MX2008003923A - Dna modular cloning vector plasmids and methods for their use - Google Patents

Abstract

Shortened abstract A group of modular cloning vector plasmids for the synthesis of transgenes or other complicated DNA constructs The invention is useful for assembling a variety of DNA fragments into a deÏÇovo DNA construct by using cloning vectors optimized to reduce the amount of manipulation needed The module vector contains at least one multiple cloning site and multiple sets of rare restriction and/or unique homing endonuclease ("H") sites, arranged in a linear pattern This arrangement defines a modular architecture that allows the user to place domain modules or inserts into a PE3 transgene vector construct without disturbing the integrity of DNA elements already incorporated into the PE3 vector in previous cloning steps The PE3 transgenes produced using the invention may be used in a vaÏÇety of organisms including bacteria, yeast, mice, and other eukaryotes with little or no further modification

Description

PLASMIDES MODULAR CLONING VECTORS OF DNA AND METHODS FOR USE FIELD OF THE INVENTION The present invention relates to the field of plasmid cloning vectors, and to the use of plasmid cloning vectors to make DNA or transgene constructs.

BACKGROUND OF THE INVENTION The basis of molecular biology is recombinant DNA technology, which can here be summarized as the modification and propagation of nucleic acids for the purpose of studying the structure and function of nucleic acids and their protein products. The individual genes, gene regulatory regions, subgroups of genes and of course the compound chromosomes in which they are contained are all comprised of antiparallel double-stranded sequences of the nucleotides adenine, thymine, guanine and cytosine, conventionally identified by the initials A, T, G and C, respectively. These DNA sequences, as well as the cDNA sequences which are copies of double-stranded DNA derived from mRNA (messenger RNA), can be excised in different fragments, isolated and inserted within a REF: 191362 vector such as a bacterial plasmid for study gene products. A plasmid is a chromosomal piece of DNA that was originally derived from bacteria, and can be manipulated and reintroduced into a host bacterium for purposes of study or production of a gene product. The DNA of a plasmid is similar to all chromosomal DNA, since it is composed of the same nucleotides A, T, G and C that code for genes and regulatory regions of genes, however, this is a relatively small molecule comprised of less than about 30,000 base pairs, or 30 kilobases (kb). In addition, the nucleotide base pairs of a double-stranded plasmid form a continuous circular molecule, also distinguishing the plasmid DNA from that of the chromosomal DNA. Plasmids increase the rapid exchange of genetic material between bacterial organisms and allow rapid adaptation to changes in the environment, such as temperature, food supply and other challenges. Any acquired plasmid must express a gene or genes that contribute to the survival of the host or it will also be destroyed or discarded by the organism, since the maintenance of unnecessary plasmids could be a wasted use of resources. A clonal population of cells contains identical genetic material, including any plasmids that it can harbor. The use of a plasmid cloning vector with a DNA insert in such a clonal population of host cells will amplify the amount of available DNA of interest. The DNA thus cloned can then be isolated and recovered for the next manipulation in the steps required for the construction of a DNA construct. In this way, it can be seen that plasmid cloning vectors are useful tools in the study of the function of genes, providing the ability to quickly produce large amounts of the DNA insert of interest. While some elements found in plasmids are naturally occurring, others have been engineered to increase the utility of plasmids as DNA vectors. These include genes for resistance to antibiotics or chemicals and a multiple cloning site (MCS), among others. Each of these elements has a role in the present invention, as well as in the prior art. The description of the role that each element plays will reveal the limitations of the prior art and will demonstrate the utility of the present invention. A plasmid-borne gene, particularly useful, which can be acquired by a host, is one that could confer resistance to antibiotics. In the daily practice of recombinant DNA technology, antibiotic resistance genes are exploited as positive or negative selection elements to preferentially increase the culture and amplification of the desired plasmid over that of the other plasmids. In order to be maintained by a host bacterium, a plasmid must also contain a segment of sequences directing the host to duplicate the plasmid. The sequences known as the origin of the replication element (ORI) direct the host to use its cellular enzymes to make copies of plasmids. When such a bacterium divides, the daughter cells each will retain a copy or copies of any such plasmid. Certain strains of E. coli bacteria have been derived to maximize this duplication, producing more than 300 copies per bacterium. In this way, the culture of a desired plasmid can be increased. Another essential element in any cloning vector is a site for the insertion of genetic materials of interest. This is a synthetic element that has been engineered within the "wild type" plasmids thus conferring utility as a cloning vector. Any typical cloning vector plasmid, commercially available, contains at least one such region, known as a multiple cloning site (MCS). An MCS typically comprises nucleotide sequences that can be cleaved by a single or a series of restriction endonuclease enzymes (hereinafter referred to as "restriction enzymes"), each of which has a different endonuclease restriction site and a different excision pattern. These endonuclease sites or endonuclease restriction sites (hereinafter referred to as "restriction sites") encoded in the DNA molecule typically comprise a double-stranded palindromic sequence. For some restriction enzymes, as few as 4-6 nucleotides are sufficient to provide a restriction site, while some restriction enzymes require a restriction site of 8 or more nucleotides. The EcoRI enzyme, for example, recognizes the hexanucleotide sequence: 5 'GAATT-C3', where 5 'indicates the end of the molecule known by convention as the "upstream" end or with the 5' and 3 ' same mode indicates the end "with direction down" or with direction 3 '. The complementary strand of the restriction site could be its antiparallel strand, 3 'G-A-A-T-T-C-5'. In this way, the double-stranded restriction site can be represented within the larger double-stranded molecule in which it appears as: 5 'GAATTC 3' 3 'CTTAAG 5' Like many other restriction enzymes, EcoRI, does not breaks exactly in the axis of symmetry of the dyad, but positions with four nucleotides of separation in the two strands of DNA between the nucleotides indicated by a "/": 5 'G / AATTC 3' 3 'CTTAA / G 5' such that the double-stranded DNA molecule is cleaved and has the resulting configuration of the nucleotides at the newly formed "ends": 5 'G3' 5'AATTC 3 '3' CTTA-A5 '3'G 5' This cleavage in stages or staggered produces DNA fragments with protruding 5 'ends. Because pairs A-T and G-C are spontaneously formed when in close proximity to each other, protruding ends such as these are called cohesive or "sticky" ends. Any of these ends can form hydrogen bonds with any other complementary ends cleaved with the same restriction enzyme Since any DNA containing a specific restriction site will be cut in the same way as any other DNA containing the same sequence, those ends splits will be complementary. Therefore, the ends of any DNA molecules cut with the same restriction enzyme "agree or mate" with one another in the way that the adjacent pieces of a "puzzle" are coupled, and can be enzymatically linked between yes. It is this property that allows the formation of recombinant DNA molecules, and allows the introduction of foreign DNA fragments into bacterial plasmids, or within any other DNA molecule. An additional general principle to consider when constructing recombinant DNA molecules is that all restriction sites that appear within a molecule will be cut with a particular restriction enzyme, not just the site of interest. The larger a DNA molecule is, the more likely it is that any restriction site will reappear. Assuming that any restriction sites are randomly distributed along a DNA molecule, a tetranucleotide site will appear, on average, once every 44 (for example, 256) nucleotides, while a hexanucleotide site will appear once every 46 (eg, 4096) nucleotides, and octanucleotide sites will appear once every 48 (eg, 114,688) nucleotides. In this way, it can easily be seen that the shorter restriction sites will appear frequently, while the longer ones will rarely appear. When the construction of transgene or another recombinant DNA molecule is planned, this is a vital problem, since such a project often requires the assembly of various pieces of DNA of various sizes. The larger these pieces are, the greater the probability that the sites you want to use will appear in several pieces of the DNA components, making the manipulation difficult. The restriction sites that frequently appear are referred to herein as common restriction sites and the endonucleases that break these sites are referred to as common restriction enzymes. Restriction enzymes with cognate sequences greater than 6 nucleotides are termed as rare restriction enzymes, and their cognate sites as rare restriction sites. However, there are some restriction sites of 6 base pairs that appear more infrequently than what would be statistically predicted, and these sites and the endonucleases that break them are also referred to as rare. Thus, the designations "rare" and "common" do not refer to the abundance or relative availability of any particular restriction enzyme, but rather to the frequency of occurrence of the nucleotide sequence that constitutes their cognate restriction site within of any DNA molecule or fragment isolated from a DNA molecule, or any gene or its DNA sequence. A second class of restriction enzyme has recently been isolated called self-directed endonuclease (HE) enzymes. HE enzymes have large asymmetric restriction enzymes (12-40 base pairs). The HE restriction sites are extremely rare. For example, the HE known as I-Scel has a restriction site of 18 base pairs (5 '... AGGGATAACAGGGTAAT ... 3'), which is predicted appears only once every 7x10,0 pairs of bases of random sequence. This rate of occurrence is equivalent to only one site in 20 mammalian-sized genomes. The rare nature of HE sites greatly increases the possibility that a genetic engineer could cut a final transgenic product without disturbing the integrity of the transgene if HE sites were included in appropriate positions in a plasmid cloning vector. Since a DNA molecule from any source organism will be cut in an identical manner by its cognate restriction enzyme, foreign pieces of DNA from any species can be cut by a restriction enzyme, inserted into a bacterial plasmid vector that was cleaved with the same restriction enzyme, and amplified in a suitable host cell. For example, a human gene can be cut at two sites with a restriction enzyme known as EcoRI and the desired fragment with the EcoRI ends can be isolated and mixed with a plasmid that was also cut with EcoRI, in what is commonly known as a ligation reaction or ligation mixture. Under the appropriate conditions in the ligation mixture, some of the isolated human gene fragments will couple with the ends of the plasmid molecules. These freshly bound ends can be linked together and the plasmid recircularized, now containing its new DNA insert. The ligation mixture is then introduced into E. coli or another suitable host, and the newly engineered plasmids will be amplified as the bacteria divide. In this way, a relatively large number of copies of the human gene can be obtained and harvested from the bacteria. These copies of genes can then be later manipulated for the purpose of research, analysis or production of their gene product protein. The recombinant DNA technology is frequently incarnated in the generation of the so-called "transgenes". Transgenes frequently comprise a variety of genetic materials that are derived from one or more donor organisms and introduced into a host organism. Typically, a transgene is constructed using a cloning vector as the starting point or "backbone" of the project, and a series of complex cloning steps are planned to assemble the final product within that vector. The elements of a transgene, comprising the nucleotide sequences, include, but are not limited to 1) regulatory and / or regulatory enhancing elements, 2) a gene that will be expressed as a mRNA molecule, 3) DNA elements that provide stabilization of the mRNA message, 4) nucleotide sequences that mimic the mammalian intronic gene regions, and 5) the signals for mRNA processing such as the poly-A tail added to the end of the naturally occurring mRNA. In some cases, an experimental design may require the addition of a localization signal to provide transport of the gene product to a particular subcellular site. Each of these elements is a fragment of a larger DNA molecule that is cut from a donor genome or, in some cases, synthesized in a laboratory. Each piece is assembled with the others in a precise order in a 5 '-3' orientation within a plasmid cloning vector. The promoter of any gene can be isolated as a DNA fragment and placed within a synthetic molecule, such as a plasmid, to direct the expression of a desired gene, assuming that the conditions necessary for the stimulation of the promoter of interest can be provided. . For example, the promoter sequences of the insulin gene can be isolated, placed in a plasmid cloning vector together with a reporter gene, and used to study the conditions required for the expression of the insulin gene in an appropriate cell type. Alternatively, the insulin gene promoter can be linked to the coding sequence of the protein encoding protein, of any gene of interest, in a plasmid cloning vector, used to drive the expression of the gene of interest in cells that express insulin, assuming that all the necessary elements are present within the transgene of DNA thus constructed. A reporter gene is a particularly useful component of some types of transgenes. A reporter gene contains the nucleotide sequences that code for a protein that will be expressed under the direction of a particular promoter of interest to which it is linked in a transgene, providing a measurable biochemical response of the promoter activity. A reporter gene is typically easy to detect or measure against the background of endogenous cellular proteins. Reporter genes commonly used include, but are not limited to, LacZ, the green fluorescent protein, and luciferase, and other reporter genes, many of which are well known to those skilled in the art. Introns, which are non-coding regions within mammalian genes, are not found in bacterial genomes, but are required for the proper formation of mRNA molecules in mammalian cells. Therefore, any DNA construct for use in mammalian systems must have at least one intron. The introns can be isolated from any mammalian gene and inserted into a DNA construct, together with the appropriate splicing signals that allow mammalian cells to remove the intron and join the remnant mRNA ends to each other. A stabilization element of mRNA is a DNA sequence that is recognized by the binding proteins that protect some mRNAs from degradation. The inclusion of a mRNA stabilizing element will often increase the level of mRNA gene expression in some types of mammalian cells and thus may also be useful in some DNA constructs or in transgenes. A stabilization element of the mRNA can be isolated from DNA or RNA of natural origin, or synthetically produced for inclusion in a DNA construct. A localization signal is a DNA sequence that codes for a protein signal for the subcellular routing of a protein of interest. For example, a nuclear signaling signal will direct a protein to the nucleus; a localization signal in the plasma membrane will direct it to the plasma membrane, etc. In this way, a localization signal can be incorporated in a DNA construct to promote the translocation of its protein product to the desired subcellular position. A marker sequence can be encoded in a DNA construct so that the protein product will have a unique bound region. This unique region serves as a protein marker that can distinguish it from its endogenous counterpart. Alternatively, this may serve as an identifier which can be detected by a wide variety of techniques well known in the art, including but not limited to, reverse transcriptase polymerase chain reaction (RT-PCR). , immunohistochemistry, or in situ hybridization. With a complex transgene, or with one that includes particularly large regions of DNA, there is an increased likelihood that multiple restriction sites will exist in these pieces of DNA. Recall that the restriction sites that code for any hexanucleotide site appear every 4096 base pairs (46). If a promoter sequence is 3000 base pairs and a gene of interest of 1500 base pairs is to be assembled within a cloning vector of 3000 base pairs, it is statistically very likely that many sites of 6 or less nucleotides will not be useful, since any usable sites should appear only in two of the pieces. In addition, the sites may appear in the appropriate areas of the appropriate molecules to be assembled. In addition, most cloning projects will need to have additional DNA elements added, which increases the complexity of the growing molecule and the probability of inopportune repetition of any particular site. Since any restriction enzyme will cut all of its restriction sites in a DNA molecule, if a restriction enzyme site reappears, all unwelcome sites will be cut along with the desired sites, disrupting the integrity of the molecule. In this way, each cloning step must be carefully planned so as not to disturb the growing molecule by cutting it with a restriction enzyme that has already been used to incorporate a preceding element. Finally, when a researcher wishes to introduce a completed transgene within a mammalian organism, the construction of the fully assembled transgene frequently must be linearized at a single restriction site at least at one end of the transgene, thus requiring another unique site found at another site in the construction. Since most DNA constructs are designed for a simple purpose, little appreciation is given to any future modifications that may need to be made, further increasing the difficulty for future experimental changes. Traditionally, transgene design and construction consume significant amounts of time and energy for several reasons, including the following: 1. There is a wide variety of restriction enzymes and HE available that will generate an array of extremes; however, most of these are not compatible with each other. Many restriction enzymes, such as EcoRI, generate DNA fragments with outstanding 5 'cohesive ends or "tails"; others (for example, PstI) generate fragments with 3 'protruding tails, while others (for example, Ball) break in the axis of symmetry to produce blunt end fragments. Some of these will be compatible with the ends formed by cleavage with other restriction enzymes and / or HE, but most of the tools will not be. The ends that can be generated with each isolation procedure of the DNA fragment must be carefully considered in the design of a DNA construct. 2. The DNA fragments necessary for the assembly of a DNA or transgene construct must first be isolated from their source genomes placed within plasmid cloning vectors, and amplified to obtain useful amounts. The step can be performed using any number of commercially available cloning vectors, or individually altered. Each of the different commercially available cloning plasmid vectors were, for the most part independently related, and thus contain different sequences and restriction sites for the DNA fragments of the genes or genetic elements of interest. The genes must therefore be individually tailored to suit each of these vectors as necessary for any given group of experiments. The same DNA fragments will often need to be further altered for subsequent experiments or cloning into other combinations for new DNA constructs or transgenes. Since each DNA or transgene construct is routinely made for a particular application without consideration or knowledge of how it will be used immediately, it must often be "backward" for subsequent applications. 3. In addition, the DNA sequence of any given gene or genetic element varies and may contain internal restriction sites that make it incompatible with currently available vectors, thereby complicating manipulation. This is especially true when several DNA fragments are assembled within a single DNA or transgene construct.

