatcatc


 


 8581 acgatttctg taatagcacc acaccgtgct ggattggcta tcaatgcgct gaaataataa


 


 8641 tcaacaaatg gcatcgttaa ataagtgatg tataccgatc agcttttgtt ccctttagtg


 


 8701 agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta


 


 8761 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc


 


 8821 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg


 


 8881 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg


 


 8941 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg


 


 9001 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa


 


 9061 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc


 


 9121 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc


 


 9181 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag


 


 9241 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct


 


 9301 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta


 


 9361 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc


 


 9421 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggC


 


 9481 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt


 


 9541 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct


 


 9601 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc


 


 9661 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca


 


 9721 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta


 


 9781 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa


 


 9841 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg


 


 9901 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg


 


 9961 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc


 


10021 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc


 


10081 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa


 


10141 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc


 


10201 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg


 


10261 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc


 


10321 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat


 


10381 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg


 


10441 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc


 


10501 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg


 


10561 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat


 


10621 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg


 


10681 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg


 


10741 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct


 


10801 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac


 


10861 atttccccga aaagtgccac


 


SEQ ID NO: 48 (cecropin prepro)


AAT TTC TCA AGG ATA TTT


 


TTC TTC GTG TTC GCT TTG


 


GTT CTG GCT TTG TCA ACA


 


GTT TCG GCT GCG CCA GAG


 


CCG AAA


 


SEQ ID NO: 49 (cecropin prepro extended)


AAT TTC TCA AGG ATA TTT


 


TTC TTC GTG TTC GCT TTG


 


GTT CTG GCT TTG TCA ACA


 


GTT TCG GCT GCG CCA GAG


 


CCG AAA TGG AAA GTC TTC


 


AAG


 


SEQ ID NO: 50 (cecropin pro)


GCG CCA GAG CCG AAA


 


SEQ ID NO: 51 (cecropin pro extended)


GCG CCA GAG CCG AAA TGG AAA GTC TTC AAG


 


SEQ ID NO: 52 (a Kozak sequence)


ACCATGT

## Claims

1. A method of producing a transgenic chicken or quail comprising, administering to an artery leading to the ovary of a chicken or quail a composition comprising a transposon-based vector, wherein the transposon-based vector comprises a prokaryotic transposase gene operably linked to a first promoter, a modified Kozak sequence positioned 3′ to the first promoter so as to encode for at least the first codon of the transposase gene, and one or more genes of interest operably-linked to one or more additional promoters, wherein a plurality of the first twenty codons of the transposase gene are modified from the wild-type sequence by changing the nucleotide at the third base position of the codon to an adenine or thymine without modifying the amino acid encoded by the codon, and wherein the one or more genes of interest and their operably-linked promoters are flanked by transposase insertion sequences recognized by a transposase encoded by the transposase gene, such that the one or more genes of interest and their operably linked promoters and transposase insertion sequences are incorporated into a germ-line cell of the chicken or quail.

2. The method of claim 1, wherein the transposase gene is operably linked to two stop codons.

3. The method of claim 1, wherein the composition is administered to the chicken between approximately 14 and 16 weeks of age.

4. The method of claim 1, wherein the composition is administered to the quail between approximately 5 and 6 weeks of age.

5. The method of claim 1, wherein the transposon-based vector comprises an avian optimized polyA sequence operably linked to the transposase gene.

6. The method of claim 5, wherein the avian optimized polyA sequence comprises between approximately 40 and 100 nucleotides that precede a polyA nucleotide string.

7. The method of claim 1, wherein the transposase is a Tn10 transposase.

8. The method of claim 1, wherein the first promoter is a constitutive promoter.

9. The method of claim 1, wherein the first promoter is an oviduct-specific promoter selected from the group consisting of ovalbumin, ovotransferrin, ovomucoid, ovomucin, g2 ovoglobulin, g3 ovoglobulin, ovoflavoprotein, and ovostatin.

10. The method of claim 1, wherein the first promoter is an ovalbumin promoter having the sequence as set forth in SEQ ID NO: 15 or nucleic acids 4050-4938 of SEQ ID NO: 44.

11. The method of claim 1, wherein the one or more genes of interest are operably-linked to a second promoter.

12. The method of claim 11, wherein the second promoter is an oviduct-specific promoter selected from the group consisting of ovalbumin, ovotransferrin, ovomucoid, ovomucin, g2 ovoglobulin, g3 ovoglobulin, ovoflavoprotein, and ovostatin.

13. The method of claim 11, wherein the second promoter is an ovalbumin promoter having the sequence as set forth in SEQ ID NO: 15 or nucleic acids 4050-4938 of SEQ ID NO: 44.

14. The method of claim 1, wherein the transposon-based vector further comprises an enhancer operably-linked to the one or more genes of interest.

15. The method of claim 14, wherein the enhancer comprises at least a portion of an ovalbumin enhancer.

16. The method of claim 1, wherein the transposon-based vector further comprises an egg directing sequence comprising an ovalbumin signal sequence or vitellogenin signal sequence operably-linked to the one or more genes of interest.

17. The method of claim 16, wherein the egg directing sequence is an ovalbumin signal sequence as set forth in SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21.