While restriction enzymes can be used to manipulate genetic material, it is known that an MCS or other components of a transgene can be created by de novo synthesis, by recombination and / or cloning of PCR terminator. Such a method of synthesis of the component elements of a transgene includes the method described by Jarret in U.S. Patent No. 6,358,712. While Jarret describes a method for "welding" the elements of a transgene to each other, the prior art does not teach a method or means to "desolder" and reassemble these elements, once they have been assembled. Therefore, it would be advantageous to provide a means to assemble each component of one transgene with the others in a precise order and a 5'-3 'orientation within a cloning vector plasmid. There is also a need for a system that could allow the user to rapidly assemble a number of DNA fragments within a molecule, despite a redundancy of the restriction sites found at the ends, and within each of these DNA fragments. . It would also be useful to provide a simple means for rapidly altering the ends of the selected DNA fragments, in order to add restriction sites thereto. The inclusion of single or opposite pairs of HE restriction sites could increase the possibility of having the unique sites for cloning. A system that could also allow easy substitutions or the removal of one or more of the fragments would add a level of versatility not currently available to users. Thus, a "modular" system that allows someone to insert or remove DNA fragments into or out of the "cassette" regions flanked by rare, fixed restriction sites within the cloning vector would be especially useful, and welcome to the field of recombinant DNA technology. Accordingly, an aspect of the present invention is to provide a method for assembling rapidly for a DNA construct or a transgene by the use of a plasmid cloning vector. Yet another aspect is to incorporate DNA fragments, also known as "inserts" or "modules" such as a promoter, expression and a 3 'regulatory nucleotide sequence, into the cloning vector plasmid, in a sequential manner.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a structure and methods for creating modular cloning vectors for the synthesis of a genetic domain module, a PE3 transgene, and other complicated DNA constructs, by providing a spinal column within modular cloning vectors having dedicated coupling points known as gene pivots in the present. The present invention relates to a domain module coupling vector, which consists of a DNA cloning vector comprising a multiple cloning mode (MC) for the subcloning of a genetic material of interest within the MC module, comprising the MC module: a) a first gene pivot (GP) comprising at least two rare, non-variable restriction sites operable to define the 5 'portion of the MC module; b) a nucleic acid sequence comprising a multiple cloning site (MCS) that includes a plurality of restriction sites selected from the common restriction sites that are unique within the coupling vector of the domain module, to provide cloning sites for the cloning of the genetic material of interest within the MC module; and c) a second gene pivot comprising at least two rare, non-variable restriction sites operable to define the 3 'position of the MC module. The present invention also relates to a domain module vector consisting of a DNA cloning vector, comprising a domain module that includes: a) a first gene pivot comprising at least two rare, non-variable restriction sites , operable to define the 5 'portion of the domain module; b) a genetic module of interest consisting of a nucleic acid sequence comprising a genetic material of interest; and c) a second gene pivot comprising at least two rare, non-variable restriction sites operable to define a 3 'portion of the domain module. The present invention also relates to a PE3 coupling vector consisting of a DNA cloning vector, comprising a cloning module of PE3 including at least one cloning module, configured to clone at least a first domain module within the cloning module PE3, the PE3 cloning module comprises: a) a first gene pivot that includes at least two rare, non-variable restriction sites, which after cloning is operable to define the 5 'portion of at least the first domain module; b) a filler module consisting of a first nucleic acid sequence comprising the filler, which after cloning is replaced by the first domain module; and c) a second gene pivot comprising at least two rare, non-variable restriction sites which, after cloning, is operable to define the 3 'portion of at least the first domain module. The present invention also relates to a coupling vector PE3 consisting of a DNA cloning vector, comprising a cloning module PE3 including a plurality of cloning modules configured to clone a plurality of domain modules within the module of cloning. cloning PE3, the PE3 cloning module comprises: a) a first gene pivot that includes at least two rare, non-variable restriction sites that after cloning is operable to define the 5 'portion of a first domain module; b) a first filler module consisting of a first nucleic acid sequence comprising the filler, which after cloning is replaced by the first domain module; c) a second gene pivot comprising at least two rare, non-variable restriction sites, which after cloning is operable to define a shared junction between the 3 'portion of the first domain module and the 5' portion of a second module Of domain; d) a second filler module consisting of a second nucleic acid sequence comprising the filler, which after cloning is replaced by the second domain module; e) a third gene pivot comprising at least two rare, non-variable restriction sites, which after cloning is operable to define a shared junction between the 3 'portion of the second domain module and the 5' portion of a third module Of domain; f) a third filler module consisting of a third nucleic acid sequence comprising the filler, which after cloning is replaced by the third domain module; and g) a fourth gene pivot comprising at least two rare, non-variable restriction sites, which after cloning is operable to define the 3 'portion of the third domain module. The present invention further relates to a multiple cloning coupling vector (MC) PE3 consisting of a DNA cloning vector that includes the cloning module PE3 configured to clear at least three domain modules within the cloning module PE3, the PE3 cloning module comprises: a) a first gene pivot including at least two rare, non-variable restriction sites, which after cloning is operable to define the 5 'portion of the first domain module; b) a first nucleic acid sequence; c) a second gene pivot comprising at least rare, non-variable restriction sites, which after cloning is operable to define a shared junction between the 3 'portion of the first domain module and the 5' portion of the second domain module; d) a second nucleic acid sequence; e) a third gene pivot comprising at least two rare, non-variable restriction sites, which after cloning is operable to define a shared junction between the 3 'portion and the second domain module and the 5' portion of the third module Of domain; f) a third nucleic acid sequence; and g) a fourth gene pivot comprising at least two rare, non-variable restriction sites, which after cloning is operable to define a shared junction between the 3 'portion of the third domain module; wherein at least one of the first, second and third nucleic acid sequences is a multiple cloning module containing a multiple cloning site (MCS) comprising a plurality of restriction sites selected from the common restriction sites that are unique within the coupling vector of PE3, to provide the cloning sites for the cloning of the genetic material of interest within the multiple cloning module, and the remaining nucleic acid sequences are filled-in sequences. The present invention further relates to a PE3 vector consisting of a DNA cloning vector, comprising a PE3 module comprising a promoter module, an expression module and a 3 'regulatory module, the PE3 module comprising: a) a first gene pivot comprising at least two rare, non-variable restriction sites operable to define the 5 'portion of a promoter module; b) the promoter module; c) a second gene pivot comprising at least two rare, non-variable restriction sites operable to define a shared junction between the 3 'portion of the promoter module and the 5' portion of an expression module; e) the expression module; e) a third gene pivot comprising at least two rare, non-variable restriction sites operable to define a shared junction between the 3 'portion of the expression module and the 5' portion of the regulatory module 3 '; f) the regulatory module 3 '; and g) a fourth gene pivot comprising at least two rare, non-variable restriction sites operable to define a 3 'portion of regulatory module 3'. The present invention also provides that a PE3 coupling vector, the PE3 vector and the PE3 (MC) multiple cloning splice vector, can comprise a means for the release of the PE3 module typically comprising the promoter domain, domain modules. of Expression and 3 'Regulator Domain flanked by gene pivots, from the PE3 vector for insertion into a multigenic coupling vector. A means for releasing and inserting by means of cloning PE3 comprises a pair of self-directed endonucleases (HE) placed in the PE3 coupling vector or the coupling vector MC of PE3, flanking the nucleic acid sequences comprising the cloning module PE3. After insertion or cloning of the promoter domain, the expression domain and the 3 'regulatory domain within the cloning module of PE3, the HE pair provides a means of removing the PE3 module from the PE3 vector for insertion into the In a compatible coupling position, within the multigenic vector, the compatible coupling position is typically defined by the same HE pair. While any self-directed endonucleases can be used either in the first position (at the 5 'end of the excised PE3 module) or the second position (at the 3' end of the excised PE3 module), a preferred HE pair consists of an I- Ceu I in the first position, and ISce-I invested in the second position. In the other embodiments of the coupling coupling vector of PE3 or the coupling vector MC of PE3, a different removal and insertion means may be used instead of HE. Such a different excision or insertion means may include a pair one or more rare, non-variable restriction sites, including a different pair of rare restriction sites from those in the gene pivots or the flanking gene pivots (GP1 and GP4). An insert means employs recombination, discussed below in the present. The present invention provides a method for constructing a modular vector PE3 comprising the steps of: a) providing a cloning vector VE3 comprising a cloning module of PE3, the cloning mode of PE3 comprising a cloning sequence; a first gene pivot comprising at least two rare, non-variable restriction sites, at least one first filler module consisting of a nucleic acid sequence comprising the filler and a second gene pivot; b) the provision of at least one domain module vector comprising in sequence: the first gene pivot, a genetic module of interest consisting of a nucleic acid sequence comprising a genetic material of interest; and the second gene pivot; c) that provides a cognate restriction enzyme for one of the expensive restriction sites of the first gene pivot, and a second restriction enzyme cognate for one of the rare restriction sites of the second gene pivot; d) excising and isolating the genetic mode of interest from the first domain module vector using the first and second cognate restriction enzymes; e) removing the first filler module from the PE3 cloning module of the PE3 cloning vector using the first and second coagulated restriction enzymes; and f) ligation of the genetic module of interest of the PE3 cloning module. The method also provides for the insertion of a second genetic module of interest into PE3 using a third gene pivot and the constrained restriction enzymes to excise and ligate the second genetic module of interest within the PE3 cloning module. Similarly, a third genetic module of interest can be inserted into PE3 using a third gene pivot and cognate restriction enzymes, to excise and ligate the second genetic module of interest within the PE3 cloning module. The method provides a sequential arrangement of genetic modules of interest within the cloning vector of PE3. Typically, the first, second and third genetic modules of interest correspond to a promoter module, the expression module and a 3 'regulatory module, respectively. Yet another embodiment of the invention is a method for making a transgene comprising the steps of providing a plasmid cloning vector that includes a backbone or backbone, the backbone has at least one first, one second, a third and a fourth coupling points, the coupling points are arranged sequentially in a 5 '-3' direction and each has at least one rare, non-variable restriction site operable to be cleaved by a restriction enzyme, cleaving the first coupling point with a first restriction enzyme corresponding to one of at least one rare, non-variable restriction site of the first coupling point, leaving the first coupling site cleaved with an exposed 3 'end, cleaving the second coupling point with a second restriction enzyme that corresponds to at least one rare restriction site, not variable from the second coupling point, leaving the A cleavage coupling point with an exposed 5 'end, providing a first insert comprising a 5' end, a nucleotide sequence of interest, and a 3 'end, wherein the 5' end of the first insert is compatible to the 3 'end. exposed from the first cleaved coupling point, and the 3 'end of the first insert is compatible to the exposed 5' end of the second cleaved coupling point, and by placing the first insert and the cleaved cloning vector plasmid, into an appropriate reaction mixture to cause ligation and self-orientation of the first insert within the spinal column between the first coupling point and the second coupling point, where the spinal column is reassembled. In this embodiment, the second coupling point can be subsequently cleaved with the second restriction enzyme, leaving the second coupling point cleaved with an exposed 3 'end, the third coupling point can be excised with a third restriction enzyme that corresponds at least a rare restriction site, not variable of the third coupling point, leaving the third coupling point excised with an exposed 5 'end, followed by the steps of providing a second insert comprising a 5' end, a nucleotide sequence of interest and a 3 'end, wherein the 5 'end of the second insert is compatible to the exposed 3' end of the second cleavage coupling point, and the 3 'end of the second insert is compatible with the exposed 5' end of the third cleavage coupling point, and placing the second insert and the cleaved cloning vector plasmid into an appropriate reaction mixture to cause ligation and self-orientation of the second insert within the spinal column between the second attachment point and the third attachment point, wherein the vertebral column is reassembled In addition, this embodiment may include the steps of subsequently excising the backbone or backbone at the third attachment point with the third restriction enzyme, leaving the third attachment site excised with an exposed 3 'end, excising the fourth attachment point. with a fourth restriction enzyme corresponding to one of at least one rare, non-variable restriction site of the fourth coupling point, leaving the fourth coupling site excised with an exposed 5 'end, providing a third insert comprising one end ', a nucleotide sequence of interest, and a 3' end, wherein the 5 'end of the third insert is compatible with the exposed 3' end of the third cleavage coupling point, and the 3 'end of the third insert is compatible with the end 5 'exposed from the fourth cleavage coupling point, and placing the third insert and the cleaved cloning vector plasmid into a mixture of Appropriate reaction to cause ligation and self-orientation of the third insert within the spinal column between the fourth coupling point and the second coupling point, where the spine is reassembled. Yet another embodiment of the invention is a method for making a modular cloning vector plasmid for the synthesis of a transgene or other complicated DNA construct, the method comprising the steps of providing a plasmid cloning vector including a spinal column, the column vertebral has at least first and second coupling points, the coupling points each have at least one rare restriction site, non-variable operable to be cleaved by a restriction enzyme, cleaving the first coupling point with a first restriction enzyme which corresponds to one of at least one rare, non-variable restriction site of the first attachment point, leaving the first attachment site excised with an exposed 3 'end and the vertebral column excised with an exposed 5' end, providing a first insert comprising a 5 'end, a nucleotide sequence of interest, and a 3' end, wherein the emo 5 'of the first insert is compatible to the exposed 3' end of the first cleavage coupling point, and the 3 'end of the insert is operable, when combined with the exposed 5' end of the excised spinal column, to form a third point of coupling having at least one rare, non-variable restriction site, operable to be cleaved with a third restriction enzyme, by placing the first insert and the cleaved cloning vector plasmid, in an appropriate reaction mixture, to cause ligation and the self-orientation of the first insert within the main chain between the first coupling point and the third coupling point, and with direction 5 'of the second coupling point, where the spine is reassembled, after which it breaks the second coupling point with the third restriction enzyme, leaving the third coupling point cleaved with an exposed 3 'end and the spinal column cleaved with an exposed 5 'end, providing a second insert comprising a 5 'end, a nucleotide sequence of interest, and a 3' end, wherein the 5 'end of the second insert is compatible with the exposed 3' end of the third cleavage coupling point, and the 3 'end of the second insert is operable, when combined with the exposed 5' end of the excised spinal column, to form a fourth coupling point comprising at least one rare, non-variable restriction site, operable to be excised by a fourth restriction enzyme, placing the second insert and the cleaved cloning vector plasmid in an appropriate reaction mixture to cause ligation and self-orientation of the second insert within the spinal column, between the third attachment point and the fourth point coupling, with 5 'direction from the second coupling point, where the spine is reassembled, after which it breaks, the fourth point of ac opulation with the fourth restriction enzyme, leaving the fourth coupling site cleaved with an exposed 3 'end, cleaving the second coupling point with a second restriction enzyme corresponding to one of at least the rare, non-variable restriction site of the second coupling point, leaving the second coupling point cleaved with an exposed 5 'end, providing a third insert comprising a 5' end, a nucleotide sequence of interest, and a 3 'end, where the 5' end of the third insert is compatible with the exposed 3 'end of the fourth cleavage coupling point, and the 3' end of the third insert is compatible with the exposed 5 'end of the second cleavage coupling site, and by placing the third insert and the plasmid cloning vector excised, in an appropriate reaction mixture to cause ligation and self-orientation of the third insert within the spinal column between the fourth coupling point and the second coupling point, where the spine is reassembled.

A further understanding of the nature and advantages of the present invention will be more fully appreciated with respect to the following figures and the detailed description.

BRIEF DESCRIPTION OF THE FIGURES The appended figures, which are incorporated in and constitute a part of this specification, illustrate the embodiments of the invention, and together with a general description of the invention given above and the detailed description given below, serve to explain the principles of the invention. Figure 1 is a schematic illustration of a coupling vector of the domain module, containing a multiple cloning site for the cloning of a genetic module. Figure 2 is a schematic illustration of a coupling vector PE3 containing a domain cloning module for cloning into a domain module. Figure 3 is a schematic illustration of an alternative embodiment of a multiple cloning coupling vector PE3 containing a multiple cloning site for cloning into the genetic module. Figure 4 is a schematic illustration of a multigenic vector containing a means for cloning into a PE3 vector. Figure 5 is another schematic illustration of the module concept of the invention. Figure 6 is a map of the coupling plasmid. Figure 7 is a linear restriction map illustrating an example of the restriction enzyme sites that can be included in the MCS coupling plasmid. Figure 8 is a multigene (or primary) coupling plasmid map. Figure 9 is a linear protection map illustrating an example of the restriction enzyme sites that can be included in the MCS multigene coupling plasmid. Figure 10 is a map of shuttle vector plasmid P ("SVP"). Figure 11 is a linear restriction site map illustrating an example of the restriction enzyme sites that can be included in shuttle vector P MCS. Figure 12 is a map of the shuttle vector plasmid E ("SVE"). Figure 13 is a linear restriction site map illustrating a restriction example that can be included in shuttle vector E MCS.

Figure 14 is a map of shuttle vector 3 ' ("SV3"). Figure 15 is a linear restriction site map illustrating an example of the restriction enzyme sites that can be included in the shuttle vector 3 'MCS. Figure 16 is a schematic illustration of a multigene coupling vector developed and used with the PE3 modules according to the invention. Figure 17 is a schematic illustration of the insertion of module PE3 into a multigene coupling vector that employs gene pivots to provide a multigenic vector with module PE3. Figure 18 is a schematic illustration of the insertion of the PE3 module into a multigenic coupling vector employing a multiple cloning site in the multigenic vector. Figure 19 is a schematic illustration of the insertion of the PE3 module into a multigene coupling vector employing BstX-1 restriction sites in the multigenic vector. Figure 20 is a schematic illustration of the insertion of the PE3 module into a multigenic coupling vector using self-directed endonucleases in the multigenic vector.

Figure 21 is a schematic illustration of the insertion of the PE3 module into a multigenic coupling vector employing recombination engineering between the PE3 vector and the multigenic vector.

BRIEF DESCRIPTION OF THE SEQUENCE LIST The appended sequence listings, which are incorporated in and constitute a part of this specification, illustrate the embodiments of the invention and, together with a general description of the invention given above, and the description given below, they serve to explain the principles of the invention. SEQ ID: 01 is an example of a nucleotide sequence for a PE3 coupling plasmid, MCS. SEQ ID: 02 is an example of a nucleotide sequence for a PE3 coupling plasmid. SEQ ID: 03 is an example of a nucleotide sequence for a primary coupling plasmid MCS. SEQ ID: 04 is an example of a nucleotide sequence for a primary coupling plasmid. SEQ: ID 05 is an example of a nucleotide sequence for an MCS plasmid of shuttle vector P. SEQ ID: 06 is an example of a nucleotide sequence for a plasmid of shuttle vector P. SEQ ID: 07 is an example of a nucleotide sequence for a CS plasmid of shuttle vector E. SEQ ID: 08 is an example of a nucleotide sequence for a plasmid of shuttle vector E. SEQ ID No: 09 is an example of a nucleotide sequence for a plasmid MSC of shuttle vector 3. SEQ ID No: 10 is an example of a nucleotide sequence for a plasmid of shuttle vector 3.

DETAILED DESCRIPTION OF THE INVENTION For the purposes of this invention, the following terms are defined as follows: "Chromatin Modification Domain" (CMD) refers to nucleotide sequences that interact with a variety of proteins associated with maintenance and / or alteration of chromatin structure. "Cloning" refers to the process of ligating a DNA molecule within a plasmid and transferring it into a host cell suitable for duplication during host propagation. The term "cloning vector" refers to a circular DNA molecule that minimally contains an origin of replication, a means for positive selection of the host cells harboring the vector such as an antibiotic resistance gene, and a site of multiple cloning A cloning vector may consist of a plasmid, a cosmid, an artificial chromosome (BAC, PAC, YAC, and others) or another backbone or backbone of the viral vector. "Cognate sequence" or "cognate sequences" refers to the minimum string of nucleotides required for a restriction enzyme to bind to and break a DNA molecule or gene. "Common" refers to any restriction site that appears relatively frequently within a genome. "Compatible with" refers to a tail or end, either 5 'or 3' of a strand of DNA that can form hydrogen bonds with any other complementary ends cleaved with the same restriction enzyme or created by some other method. Since any DNA containing a restriction site specific for a restriction enzyme will be cut in the same manner as any other DNA comprising the same sequence, those cleaved ends will be complementary and thus compatible. Therefore, the ends of any DNA molecules cut with the same restriction enzyme "are coupled" with one another, in the form of adjacent pieces of a puzzle-like "coupling", and can be enzymatically linked together. The compatible ends will form a restriction site for a particular restriction enzyme when combined with each other. "De novo synthesis" refers to the process of synthesizing double-stranded DNA molecules of any length by binding compatible protruding portions of complementary single-stranded DNA molecules representing subsequences of the DNA molecule total, desired. "DNA construction" refers to a DNA molecule synthesized by consecutive cloning steps within a plasmid cloning vector, and is commonly used to direct expression of the gene in any appropriate host cell such as cells cultured in vitro or a transgenic mouse in vivo. A transgene used to make such a mouse can also be referred to as a DNA construct, especially during the period of time when the transgene is being designed and synthesized. "DNA fragment" refers to any molecule isolated from DNA, including but not limited to a protein coding sequence, reporter gene, promoter, enhancer, intron, exon, poly-A tail, multiple cloning site, localization signal nuclear, or stabilization signal of mRNA, or any other DNA molecule of natural or synthetic origin. Alternatively, a DNA fragment can be completely of synthetic origin, produced in vitro. In addition, a DNA fragment can comprise any combination of fragments of isolated natural and / or synthetic origin. "Coupling plasmid" refers to a specialized cloning vector plasmid used in the invention to assemble the DNA fragments with a DNA construct. "Endonuclease" or "endonuclease enzyme" also commonly referred to herein as a "restriction enzyme," refers to a member or members of a classification of catalytic molecules that bind to a restriction site encoded in a DNA molecule and break the DNA molecule in a precise position in or near the sequence. The terms "endonuclease restriction site" or "restriction site" (as well as "cognate sequence" or "cognate sequences" described above) refer to the minimum set of nucleotides required for a restriction enzyme to bind to, and break a DNA molecule or a gene. "Augmentation region" refers to a sequence of nucleotides that is not required for the expression of a target gene, but will increase the level of expression of the gene under appropriate conditions. "Genetic expression host gene selector" (GEH-S) refers to a genetic element that can confer on a host organism a trait that can be selected, tracked or detected by optical sensors, PCR amplification, biochemical assays or by assays of cell / organism survival (resistance or toxicity to cells or organisms when treated with an appropriate antibiotic or chemical). The terms "gene promoter" or "promoter" (P) can be used interchangeably and refer to a nucleotide sequence required for the expression of a gene. The terms "insert" and "module" are essentially interchangeable, with the only final distinction being that an "insert" is inserted into the vector, and once it is inserted, it is more commonly called a "module". A module can then be removed from the vector. Also, the term "insert" is commonly used for an isolated module used as an insert as a modular acceptor vector. "Intron" refers to the nucleotide sequences of a non-coding region of a gene protein found between two regions or exons that encode the protein. "Localization signal" (LOC) refers to the nucleotide sequences that code for a signal for the subcellular routing of a protein of interest.

"Multiple cloning site" (MCS) refers to nucleotide sequences that comprise at least one single restriction site, and more typically, a grouping of unique restriction sites, for the purpose of cloning the DNA fragments within a plasmid cloning vector. The term "mRNA stabilization element" refers to a DNA sequence that is recognized by the binding proteins considered to protect mRNAs from degradation. The term "operable to define" when referring to the group of restriction sites means that when any of the restriction sites in the group is cut with one of the constrained restriction enzymes, the salient residue represents the 5 'portion for the 3 'nucleotide sequences from the group of restriction sites. The term "origin of replication" (ORI) refers to the nucleotide sequences that direct the replication or duplication of a plasmid within a host cell. "PCR terminator outstanding cloning technology" refers to the process of amplification of genetic modules using the polymerase chain reaction in conjunction with the single-stranded DNA primers with the protruding, protected 5 'nucleotides that can serve as binding sites with the protruding portions of complementary DNA. "Poly-A-tail" refers to a sequence of adenine nucleotides (A) commonly found at the end of messenger RNA molecules (mRNA). A poly-A tail signal is incorporated within the 3 'ends of the DNA constructs or the transgenes to facilitate expression of the gene of interest. "Primer site" refers to the nucleotide sequences that serve as DNA templates on which the oligonucleotides of single-stranded DNA can be annealed for the purpose of initiating DNA sequencing, PCR amplification and / or RNA transcription . The term "pUC19" refers to a plasmid cloning vector well known to those skilled in the art, and can be found in the NCBI Genbank database as access # L09137. "Random nucleotide sequences" refer to any combination of nucleotide sequences that do not duplicate the sequences encoding other elements specified as components of the same molecule. The number of nucleotides required in the random sequences is dependent on the requirements of the restriction enzymes that flank the random sequences. Most endonucleases require a minimum of 2-4 additional random sequences to stabilize the DNA binding. It is preferred that the number of random sequences could be a multiple of 3, corresponding to the number of nucleotides that constitute a codon. The preferred minimum number of random sequences is therefore 6, however, more or less nucleotides can be used. "Rare" refers to a restriction site that appears relatively infrequently within a genome. "Recombination arm" refers to nucleotide sequences that facilitate homologous recombination between transgenic DNA and genomic DNA. Successful recombination requires the presence of a left recombination arm (LRA) and a right recombination arm (RRA) flanking a region of transgenic DNA that is to be incorporated into a host genome via homologous recombination. "Recombination or recombination engineering" refers to the process of using random or site-selective recombinase enzymes in conjunction with DNA sequences that can be driven by recombinase enzymes to translocate a portion of the genetic material from a DNA molecule to a different DNA molecule. "Reporter gene" refers to a nucleotide sequence that codes for a protein useful for monitoring the activity of a particular promoter of interest "Restriction endonuclease" or "restriction enzyme" refers to a member or members of a classification of catalytic molecules that bind to a cognate DNA sequence and break the DNA molecule at a precise site in that sequence. "Shuttle vector" refers to a specialized cloning vector plasmid, used in the invention to make an intermediate molecule that will modify the ends of a DNA fragment. "Marker sequence" (TAG) refers to the nucleic acid sequences encoding a unique protein region that allows it to be detected, or in some cases, distinguished from any endogenous counterpart. "Untranslated region" (UTR) refers to the idiodal nucleotide sequences that span the region that does not code for the protein of a mAR molecule. These untranslated regions may reside at the 5 'end (5' UTR) or at the 3 'end (3' UTR) of an mAR molecule. "Unique" refers to any restriction endonuclease or HE site that is not found elsewhere within a particular DNA molecule.

The present invention provides the structures and methods for creating a multigene coupling vector, typically a plasmid vector and also referred to as a modular cloning vector for the synthesis of a PE3 transgene or other complicated DNA construct, by providing a spine or main chain that has modular cloning or coupling points in it. The invention is useful for assembling a variety of DNA fragments in a de novo DNA construct or a transgene, by using cloning vectors optimized for > reduce the amount of manipulation frequently needed. The transgenic vector of PE3, referred to herein as a PE3 coupling vector, or Coupling Plasmid, typically contains at least one multiple cloning site (MCS) and multiple groups of rare restriction endonuclease and / or endonuclease sites. self-directed ("HE"), arranged in a linear pattern. This arrangement defines a modular architecture that allows the user to place domain modules as inserts in a PE3 transgene construct without disturbing the integrity of the DNA elements already incorporated within the coupling plasmid in previous cloning steps. While the present invention describes and exemplifies the modular vectors and methods for constructing the genetic domain modules within the PE3 transgene modules, and the PE3 transgene modules for insertion into the multigenic vectors, these are vectors of cloning plasmids, similar methods can be used to construct the genetic domain media and PE3 transgene modules in larger extrachromosomal DNA molecules such as cosmids or artificial chromosomes, including bacterial artificial chromosomes (BACs). The wide variety of genetic elements that can be incorporated into the plasmid cloning vectors also allows the transfer of the final transgene PE3 products within a wide variety of host organisms with little or no additional manipulation. The present invention provides that the ends 'and 3' of each of the coupling points (gene pivots) and each of the domain modules or inserts are all compatible with a corresponding end or another point or coupling insert. For example, if a first coupling point contains a restriction site for a rare, non-variable restriction enzyme, such as SgrAI and that the coupling point is thereafter excised, then a first insert intended to be inserted at the end 3 'of the first cleaved coupling point will contain a compatible 5' end to create a restriction site for SgrAI when the insert has been combined with the first coupling point. A second coupling point within the plasmid can, for example, having a restriction site for a non-variable restriction enzyme such as SwaI. Any second insert will have at its 3 'end a compatible nucleotide sequence to be combined with the cleaved 5' end of the second cleavage coupling site to create a restriction site for SwaI. In addition, the 3 'end of the first insert and the 5' end of the second insert, in order to be comfortably inserted into the modular cloning vector plasmid and also thereafter to be removed at the same point, must contain compatible ends for create a third restriction site for a third, rare, non-variable restriction enzyme, such as, for example, Pací or Salí. The coupling vectors of the domain module of the invention, also known herein as "shuttle vectors" for launching into the domain modules in the PE3 coupling vector, contain a multiple cloning site with common restriction sites, which it is flanked by at least one rare restriction site, and optionally with HE sites. Shuttle vectors are designed for the cloning of DNA fragments within the common restriction sites between the rare restriction sites. The cloned fragments can be subsequently released by excision at the site or rare restriction sites (or HE) and incorporated within the Pe3 coupling vector and using the same or same rare restriction sites and / or HEs found in the shuttle vectors. Thus, contrary to conventional cloning vectors, the CS design allows domain modules ("cassettes" or modules of DNA fragments) to be inserted into the modular regions of the PE3 coupling vector (plasmid). of coupling), using pivots of defined genes comprising a group of rare restriction sites. Similarly, each can be easily removed using the same rare restriction enzymes, and / or HE and replaced with any other DNA fragment of interest. This feature allows the user to change the direction of an experimental project quickly and easily without having to reconstruct the complete DNA construction. Therefore, the domain module and the PE3 cloning vectors of the present invention allow the user to clone a DNA fragment into an intermediate domain module vector using common restriction sites, creating a cartridge acceptance module. , and then transferring that modular fragment to the desired modular point in the final construction, by means of rare restriction sites. In addition, this allows future alterations to the module to replace individual modules in the PE3 Coupling Plasmid with other cassette modules. The following descriptions show the distinctions of the present invention compared to the prior art. Each coupling point (defined by a gene pivot) represents an area in which there are preferably at least two rare, non-variable, fixed restriction sites, and more preferably fixed groupings of at least three rare, non-variable restriction sites , and more preferably fixed clusters of no more than 4 rare, non-variable restriction sites. A particular restriction site of each coupling point is cleaved by its cognate restriction enzyme. This will create either a desired 5 'or 3' end which is compatible with the complementary 5 'or 3' end of one of the preconstructed inserts containing a nucleotide sequence of choice, such as an Expression Promoting or Regulating nucleotide sequence. At least two inserts, each of which have 5 'and 3' ends which are compatible with the excised coupling point of interest, can be added together with the cleaved cloning vector plasmid to an appropriate reaction mixture and, assuming the appropriate thermodynamic medium, the inserts can simultaneously, for example, in a simple step, become integrated into the plasmid cloning vector. During this unique addition and ligation reaction, the coupling points are reformed and the plasmid cloning vector becomes modular, since the coupling points and the connection between the two modules can be re-cleaved with the appropriate restriction enzymes. The module can then be later removed, and a new module can be placed in its place. While it is possible to employ a simple restriction site at the pivot point flanking the modules, there is a distinct possibility that a simple "rare" restriction site is very frequent within the particular DNA molecule. Recall that the frequency of the restriction enzyme sites is a function of 4n, as explained above. For example, a particular promoter module in a plasmid cloning vector may contain a rare restriction site within its DNA sequence that is also present at its pivot point, thereby making it advantageous to have more than one restriction site in the pivot points. Thus, it is preferable to use more than one rare restriction site at a single pivot point, due to the statistical probability that more than one of the restriction sites that exist within the DNA sequence of the module of interest will be much less. However, it is also preferable that no more than three or four restriction sites are located at a single pivot point, or even the "pooling" of the restriction sites will begin to affect the transcription / translation of the target molecule. With this number, the combination of restriction sites grouped together will be at most 18-24 base pairs in length.

Domain Module Vectors: Figure 1 shows a simplified representation of a first embodiment of the present invention, of a domain module coupling vector 1. Vector 1 consists of a strand of DNA having the multiple cloning site 2, and is typically a plasmid. The coupling vector of the domain module comprises a multiple cloning module (MC module) consisting of five cloning sites arranged in sequence, MC-1, MC-2, MC-3, MC-4, and MC-5. The multiple cloning site comprises a plurality of restriction sites that are independently selected from common restriction sites, as described hereinafter. Two of the restriction sites define a coupling position for the genetic material of interest, illustrated as a gene of interest 3. The MC module makes it possible to subclonate a genetic material of interest between two of the restriction sites at the site of multiple cloning of the MC module. The gene of interest 3 is typically released from a gene of the vector of interest (not shown), and includes a pair of cloning sites 4a and 4b, shown as MC-1 and MC-3. The domain module coupling vector also comprises a pair of restriction sites, designated as gene pivots 5 and 6, flanking the multiple cloning site 2. The gene pivots each comprise at least two rare, non-variable restriction sites , as defined later in this. The gene pivots 5 and 6 operate to define the 5 'and 3' portions, respectively, of the MC module. In a second embodiment of the invention, the coagulated restriction enzymes MC-1 and MC-3 (not shown) can cut the gene of interest from its vector, and open the MC module at the MC sites, namely MC-1. and MC-3, whereby ligation of the gene of interest between MC, MC-1 and MC-3 sites is predicted, whereby a domain module vector 7 is formed. In the illustrated embodiment, the gene of interest comprises an expression domain, such that the domain module is an expression module and the domain module vector is more particularly an expression module vector. The expression module vector comprises an expression module 8, which comprises the first and second gene pivots 5 and 6 flanking a nucleic acid sequence comprising the subcloned gene of interest 3 that includes the expression domain. In the illustrated embodiment, the gene of interest comprises an expression domain, wherein the first gene pivot (or the 5 'portion of the expression domain) is hereinafter referred to as GP2 and the second gene pivot (or portion 3). 'of the expression domain) as GP3. It can be understood that in alternative embodiments, the gene of interest may comprise a promoter domain or a 3 'regulatory domain whereby its subcloning provides a promoter module within a promoter module vector and a 3' regulatory module within a vector of regulating module 3 ', respectively. For the promoter module coupling vectors, the first gene pivot (or the 5 'portion of the promoter domain) is hereinafter referred to as GP1 and the second gene pivot (or the 3' portion of the promoter domain) as GP2. For the coupling vectors of the 3 'regulatory module, the first gene pivot (or the 5' portion of the regulatory domain 3 ') is hereinafter referred to as GP3 and the second gene pivot (or the 3' portion of the regulatory domain 3). ') as GP4. The gene pivots for any of the coupling vectors of the domain module, including the gene pivots GP1, GP2, GP3 and GP4, can comprise rare restriction sites selected from the group consisting of AsiS I, Pac I, Sbf I, Fse I , Ase I, Mlu I, SnaB I, Not I, Sal I, Swa I, Rsr II, BSiW I, Sfo I, Sgr AI, AflII, Pvu I, Ngo MIV, Ase I, FIp I, Pme I, Sda I , Sgf I, Srf I, and Sse8781 I, and more typically from the group selected from AsiS I, Pac I, Sbf I, Fse I, Ase I, Mlu I, SnaB I, Not I, Sal I, Swa I, Rsr II, BSiW I. The most typical modalities of the gene pivot contain a series of at least 3, and no more than 4, rare restriction sites, and typically any gene pivot does not include a rare restriction site from any other gene pivot. In a preferred embodiment, a gene pivot GP1 is selected from the group consisting of at least AsiS I, Pac I, and Sbf I, the gene pivot GP2 is selected from the group consisting of at least Fse I, Ase I, and Mlu I , the gene pivot GP3 is selected from the group consisting of at least SnaB I, Not I, and Sal I, and the gene pivot GP4 is selected from the group consisting of at least Swa I, Rsr II, and BSiW I. In a Particular preferred embodiment, the gene pivot GP1 consists of, in order, AsiS I, Pac I, and Sbf I; the gene pivot GP2 consists of, in order, Fse I, Ase I, and Mlu I; the GP3 gene pivot consists of, in order, SnaB I, Not I, and Sal I; and the GP4 gene pivot consists of, in order, Swa I, Rsr II, and BSiW I. It can be understood that, the multiple cloning site may comprise three combinations of the restriction site, in larger or smaller numbers of sites, in reverse order (eg, MC-5, MC-4, MC- 3, MC-2, and MC-1), and using two types of cloning sites. The multiple cloning sites can be varied to accommodate the pair of cloning sites for a particular gene of interest. Also contemplated in an alternative embodiment is a library of domain module coupling vectors, which can be used to easily subclone a wide variety of genes of interest. The domain module coupling vector library can be configured as a dedicated promoter domain module vector, the expression domain module vector, or the 3 'regulatory module vector depending on the type of gene pivots selected for the vector coupling of particular domain module. Each sub-library of promoter, expression and 3 'regulatory domain module vectors has its own dedicated pair of gene pivots (namely, GP1 and GP2, GP2 and GP3, and GP3 and GP) to ensure proper cloning of the respective domain modules in higher order PE3 coupling vectors which are described hereinafter.

Coupling Vectors to PE3 and Coupling to PE3 MC Figure 3 shows a simplified representation of a third embodiment of the present invention, of a first coupling vector 10 of PE3. The coupling vector 10 of PE3 consists of a strand of DNA having at least a first cloning module 11b and is typically a plasmid. At least one first cloning module comprises at least a first and a second gene pivot, illustrated as GP2 and GP3, which flank a nucleic acid sequence comprising the filler 18b. A DNA filler domain is a random nucleotide sequence that does not code for a restriction site or any other biological function resident within the PE3 coupling vector. The filler DNA serves to increase the efficiency of the restriction enzyme cleavage activity by providing longer stretches of DNA to which the restriction enzyme can bind. This is important because many restriction enzymes can not bind to, and cut their cognate recognition sites if the DNA lengths are limiting. The first and second gene pivots are as described hereinabove. The first cloning module 11b is configured to clone at least a first domain module within the cloning module of PE3. In the illustrated embodiment, where the first cloning module is an expression domain cloning module, the first and second gene pivots are represented as GP2 and GP3, as defined hereinabove. The first cloning module 11b comprising the filler, also referred to as an expression filler module, provides a coupling position for the insertion of an expression domain module comprising compatible gene pivots. A compatible gene pivot is one that has identical restriction sites, or that comprises at least one rare restriction site, unique in common with the gene pivot of the cloning module. Also illustrated is a second cloning module a, which is a promoter filler module having its first gene pivot 12 as GP1, and its second gene pivot 13 as GP2 which is a shared junction with the portion 5 'of the cloning module 11b. The promoter filler module lia comprises the filler and provides a coupling position for the insertion of a promoter domain module having compatible gene pins, which replaces the promoter filler module after cloning. The third cloning module 11c is a regulating filler module 3, having its first gene pivot 14 as GP3 which is a joint shared with the 3 'portion of the cloning module 11b, and its second gene pivot 15 as GP4. The regulator filler module 3 '11c comprises the filler and provides a coupling position for the insertion of a regulatory domain module 3' having compatible gene pivots, which replaces the regulatory filler module 3 'after cloning. In a fourth embodiment of the invention, the restriction enzymes cognate for one of the rare restriction sites contained in each gene pivot for one of the domain module vectors, illustrated as expression module 7, can cut the rare restriction site of its residual vector, and likewise opens the coupling of PE3 in the same rare restriction sites in the corresponding gene pivots GP2 and GP3, whereby the expression filler module is replaced with the expression module. In a separate cloning event, the cognate restriction enzymes suitable for the gene pins for the promoter module (not shown) and the corresponding gene pins 12 and 13 of the promoter filler module can be used to ligate the promoter module into the PE3 module, whereupon the promoter filler module is replaced with the promoter module. In the same way, the regulating module 3 'can be linked inside the module PE3. In the present invention at least two, and more typically at least three, rare restriction sites are used as coupling pins or gene pivots. The rarity of a rare restriction site in the genetic sequences makes it unlikely that both rare restriction sites in a two-site gene pivot, and very unlikely that the three rare restriction sites in a three-site genetic pivot, could be found in the coupling vector. In the case where the DNA cloning vector is found to include one of the rare restriction sites of the gene pivot, the skilled user can cut the gene pivot with one of the other rare, cognate restriction enzymes. As an illustration, if the gene pivot GP2 consists of Fse I, Ase I and Mlu I, and the coupling vector PE3 or the E domain itself has an Ase I within its sequence, then the person skilled in the art can use either Fse I or Mlu I to cut the gene pivots. The DNA cloning vector of the PE3 vector also typically comprises a means for releasing the cloning module PE3 from the PE3 coupling vector, for cloning into a multigene coupling vector, as will be described hereinafter. Figure 3 shows an alternative modality for cloning into domain modules within a multiple cloning coupling vector of PE3 (MC). A first coupling vector MC of PE3, designated as the coupling vector 20a of the MC promoter of PE3, consists of a strand or strand of DNA having at least one first cloning module 21a, and is typically a plasmid. The first cloning module 21a comprises at least a first and a second gene pivot, illustrated as GP1 and GP2, flanking a nucleic acid sequence comprising a multiple cloning site (MCS) comprising a plurality of independently selected restriction sites. of common restriction sites, as described later in this. In the illustrated embodiment, the coupling vector 20 of the MC3 promoter of PE3 has three cloning modules, 21a, 21b and 21c, each linked by gene pivots GP1, GP2, GP3 and GP4, as described herein. The second and third cloning modules are shown as filler modules containing a nucleic acid sequence comprising the filler. The multiple cloning site consists of five cloning sites arranged in sequence, MC-1, MC-2, MC-3, MC-4, MC-5 and MC-6. Any two of the restriction sites can define a coupling position for a genetic material of interest. In the coupling vector 20a the MC of PE3, the genetic material of interest is typically a promoter domain. The selection of the two cloning sites of interest may be based on the understanding that these restriction sites are unique within the mating vector 20a MC of PE3. Alternative embodiments include an MC expression vector of PE3, wherein the second cloning module 21b comprises the multiple cloning site, with the first and third remainer cloning modules 21a and 21c comprising the filler, and a vector of coupling regulator MC-3 of PE3, wherein the third cloning module 21c comprises the multiple cloning site, with the first and second cloning modules 21a and 21b remaining comprising the filler. In other alternative embodiments, two of the three cloning modules may comprise the multiple cloning site. Subcloning the multiple cloning site with a genetic material of interest and cloning the domain modules into the filler modules in the coupling vectors PE3 can be accomplished as described hereinabove. The gene pivots GP1, GP2, GP3 and GP4 are as described hereinabove with respect to the position of the respective modules in the promoter, expression and 3 'regulatory domain modules. The present invention also provides for the elaboration of the PE3 vectors from the modular domain vectors and a modular cloning vector of PE3. A first method for constructing a modular PE3 vector comprises the steps of: a) providing a cloning vector of PE3 comprising a PE3 cloning module, the cloning module of PE3 comprises in sequence: a first gene pivot comprising minus two rare non-variable restriction sites, at least one first filler module consisting of a nucleic acid sequence comprising the filler, and a second gene pivot; b) the provision of at least a first domain module vector comprising in sequence: the first gene pivot, a genetic module of interest consisting of a nucleic acid sequence comprising a genetic material of interest; and the second gene pivot; c) the provision of a first restriction enzyme cognate for one of the rare restriction sites of the first gene pivot and a second restriction enzyme cognate for one of the rare restriction sites of the second gene pivot; d) removing and isolating the genetic module of interest from the first domain module vector using the first and second cognate restriction enzymes; e) removing the first filler module from the PE3 cloning module of the PE3 cloning vector using the first and second coagulated restriction enzymes; and f) ligation of the genetic module of interest within the cloning module of PE3. In addition, the method also provides, wherein the PE3 cloning module provided further comprises in sequence after the second gene pivot, a second filler module and a third gene pivot, with the additional steps of: g) providing a second module vector of domain comprising in sequence the second gene pivot, a second genetic module of interest consisting of a nucleic acid sequence comprising a second genetic material of interest, and the third gene pivot; h) the provision of a third cognate restriction enzyme for one of the rare restriction sites of the third gene pivot; i) removing and isolating the second genetic module of interest from the second domain module vector using the second and third cognate restriction enzymes; j) removing a second filler module from the PE3 cloning module of the cloning vector PE3 using the second and third cognate restriction enzymes; and k) the ligation of the second genetic module of interest within the PE3 cloning module. Similarly, a third genetic module of interest can be inserted into PE3 using a third gene pivot and the constrained restriction enzymes to excise and ligate the second genetic module of interest within the PE3 cloning module. The method provides a sequential arrangement of genetic modules of interest within the cloning vector of PE3. Typically, the first, second and third genetic modules of interest correspond to a promoter module, the expression module, and a 3 'regulatory module, respectively. In a typical embodiment, the first gene pivot is GP1, the second gene pivot is GP2, the third gene pivot is GP3, and the fourth gene pivot is GP4, as described hereinabove. In yet another embodiment, the method for making a PE3 cloning vector comprises the steps of: a) providing a PE3 cloning vector having a backbone, the backbone comprising at least a first, a second, a third and a fourth gene pivots, the gene pivots are arranged sequentially in a 5 '-3' direction and each has at least two rare, non-variable restriction sites operable to be cleaved by a cognate restriction enzyme; b) cleavage of the first gene pivot with a first restriction enzyme cognate to one of the non-variable rare restriction site of the first gene pivot, leaving the first gene pivot excised, with a 3 'end exposed; c) cleavage of the second gene pivot with a second restriction enzyme cognate to one of the non-variable rare restriction site of the second gene pivot, leaving the second gene pivot excised, with a 5 'end exposed; d) the provision of a first domain module comprising a 5 'end, a first genetic material of interest, and the first gene pivot a 3' end, wherein the 5 'end of the first domain module is compatible with the end 3'. exposed from the first excised gene pivot, and the 3 'end of the first domain is compatible with the exposed 5' end of the second excised gene pivot; and e) the placement of the first domain module and the cloning vector of PE3 into an appropriate reaction mixture to cause self-targeting ligation of the first domain module within the backbone between the first gene pivot and the second gene pivot , where the backbone or main chain is reassembled. The method further comprises the steps of: f) thereafter, excising the second gene pivot with the second cognate restriction enzyme, leaving the second gene spindle excised, with an exposed 3 'end; g) the third gene pivot is broken with a third restriction enzyme cognate for one of the rare restriction sites of the third gene pivot, leaving the third gene pivot excised, with a 5 'end exposed; h) the provision of a second domain module comprising a 5 'end, a first genetic material of interest and a 3' end, wherein the 5 'end of the second domain module is compatible with the exposed 3' end of the second spliced gene pivot, and the 3 'end of the second domain module is compatible with the exposed 5' end of the third splinted gene spindle; and i) placing the second domain module and the cleaved PE3 cloning vector in an appropriate reaction mixture, to cause ligation and self-orientation of the second domain module within the backbone between the second gene pivot and the third gene pivot, where the spine is reassembled. The method can further provide the steps of: j) thereafter, the excision of the spinal column at the third gene pivot with the third cognate restriction enzyme, leaving the third gene pivot excised with an exposed 3 'end; k) excision of the fourth gene pivot with a fourth restriction enzyme cognate to one of the rare restriction sites of the fourth gene pivot, leaving the fourth gene pivot excised with an exposed 5 'end; 1) the provision of a third domain module comprising a 5 'end, a third genetic material of interest and a 3' end, wherein the 5 'end of the third domain module is compatible with the exposed 3' end of the third spliced gene pivot, and the 3 'end of the third domain module is compatible with the exposed 5' end of the fourth splinted gene pin; and m) placing the third domain module and the cleaved PE3 cloning vector within an appropriate reaction mixture to cause ligation and self-orientation of the third domain module, within the backbone between the third gene pivot and the fourth gene pivot, where the spine is reassembled. Typically, the first, second and third generic modules of interest correspond to a promoter module, expression module and a 3 'regulatory module, respectively. In a typical embodiment, the first gene pivot is GP1, the second gene vector is GP2, the third The gene pivot is GP3 and the fourth gene pivot is GP4, as described hereinabove.

Multigenic Cloning Vectors The coupling vectors PE3 and coupling MC of the present invention provide a means to easily and quickly assemble one or a plurality of transgenes PE3 of interest. The invention also provides a means to quickly and easily insert one or a plurality of PE3 modules into a modular multigenic vector. Figure 4 shows a simplified diagram of the insertion of a PE3 module into a multigene coupling vector comprising one or (as illustrated) a plurality of PE3 filler modules that can be easily released to make it possible to insert one or more PE3 modules. The multigenic vectors are described in more detail later and in Figures 16-21. The restriction sites used in the invention are chosen according to an appearance hierarchy. In order to determine the frequency of the appearance of the restriction site, the information of the DNA sequence corresponding to 19 different genes was analyzed using the Vector NTI software. This search covered a total of 110,530 nucleotides of the DNA sequence. The results from these analyzes were calculated according to the number of instances of a restriction site that appears within the 110,530 nucleotides analyzed. Restriction sites were then assigned with a hierarchical designation according to four classifications, where "common" sites appear more than 25 times per 110,530 nucleotides, "sites of 6 lower frequency base pairs" appear between 6-24 times by 110,530 nucleotides, and the "rare" sites appear between 0-5 times by 110,530 nucleotides. A partial view of enzymes, "suitable", is listed here according to their appearance classifications: Common restriction sites Suitable common restriction enzymes may include, but are not limited to, Ase I, BamH I, Bgl II, BIp I, BstX I, EcoR I, Hinc II, Hind III, Neo I, Pst I , Sac I, Sac II, Sph I, Stu I, Xba I. Suitable restriction sites that have a recognition site of 6 base pairs, but have a lower frequency of appearance, may include but are not limited to, Aar I, Aat II, Afl II, Age I, ApaL I, Avr II, BseA I, BspD I, BspE I, BstB I, Cia I, Eag I, Eco0109 I, EcoR V, Hpa I, Kpn I, Mfe I , Nar I, Nde I, NgoM IV, Nhe I, Nsi I, Pml I, SexA I, Sma I, Spe I, Xho I.

Rare Restriction Sites: Suitable rare enzymes may include, but are not limited to, Acl I, Nru I, Pac I, Pme I, Sbf I, Sfi I, PI-Sce I, I-Sce I, I-Ceu I, PI-Psp I, I-Tli I, Fse I, Sfo I, So YES, Sgr AI, Ase I, MIu I, Sna BI, Not I, Sal I, Swa I, Rsr II, Bsi WI, AflIII, Pvu I, Ngo MIV, Ase I, FIp I, Pme I, Sda I, Sgf I, Srf I, and Sse8781 1.

Self-directed endonucleases: Suitable self-directed endonucleases may include, but are not limited to, I-Scel, PI-Sce I, I-Ceu I, PI-Psp I, I-Chu I, I-Cmoe I, I-Cpa I, I-Cpa H, I-Cre I, I-Cvu I, I-Dmo I, I-LIa I, I- so I, I-Nan I, I-Nit I, I-Nja I, I-Pak I, I-For I, I-Ppo I, I-Sca I, I-Sce II, I-Sce III, I-Sce IV, I-Sce V, I-Sce VI, I-Ssp68031, I-Tev I , I-Tev II, I-Two I, PI-Mga I, PI-Mtu I, PI-Pfu I, Pl-Pfu II, Pl-Pko I, PI-Pko II, PI-TfU I, PI-Tfu D , PI-Thy I, and PI-Tii. The individual components of a PE3 vector or a transgene are the promoter enhancer module (designated as "P"), the expressed protein module ("E"), and / or the 3 'regulatory region module ("3") , and can be assembled as modules transferred from the domain module vectors (or "shuttle") into a PE3 coupling vector (which may also be referred to as a docking station). Figure 5 shows a promoter module, within a promoter vector, which can be excised and inserted into a PE3 coupling vector as a predetermined coupling position. As shown also in Figure 5, if higher orders of complexity are required, assembled PE3 modular transgenes, or other nucleotide sequences, may then be transferred into a multigene coupling vector (also referred to as a Primary Coupling Station Plasmid) or into a coupling vector that is directed to the locus. Each of the five types of cloning vector plasmids (promoter module, expression module and 3 'regulatory module vectors, and the coupling vector of PE3 and multigenic vectors) will be explained in greater detail to illustrate the components incorporated within each. Domain module coupling vectors, also known as "shuttle vectors" that contain a multiple cloning site, typically comprising common restriction sites, and flanked by a gene pivot, typically comprise a rare restriction site and / or sites HE. The domain module coupling vector is constructed from a pUC19 backbone, and has the following modifications to the pUC19 backbone, where the sequences are numbered according to the Genbank sequence file of pUC19, access # L09137: 1 Only sequences from 806 to 2617 (Afl3-Aat2) are used in the coupling plasmid, 2. The BspHl site in 1729 in pUC19 is mutated from TCATGA to GCATGA, 3. The Acll site in 1493 in pUC19 is mutated from AACGTT to AACGCT, 4. The Acll site at 1120 in pUCl 9 is mutated from AACGTT to CACGCT, 5. The Ahdl site in pUC19 is mutated from GACNNNNNGTC to CACNNNNNGTC, The sequences' coding for BspHl / I-Ppo 1 / BspHl are inserted only in the BspHl site remaining in pUC19 after the mutation step 2 in the previous list. The three individual shuttle vectors of the present invention, the coupling vector of the promoter module, the coupling vector of the expression module, and the coupling vector of the regulatory module 3 ', are described in particular embodiments, and identified as Promoter / Intron Vector Shuttle ("SVP"), Vector Shuttle Expression ("SVE"), and 3 'Regulator of the Shuttle Vector ("SV3"), respectively. Each one is described more fully later.

Vector Shuttle P An SVP is shown in Figure 10, and this is a plasmid cloning vector that can be used to prepare the promoter and intron sequences for assembly into a PE3 coupling vector (transgene construct) as shown in FIG. described above in the present.

Figure 11 shows an example of an SVP plasmid comprising the following sequential elements in an MCS, in the listed order: 1. Two common, non-variable and unique restriction sites that define a 5 'insertion site for the mutated pUC19 vector described above (e.g., AatlI and BIpI), 2. A T7 primer site, 3. A common, non-variable, and unique restriction site that allows efficient cloning of a shuttle vector module with 3 'of the T7 primer site ( for example, Eco0109l), 4. A fixed cluster of the rare, non-variable restriction site that defines the 5 'portion of the promoter module (eg, AsiSI and SgrAI), 5. A variable MCS comprising any grouping of sites of common or rare constraints that are unique to the shuttle vector (eg, the series of restriction sites illustrated in Figure 11); 6. A fixed grouping of rare, non-variable restriction sites that define portion 3 'of the promoter module (for example, Pad, AscI, and MluI), 7. A common, non-variable and unique restriction site that allows efficient cloning of a shuttle vector module with 5' direction from the T3 primer site (by example, BspEI) 8. A T3 primer site in reverse orientation, and 9. Two common, non-variable and unique restriction sites that define a 3 'insertion site for the mutated pUC19 vector described above (e.g., Pmel and SapI). In an alternative example, the fixed grouping of the rare, non-variable restriction sites that define the 5 'portion of the promoter module (the GP1 gene pivot) comprise AsiS I, Pac I and Sbf I, and the rare restriction sites that define the 3 'portion of the promoter module (the GP2 gene pin) comprises Fse I, Ase I and Mlu I. In a more specific example, the GP1 gene pivot consists of the sequence of AsiS I, Pac I and Sbf I, and the pivot Gene GP2 consists of the sequence Fse I, Ase I and Mlu I.

Vector Shuttle E (SVE) An EVS shown in Figure 12, is a plasmid cloning vector that can be used to prepare sequences that are to be expressed by the transgene, for the modular assembly within the vector PE3 (construction of the transgene) as is described hereinabove. Figure 13 shows an example of an SVE plasmid comprising the following sequential elements in the MCS, in the listed order: 1. Two common, non-variable and unique restriction sites, which define a 5 'insertion site for the vector pUC19 mutant, described above (eg, BlpI), 2. A primer site T7, 3. A common, non-variable and unique restriction site, allowing efficient cloning of a shuttle vector module with 3 'direction of the primer site T7 (e.g., Eco0109I), 4. A fixed pool of rare, non-variable restriction sites that define the 5 'portion of the expression module (eg, Pac, Ascl, and MluI), 5. A variable MCS that consists of any grouping of common or rare restriction sites that are unique to the shuttle vector (eg, the series of restriction sites illustrated in Figure 13), 6. A fixed grouping of rare, non-variable restriction sites, defining the 3 'portion of the expression module (eg, SnaBI, Notl, and Sali), 7. A common, non-variable, and unique restriction site that allows efficient cloning of a shuttle vector module with a 5' site address of the T3 primer (e.g., BspEI) 8. A reverse orientation T3 primer site, and 9. Two common, non-variable and unique restriction sites that define a 3 'insertion site for the mutated pUC19 vector, described above (for example, Pmel). In an alternative example, the fixed pooling of the rare, non-variable restriction sites that define the 5 'portion of the expression module (the GP2 gene pivot) comprise Fse I, Ase I and Mlu I, and the rare restriction sites that define the 3 'portion of the expression module (gene pin GP3) comprise SnaB I, Not I, and Sal I. In a more specific example, the gene pivot GP2 consists of the sequence Fse I, Ase I and Mlu I, and The GP3 gene pivot consists of the sequence SnaB I, Not I, and Sal I.

Vector Shuttle 3 (SV3) SV3, shown in Figure 14, is a cloning vector plasmid that can be used to prepare the 3 'regulatory sequence, for assembly within a PE3 vector (transgene construct) as described above in the present.

In Figure 15, an example of a SV3 plasmid can comprise the following elements in the MCS, in the listed order: 1. Two common, non-variable and unique restriction sites, which define a 5 'insertion site for the vector pUC19 mutant, described above (for example, BlpI), 2. A T7 primer site, 3. A common, non-variable and unique restriction site, allowing efficient cloning of a shuttle vector module with 3 'address from the T7 primer site (eg, Eco0109l), 4. A fixed grouping of the rare, non-variable restriction sites that define the 5 'portion of the expression module (eg, SnaBI, Notl, and I left), 5. A variable MCS that 'consists of any grouping of common or rare restriction sites that are unique to the shuttle vector (eg, the series of restriction sites illustrated in the figure ), 6. A fixed grouping of rare, non-variable restriction sites that define the 3 'portion of the expression module (eg, Swal, RsrII, and BsiWI), 7. A common restriction site, not variable and only one that allows efficient cloning of a shuttle vector module with 5 'direction from the T3 primer site (e.g., BspEI) 8. A reverse orientation T3 primer site, and 9. Two common, non-variable and unique restriction sites that define a 3 'insertion site for the mutated pUC19 vector, described above (eg, Pmel). In an alternative example, the fixed grouping of the rare, non-variable restriction sites that define the 5 'portion of the expression module (the GP3 gene pivot) comprise SnaB I, Not I, and Sal I, and the rare restriction sites defining the 3 'portion of the expression module (the GP4 gene pin) comprise Swa I, Rsr II and BsiW I. In a more specific example, the GP3 gene pivot consists of the sequence SnaB I, Not I, and Sal I and the GP4 gene pivot consists of the Swa I, Rsr II and BsiW I sequence. The PE3 coupling vector (PE3 coupling plasmid) shown in Figure 6, comprises a pUC19 backbone modified as in. The above example The multiple cloning site (CS) in the PE3 coupling plasmid, shown in Figure 7, comprises the following sequential elements, in the listed order: 1. Three common, non-variable and unique restriction sites, which define a 5 'insertion site for the mutated pUC19 vector, described above (e.g., Aat II, BIp I, and EcoO109 I), 2. A T7 primer site, 3. A first single HE site (e.g. -Scel (here, in a forward orientation), 4. A pair of common non-variable and unique restriction sites, flanking the random nucleotide sequences that can serve as an acceptor module of the chromatin modification domain (RNAS-C DI) (for example, Kpn I and Avr II), 5. A fixed grouping of non-variable rare restriction sites that define the 5 'portion of the promoter module Examples of rare, non-variable restriction sites for use as pivots in the 5 'end of the pr module omotor preferably include AsiS I, PacI, and Sfo I, but may also include Pvul, AsiSI, and SgrA I, 6. The random nucleotide sequences that can serve as an promoter / intron acceptor module (RNAS-P), 7. A Fixed clustering of rare, non-variable restriction sites that define the shared junction between the 3 'portion of the promoter / intron module and the 5' portion of the expression module. Examples of rare, non-variable restriction sites for use as pivots at the 3 'end of the promoter module preferably include Fsel, Ascl and MluI, but may also include Pac, 8. The random nucleotide sequences that can serve as a module expression acceptor (RNAS-E), 9. A fixed grouping of rare, non-variable restriction sites that define the union of the portion 3 'of the expression module and the 5' portion of the 3 'regulatory module (eg, SnaB I, Not I, and Sal 1), 10. The random nucleotide sequences that can serve as a 3' regulatory domain acceptor module. (RNAS-3), 11. A fixed grouping of rare, non-variable restriction sites that define the 3 'portion of the 3' regulatory module (eg, Swa I, Rsr II, and BsiW I), 12. A pair of common, non-variable and unique restriction sites, flanking a random DNA nucleotide sequence, which can serve as an acceptor module of the chromatin modification domain (RNAS-C D-2) (eg, Xho I and Nhe I) , 13. A second unique HE site (here, identical to the one in section 3 above (IScel), and also in the reverse orientation). It has recently been observed, however, that if the two unique HE sites are identical to each other and in reverse orientation, an unfavorable recombination event may occur. Therefore, it may be preferred to use a second unique HE site that is not identical to the first single HE site. 14. A T3 primer site in reverse orientation, and 15. Four common non-variable and unique restriction sites that define a 3 'insert site for the mutated pUC19 vector described above (e.g., BspE I, Pme I, Sap I , and BspH I). In an alternative example, the fixed grouping of the rare, non-variable restriction sites that define the 5 'portion of the promoter module (the gene pivot GP1) comprise AsiS I, Pac I and Sbf I; the rare restriction sites that define the 3 'portion and the promoter module in the 5' portion of the expression module (the gene pin GP2) comprise Fse I, Ase I and Mlu I; the rare restriction sites that define the 3 'portion of the expression module and the 5' portion of the 3 'regulatory module (the GP3 gene pin) comprise SnaB I, Not I, and Sal, and the rare restriction sites that define the 3 'portion of the 3' regulatory module (the GP4 gene pivot) comprise Swa I Rsr II and BsiW I. In a more specific example, the GP1 gene pivot consists of the sequence of AsiS I, Pac I and Sbf I, the gene pivot GP2 consists of the sequence Fse I, Ase I and Mlu I, the gene pivot GP3 consists of the sequence SnaB I, Not I, and Sal I, and the gene pivot GP4 consists of the sequence Swa I Rsr II and BsiW I.

Multigenic Coupling Vector: Figure 16 shows a multigene coupling vector comprising a pUC19 backbone identical to that of the PE3 coupling vector except for the inclusion of additional restriction sites to create module limits to flank the spinal modules of vector. These spine modules include a Replication Host Selector (RHS) gene module and a Replication Origin (ORI) gene module. The RHS is defined by the vector vertebral column pivot (VB) Aat II, a DNA sequence that codes for a Replication Host Selector Gene, and two additional vector spinal pivots Age I and Avr II. The RHS module in Figure 16 is defined by the vector vertex pivot VB-I = Aat II, the RHS module = Ampicillin resistance gene, and the vertebral column pivots of vector VB-2 = Age I and VB-3 = Avr II. The ORI is defined by the vertebral column pivots of vector Age I and Avr II, a DNA sequence, which codes for an origin of replication, and the vertebral column pivot of vector Pme I. The ORI module in figure 16 is defined by the vertebral column pivots of vector VB-2 = Age I and VB-3 = Avr II, the ORI module = pUClO origin of replication and the vertebral column pivots of vector VB-4 = Pme I. The coupling vector multigene is defined by the precise placement of random nucleotide sequences and non-random nucleotide sequences that define common, rare, and self-directed endonuclease sites for purposes of creating a diverse array of domain acceptor regions. These module acceptors are defined by the following classes: GHS, BRD, SRS, CMD, (CMD / BRD), PE3 filler, PE3 MCS. These modules are defined as: A. GHS = Genome Expression Host Generator (example: NEO or PURO) B. BRD = Non-random nucleic acid sequence that can be used as a site for homologous recombination in cell lines "Recombinant-competent" bacterial C. SRS = Non-random nucleic acid sequence that can be used as a site for site-specific homologous recombination, mediated by a product of the site-specific recombinase gene (example: Cre / Lox or Flp) / Frt) D. CMD = A non-random nucleic acid sequence that can alter the chromatin structure of a host genome (eg: isolate of chicken beta-globin HS4) E. CMD / BRD = A nucleic acid sequence non-random coding for a chromatin modification domain that can serve as a DNA sequence for homologous recombination in lines of "recombination competent" bacterial cells F. Filling r of PE3 = A random nucleotide sequence that does not code for a restriction site or any other resident of biological function within the multigene coupling vector G. PE3 MCS = A non-random nucleotide sequence consisting of a plurality of unique restriction sites for the multigene coupling vector. These modules are flanked by restriction sites that can be used to make module coupling vectors. These restriction sites associated with the module can be defined according to the following classifications: A. VB = restriction sites that define the limits of a vertebral column of vector B. ES = restriction sites that define the limits of a module host selector for genome expression C. TX = synthetic BstX I sites that generate compatible protruding ends at the specific and self-directed endonuclease sites (example I-Ceu I, I-Sce I, Sbf I) D. CM = restriction sites that define the limits of a modulus of chromatin modification E. SB = restriction sites that define the boundaries in a site-specific recombination module F. BL = restriction sites that produce blunt ends One embodiment of the invention can be defined by the nucleotide sequence residing between the vertebral column pivots of vector VB-I and VB-5, where VB-I = Aat II and VB-5 = BspE I. 1. VB-I is Aat II 2. No sequence or random nucleotide sequence 3. ES-I = SexA I 4. Filler of random nucleotide sequence (or GHS module) 5. ES-2 = Srfl 6. Random nucleotide sequence filler (or BRD module) 7. HE-3 = I-Ppo I 8. Random nucleotide sequence filler (or SRS module) 9. No random nucleotide sequence or filler 10. CM-1 = Kpn I 11. Random nucleotide sequence (or CMD module) 12. CM-2 = Sac I 13. No sequence or filler of random nucleotide sequence 14. TX-I = Bst X I (I-Ceu I) front orientation . Random nucleotide sequence filler (or PE3 module) 16. TX-2 = Bst XI (I-Sce I) reverse orientation 17. No sequence or random nucleotide sequence filler 18. CM-3 = Mfe I 19. Nucleotide sequence filler random (or CMD module) 20. CM-4 = Sac II 21. No sequence or random nucleotide sequence filler 22. GP- 1 = (AsiS I / Pac I / Sbf I) 23. Filler of random nucleotide sequence (or module) PE3) 24. G-4 / CM-5 = (Swa I / Rsr II / BsiW I) 25. Filler of random nucleotide sequence (or CD module) 26. C -6 = Nar I, which has the same portion as Kas I 27. No sequence or filler of random nucleotide sequence 28. SB-I = Nsi I, the sticky protruding portion is compatible with Sbf I 29. No sequence or filler of random nucleotide sequence 30. BL-I = Sfo I 31. No sequence or filler of random nucleotide sequence 32. BL-2 = Pvu II 33. No sequence or filler of random nucleotide sequence 34. BL-3 = Nru I 35. No sequence or filler of random nucleotide sequence 36. SB-2 = BsrG I, sticky protruding portion is compatible with BsiW I 37. No sequence or random nucleotide sequence filler 38. CM-7 = Spe I 39. Random nucleotide sequence filler (or CMD module) 40. CM-8 = Sph I 41. Random nucleotide sequence filler (or SRS module) 42. HE-4 = PI-Sce I (front orientation) 43. Random nucleotide sequence filler (or BRD module) 44. VB-5 = BspE I Figures 17, 18, and 19 illustrate how the The multigene coupling vector in Figure 16 can serve as a DNA backbone using conventional subcloning methodologies, to construct a multigenic vector having two or more PE3 modules. This example of building a multigenic vector with three different PE3 modules employs the preferred order of subcloning events based on the statistical probability that the pivots necessary to create a PE3 acceptor domain in the coupling vector will not be present in any of the PE3 modules already included within the backbone of the multigenic vector. The preferred order of subcloning of the PE3 modules into the multigene coupling vector illustrated in Figure 16 is as follows: 1. A PE3 module must be subcloned into the PE3-2 filler domain first, and this PE3 module must be chosen according to the ordered absence of the following sites: i. Nsi I or BsrG I ii. Sfo I, Pvu II, or Nru I iii. BstX I 2. The second module of PE3 must be subcloned into the next domain of PE3-3, and this module of PE3 must be chosen according to the ordered absence of the following sites: iv. Sbf I or BsiW I v. Sbfl only saw. BsiW I vii. BstX I 3. The third module of PE3 must be subcloned into the PE3-1 domain. A PE3 module that meets the criteria listed in part I can be subcloned into the multigenic coupling vector by independently preparing the vector backbone and the PE3 module by digestion with two restriction enzymes, wherein one enzyme recognizes a single restriction site in the GP-I domain and the second enzyme recognizes a single restriction site in the GP-4 domain. The selected enzymes must not cut into the PE3 module. An example of this process is illustrated in Figure 17. A method for subcloning the PE3-A module in Figure 17 into the filler domain of PE3-2 requires the preparation of a linearized vector backbone produced by cutting the multigene coupling vector with the enzymes AsiS I and Rsr II. This molecular biology protocol produces the DNA coupling points with Sticky protruding portions specific to AsiS I and Rsr II. A linear DNA fragment containing the desired PE3-A module can also be prepared by digestion with AsiS I and Rsr II. The backbone of the resulting vector and the PE3-A module can be biochemically linked together using a DNA-ligase enzyme to generate a multigenic vector containing the PE3-A module. Figure 18 introduces a methodology by which the PE3 module that meets the criteria in part 2 can be subcloned into the multigenic coupling vector containing the PE3-A module at the multiple cloning site PE3-3 (PE3-3 MCS ). In this example, module PE3-A does not contain any Nsi I or BsrG I sites (designated in Figure 16 as SB-I and SB-2, respectively), and module PE3-B does not contain an Sbf I or BsiW site I. As described in Figure 18, a linearized PE3-B module is produced by digestion of the PE3-B module vector with Sbf I and BsiW I. The backbone of the linearized multigenic vector is prepared by digestion with Nsi I and BsrG I. The complementary sticky cohesive ends in the PE3-B module and the backbone of the multigenic vector can be linked together biochemically using a DNA-ligase enzyme. The resulting DNA molecule consists of the multigenic vector where the PE3-A and PE3-B modules are now a contiguous part of the DNA molecule. Figure 18 represents only one of the numerous strategies for subcloning a PE3 module within the PE3-3 filler domain in the multigene coupling vector. Other subcloning strategies include the production of linearized PE3 modules and linearized multigene coupling vectors that show sticky / blunt, blunt / sticky or blunt / blunt coupling points. It is predicted that these alternative subcloning strategies show lower success ratios to generate ligature products than those that would generate sticky / sticky strategies. These alternative strategies may be chosen if the structure of any of the PE3-A or PE3-B modules does not meet one or more of the criteria listed in subsection 2.

Figure 19 illustrates how a PE3-C module can be subcloned into the multigenic vector comprising the modules PE3-A and PE3-B. This strategy requires that neither PE3-A nor PE3-B contain a BstX I site. In this strategy, the multigene coupling vector is linearized by digestion with BstX I. The PE3-C module is linearized by digestion with the self-directed endonucleases I- Ceu I and I-Sce I. The resulting linear PE3-C module and the multigene coupling vector are linked together biochemically using a DNA ligase enzyme. The resulting DNA molecule consists of the multigenic vector in which the PE3-A, PE3-B, and PE3-C modules are now a contiguous part of the DNA molecule. Figure 21 illustrates how a PE3 module flanked by chromatin modification domains (CMD) or bacterial recombination domains (BRD) can be introduced into a multigenic coupling vector using bacterial recombination methodologies. In this example, the PE3 module replaces the PE3-1 filler domain through the action of homologous recombination between the flanking CMD / BRD domains of the PE3 module in the PE3 module vector and the PE3-1 filler domain in the PE3-1 vector. multigene coupling. This strategy requires the use of auxiliary DNA vectors or special bacterial strains, where the expression of the recombination enzymes can be induced by environmental changes (chemical, temperature or metabolic). Another embodiment of the invention, illustrated in Figure 16, can be defined by the nucleotide sequence residing between the vertebral column pivots of vector VB-I and VB-5, where VB-1 = Aat II and VB-5 = BspE I. 1. VB-I is Aat II 2. No sequence or random nucleotide sequence 3. ES-I = SexA I 4. Filler of random nucleotide sequence (or GHS module) 5. ES-2 = Srfl 6. Filler random nucleotide sequence (or BRD module) 7. HE-3 = I-Ppo I 8. Random nucleotide sequence filler (or SRS module) 9. No sequence or random nucleotide sequence filler 10. CM-1 = Kpn I 11. Random nucleotide sequence (or CMD module) 12. C -2 = Sac I 13. No sequence or filler of random nucleotide sequence 14. HE-I = I-Ceu I front orientation 15. Filler of random nucleotide sequence (or PE3 module) 16. HE-2 = I-Sce I reverse orientation 17. No sequence or random nucleotide sequence filler 18. CM-3 = Mfe I 19. Random nucleotide sequence filler (or CMD module) 20. CM-4 = Sac II 21. No sequence or random nucleotide sequence filler 22. GP- 1 = (AsiS I / Pac I / Sbf I) 23. Filler of random nucleotide sequence (or module PE3) 24. G-4 / CM-5 = (Swa I / Rsr II / BsiW I) 25. Filler of random nucleotide sequence (or CMD module) 26. CM-6 = Nar I, which has the same portion as Kas I 27. No sequence or filler of random nucleotide sequence 28. TX-I = Bst XI (I-Ceu I Front orientation 29. SB-I = Nsi I, the sticky protruding portion is compatible with Sbf I 30. No sequence or random nucleotide sequence filler 31. BL-I = Sfo I 32. No sequence or random nucleotide sequence filler 33. BL-2 = Pvu II 34. No sequence or filler of random nucleotide sequence 35. BL-3 = Nru I 36. No sequence or filler of random nucleotide sequence 37. SB-2 = BsrG I, the sticky protruding portion is compatible with BsiW I 38. No sequence or random nucleotide sequence filler 39. TX-2 = Bst XI (I-Sce I) inverse orientation 40. No sequence or random nucleotide sequence filler 41. CM-7 = Spe I 42. Random nucleotide sequence filler (or CMD module) 43. CM-8 = Sph I 44. Random nucleotide sequence filler (or SRS module) 45. HE-4 = PI-Sce I (front orientation) 46. Random nucleotide sequence filler (or BRD module) 47. VB-5 = BspE I Figure 20 illustrates how self-directed endonucleases can be used to introduce a PE3 module into the multigenic coupling vector described above. In this example, the PE3 module and the multigene coupling vector are linearized by digestion with the self-directed endonucleases I-Ceu I and I-Sce I. The resulting linear PE3 module can be linked to the multigene coupling vector biochemically using a DNA-ligaseA enzyme. This subcloning strategy can be used without a PE3 module inside a multigenic coupling vector, or that is introduced into a coupling vector, it does not meet one or more of the criteria listed in paragraphs 1 to 3. The coupling vector multigenic (primary coupling plasmid) shown in Figure 8 can be used to assemble two completed PE3 transgenes, which are first constructed in the plasmids of the PE3 coupling station, or two homology arms needed to build urt transgender to the gene or to introduce two types of elements of positive or negative selection. A non-limiting example of a multiple cloning site (MCS) in a multigene (or primary) coupling plasmid is shown in Figure 9, and comprises the following sequential elements, in the listed order: 1. Two common restriction sites do not variables and unique ones that define a 5 'insertion site for the mutated pUC19 vector described above (eg, Aat II and BIp I), 2. A M13 primer site Rev., 3. A pair of unique HE sites in opposite orientation that flanking a random nucleotide sequence of DNA that can serve as the acceptor module of the host selector gene, genomic expression (RNAS-GEH-S1) (eg, PI-SceI (forward orientation) and PI-SceI (reverse orientation)), 4. A common, non-variable and unique restriction site that allows the cloning of a shuttle vector module with 3 'address of the HE pair (eg, Eco0109l), 5. A fixed grouping of rare, non-variable restriction sites, Which defines n the 5 'portion a module of the left recombination arm (for example, SgrA I and AsiS T), 6. The random nucleotide sequences that can serve as an acceptor module of the left recombination arm (RNAS-LRA), 7. A fixed clustering of rare, non-variable restriction sites that define the 3 'portion of the acceptor module of the left recombination arm (e.g., Pací, MluI, and Ascl), 8. A single HE site (e.g., I-Ceu I (front orientation)), 9. A pair of common, non-variable, and unique restriction sites that flank a random DNA nucleotide sequence that can serve as an acceptor module of the chromatin modification module (RNAS-C DI) (e.g. , Kpn I and Avr II), 10. A primer site T7, 11. A pair of unique BstX I sites in opposite orientation (where the variable nucleotide region at the BstX I restriction site is defined by nucleotides identical to queues do not complement s generated by the arrangement of the two identical HE restriction sites accommodated in reverse-complementary orientation; for example, PI-SceI (forward orientation) and PI-SceI (reverse orientation)) flanking a random DNA nucleotide sequence that can serve as a complex transgene acceptor module (RNAS-PE3-1), 12. A pair of unique HE sites in opposite orientation that flank a random DNA nucleotide sequence that can serve as a complex transgene acceptor module (RNAS-PE3-2) (eg, I-Scel (forward orientation) and I-Scel (reverse orientation) ), 13. A T3 primer site in reverse orientation, 14. A pair of common, non-variable and unique restriction sites, flanking a random DNA nucleotide sequence that can serve as a chromatin modifying domain acceptor module (RNAS-CMD-2) (eg, Xho I and Nhe I ), 15. A unique HE site (here, identical to that in the preceding paragraph 8 (IScel), and also in reverse orientation). 16. A fixed grouping of rare, non-variable restriction sites that define in the 5 'portion a Right Recombination Arm module (eg, SnaB I, Sal I, and Not I), 17. The random nucleotide sequences that can be serve as a Right Recombination Arm acceptor module (RNAS-RRA), 18. A fixed grouping of rare, non-variable restriction sites that define in the 3 'portion of the Right Recombination Arm acceptor module (eg, Rsr II, Swa I, and BsiW I), 19. A common, non-variable and unique restriction site that allows the cloning of a shuttle vector module with a 5 'address of a HE pair (for example, BspE I), 20. A pair of unique HE sites in opposite orientation that flank a random DNA nucleotide sequence that can serve as a gene acceptor module, host selector of genome expression (RNAS-GEH-S2) (eg, PI-Psp I ( front orientation) and PI-Psp I (orientation reverse)), 21. A forward primer site 13 placed in reverse orientation, 22. The common non-variable and unique restriction sites, which define a 3 'insertion site for the mutated pUC19 vector described above (e.g., Pme I , Sap I, and BspH I). As an example of the method of practicing the present invention, a transgene containing these elements can be constructed: 1. The nucleotide sequences of the human promoter for surfactant proteins C (SP-C), 2. The sequences encoding the protein product of the beta-c receptor of the granulocyte-macrophage colony stimulating factor of the mouse gene (GMR / c) 3. The rabbit beta-globin intron sequences, and 4. A poly-A signal of the SV40. The SP-C sequence contains the internal BamHI sites and can be released from its parent plasmid only with Notl and EcoRI. GMR c has an internal Notl site, and can be cut from its parent plasmid with BamHI and Xhol. The rabbit beta-globin intron sequences can be cut from their parent plasmid with EcoRI. The poly-A tail of the SV-40 can then be cut from the parent plasmid with Xhol and Sacl. Due to the redundancy of several restriction sites, none of the parental plasmids can be used to assemble all the necessary fragments. The steps used to construct the desired transgene in the PE3 coupling plasmid of the invention are as follows: 1. Since Notl and PspOMl generate compatible cohesive ends, the sequences of the human SP-C promoter are excised with NotI and EcoRI and cloned within the PspOMl and EcoRI sites of the shuttle vector P. The product of this reaction is called pSVP-SPC 2. After the propagation and recovery steps well known to those skilled in the art, the beta-globin intron sequences of Rabbit are cloned into the EcoRI site of pSVP-SPC. The orientation of the intron in the resulting intermediate construct is modified by sequencing the product, called pSVP-SPC-rpG. 3. The promoter and the intron are excised and isolated as a contiguous fragment from pSVP-SPC-rG using AsiSl and Ascl. Concurrently, the PE3 coupling plasmid is cut with AsiSl and Ascl in preparation for ligation with the promoter / intron segment. The promoter / intron fragment is ligated into the Coupled Plasmid, propagated and recovered. 4. The Xho 1 site of the GMF ^ c fragment is filled in to create a blunt 3 'end, using techniques well known to those skilled in the art. This is then cloned into the BamHI site and the Pvu2 site of the blunt end of pSVP-SPC-rpG. The resulting plasmid (pDP-SPC-GMRPc-rG) is recovered and propagated. 5. The final cloning step is the addition of the poly-A tail of the SV-40. The SV40-polyA fragment is cut with Xhol and SacI, as is the vector container pDSl-SPC-GMRpc-rb G. Both pieces of DNA are gel purified and recovered. A ligation mixture is prepared with a 10: 1 molar ratio of SV-40-polyA to pDSl-SPC-GMR c- ^ G. The ligature products are propagated and harvested.

The new plasmid, pDSl-SPC-GMRp-r G-pA contains all the elements required for the transgene, including a unique restriction site at the 3 'end with which the plasmid pDSl-SPC-G R c-r3G-pA complete can be linearized for transfection within eukaryotic cells or microinjection within the pronucleus of a fertilized egg. Regarding the HE sites, typically at least two HE restriction sites, each capable of being cleaved by at least one HE restriction enzyme, are placed flanking the modular regions, for the purpose of creating a cassette acceptor site of the gene that He can not self-restrain himself. Also, if desired, it is possible but it is not required to place these HE sites in opposite orientation to one another. That is, because the HE sites are asymmetric and non-palindromic, it is possible to generate cohesive 3 'tails that protrude, not complement, by placing two HE restriction sites in opposite orientation. For example, I-Scel of HE cuts its cognate restriction site as indicated by "/": 5 '... TAGGGATAA / CAGGGTAA T ... 3' 3 '... ATCCC / TATTGTCCCATT A ... 5' Reverse placement of a second site within an MCS could generate two cohesive non-complementary cohesive tails: 5 '... TAGGGATAA CCCTA ... 3' 3 '... ATCCC AATAGGGAT ... 5' This is particularly useful when They want to include large transgenes within a vector. Due to the large size of the insert, it is thermodynamically more favorable for a self-annealing vector instead of accepting a large insert. The presence of the non-complementary tails generated by the placement of the HE restriction sites provides favorable chemical forces to counteract the thermodynamic inclination for self-ligating. In addition, the asymmetric nature of most of the HE protruding tails also creates a powerful cloning tool when used in combination with the BstX I restriction enzyme site (5 'CCANNNNN / NTGG 3') · The neutral domain in sequence of BstX I ("N" can be any nucleotide) can be used to generate compatible cohesive ends for two overhanging HE tails of reverse orientation, while auto-annealing is excluded.

BstX I (I-Sce I Del.) I-Sce I Front I-Sce I Inverse BxtX I (I-Sce I Inv.) '-CCAGATAA CAGGGTAAT / / ATTACCCTGTTAT GTGG-3 3 '-GGTC TATTGTCCCATTA / XTAATGGGAC AATACACC-5' Other endonucleases not included in these listings can also be used, maintaining the same functionality and the spirit and intent of the invention. Among the many advantages of the present invention, it can be readily appreciated that transgenes can be assembled rapidly, typically containing the 3 'promoter, expression and regulator modules in a very short period of time, as well as rapidly and easily varying or redesigning a transgen freshly assembled. Conventional efforts to vary an assembled transgene using known methods could usually take a year or more of laboratory time. Using the methods of the present invention, desired transgenes can be made within days or weeks, and then the desired test of each is performed, thereby saving significant researcher time and expense. The shuttles that were originally created by de novo synthesis, recombination and protruding PCR terminator portion cloning methods can be taken and used with the coupling tip technology of the present invention to quickly assemble these pre-processed elements into a plurality of transgenes. The transgenes of PE3 produced using the invention can be used in a single organism, or in a variety of organisms including bacteria, yeasts, mice and other eukaryotes with little or no further modification. While the present invention has been illustrated by the description of the embodiments thereof, and while the modalities have been described in considerable detail, it is not intended to restrict or in any way limit the scope of the appended claims to such detail. The advantages and additional modifications will be readily apparent to those skilled in the art. The invention in its broader aspects is therefore not limited to the specific details, the representative structure methods, and the illustrated examples shown and described. Accordingly, deviations from such details may be made without departing from the scope of the invention as claimed. It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (18)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A domain module coupling vector consisting of a DNA cloning vector, characterized in that it comprises a multiple cloning module (MC) for the subcloning of a genetic material of interest within the MC module, the MC module comprises: a. a first gene pivot (GP) comprising at least two rare, non-variable restriction sites operable to define the 5 'portion of the MC module; b. a nucleic acid sequence comprising a multiple cloning site (MCS) that includes a plurality of restriction sites selected from the common restriction sites, which are unique within the coupling vector of the domain module, to provide cloning sites for the cloning of the genetic material of interest within the MC module; and c. a second gene pivot comprising at least two rare, non-variable restriction sites operable to define the 3 'portion of the MC module.
2. 2. The domain module coupling vector according to claim 1, characterized in that the first gene pivot and the second gene pivot independently comprise at least 3, and not more than 4, rare, non-variable restriction sites.
3. 3. The domain module coupling vector according to claim 1, characterized in that the genetic material of interest is selected from the group consisting of a promoter domain, an expression domain and a 3 'regulatory domain.
4. 4. The domain module coupling vector according to claim 1, characterized in that the rare, non-variable restriction site is selected from the group consisting of AsiS I, Pac I, Sbf I, Fse I, Ase I, Mlu I, SnaB I, Not I, Sal I, Swa I, Rsr II, BSiW I, Sfo I, Sgr AI, AflIII, Pvu I, Ngo MIV, Ase I, FIp I, Pme I, Sda I, Sgfl, Srf I , and Sse8781 I.
5. 5. The domain module coupling vector according to claim 4, characterized in that when the genetic material of interest is a promoter domain, the first group of rare, non-variable restriction sites is selected from the group consisting of at least AsiS I, Pac I, and Sbf I, and the second group of rare, non-variable restriction sites are selected from the group consisting of at least Fse I, Ase I, and Mlu I; when the genetic material of interest is an expression domain, the first group of rare, non-variable restriction sites is selected from the group consisting of at least Fse I, Ase I, and Mlu I, and the second restriction group sites rare, not variable, is selected from the group consisting of at least SnaB I, Not I, and Sal I; and when the genetic material of interest is a 3 'regulatory domain, the first group of rare, non-variable restriction sites is selected from the group consisting of at least SnaB I, Not I, and Sal I, and the second group of non-variable restriction sites is selected from the group consisting of at least Swa I, Rsr II, and BSiW I.
6. 6. The domain module coupling vector according to claim 5, characterized in that when the genetic material of interest is the promoter domain, the first group of rare, non-variable restriction sites, consists of, in order AsiS I, Pac I, and Sbf I, and the second group of rare, non-variable restriction sites consists of, in order, Fse I, Ase I, and Mlu I; when the genetic material of interest is the expression domain, the first group of rare, non-variable restriction sites consists of, in order, Fse I, Ase I, and Mlu I, and the second group of restriction sites given, non-variables consists of, in order, SnaB I, Not I, and Sal I; and when the genetic material of interest is the 3 'regulatory domain, the first group of rare, non-variable restriction sites consists of, in order, SnaB I, Not I, and Sal I, and the second group of rare restriction sites, non-variables consists of, in order, Swa I, Rsr II, and BSiW I.
7. 7. A PE3 coupling vector consisting of a DNA cloning vector, characterized in that it comprises a cloning module PE3 comprising a plurality of cloning modules, configured to clone a plurality of domain modules within the cloning module of PE3, the cloning module of PE3 comprises: a. a first gene pivot comprising at least two rare, non-variable restriction sites that after cloning is operable to define the 5 'portion of a promoter module; b. a first filler module consisting of a first nucleic acid sequence comprising the filler, which after the cloning is replaced by the promoter module; c. a second gene pivot comprising at least two rare, non-variable restriction sites, which after cloning is operable to define a shared junction between the 3 'portion of the promoter module and the 5' portion of an expression module; d. a second filler module consisting of a second nucleic acid sequence comprising the filler, which after the cloning is replaced by the expression module; and. a third gene pivot comprising at least two rare, non-variable restriction sites that after cloning is operable to define a shared junction between the 3 'portion of the expression module and the 5' portion of the regulatory module 3 '; F. a third filler module consisting of a third nucleic acid sequence comprising the relielor, which after cloning is replaced by the regulatory module 3 '; and g. a fourth gene pivot comprising at least two rare, non-variable restriction sites that after cloning is operable to define the 3 'portion of regulatory module 3'.
8. 8. The coupling vector PE3 according to claim 7, characterized in that the first gene pivot and the second gene pivot independently comprise at least 3, and not more than 4, rare, non-variable restriction sites.
9. 9. The PE3 coupling vector according to claim 7, characterized in that the rare, non-variable restriction site is selected from the group consisting of AsiS I, Pac I, Sbf I, Fse I, Ase I, Mlu I, SnaB I, Not I, Sal I, Swa I, Rsr II, BSiW I, Sfo I, Sgr AI, AflII, Pvu I, Ngo IV, Ase I, FIp I, Pme I, Sda I, Sgf I, Srf I, and Sse8781 1.
10. The domain module coupling vector according to claim 9, characterized in that when the genetic material of interest is a promoter domain, the first group of rare, non-variable restriction sites is selected from the group which consists of at least AsiS I, Pac I, and Sbf I, and the second group of rare, non-variable restriction sites is selected from the group consisting of at least Fse I, Ase I, and Mlu I; when the genetic material of interest is an expression domain, the first group of rare, non-variable restriction sites is selected from the group consisting of Fse I, Ase I, and Mlu I, and the second group of rare restriction sites , non-variable is selected from the group consisting of at least SnaB I, Not I, and Sal I; and when the genetic material of interest is a 3 'regulatory domain, the first group of rare, non-variable restriction sites is selected from the group consisting of at least SnaB I, Not I, and Sal I, and the second group of Rare, non-variable restriction sites are selected from the group consisting of at least Swa I, Rsr II, and BSiW I.
11. 11. The domain module coupling vector according to claim 10, characterized in that when the genetic material of interest is the promoter domain, the first group of rare, non-variable restriction sites, consisting of, in order AsiS I, Pac I, and Sbf I, and the second group of rare, non-variable restriction sites, consists of, in order, Fse I, Ase I, and lu I; when the genetic material of interest is the expression domain, the first group of rare, non-variable restriction sites consists of, in order, Fse I, Ase I, and Mlu I, and the second group of restriction sites given, non-variables consists of, in order, SnaB I, Not I, and Sal I; and when the genetic material of interest is the 3 'regulatory domain, the first group of rare, non-variable restriction sites consists of, in order, SnaB I, Not I, and Sal I, and the second group of rare restriction sites , non-variables consists of, in order, Swa I, Rsr II, and BSiW I.
12. 12. The coupling vector PE3 according to claim 7, characterized in that it comprises a means for inserting the PE3 module into a multigenic coupling vector.
13. 13. A multiple cloning coupling vector of PE3 (MC) consisting of a DNA cloning vector, characterized in that it comprises a cloning module PE3 configured to clone the promoter, expression, and 3 'regulator modules within the module of cloning PE3, the PE3 cloning module comprises: a. a first gene pivot comprising at least two rare, non-variable restriction sites that after cloning is operable to define the 5 'portion of a promoter module; b. a first nucleic acid sequence; c. a second gene pivot comprising at least two rare, non-variable restriction sites, which is then operable to define a shared junction between the 3 'portion of the promoter module and the 5' portion of the expression module; d. a second nucleic acid sequence; e. a third gene pivot comprising at least two rare, non-variable restriction sites that after cloning is operable to define a shared junction between the 3 'portion of the expression module and the 5' portion of the regulatory module 3 '; F. a third nucleic acid sequence; and g. a fourth gene pivot comprising at least two rare, non-variable restriction sites, which after cloning is operable to define the 3 'portion of regulatory module 3'; wherein at least one of the first, second and third nucleic acid sequences is a multiple cloning module comprising a multiple cloning site (MCS) comprising a plurality of restriction sites selected from the common restriction sites that are unique within the coupling vector PE3, to provide cloning sites for the cloning of a genetic material of interest within the multiple cloning module, and the remaining nucleic acid sequences are fillers.
14. 14. The coupling vector MC of PE3 according to claim 13, characterized in that the first nucleic acid is the multiple cloning module, and wherein the first gene pivot is selected from the group consisting of at least AsiS I, Pac I , and Sbf I, and the second gene pivot is selected from the group consisting of at least Fse I, Ase I, and Mlu I.
15. 15. The coupling vector MC of PE3 according to claim 13, characterized in that the second acid nucleic is the multiple cloning module, and wherein the second gene pivot is selected from the group consisting of at least Fse I, Ase I, and Mlu I, and the third gene pivot is selected from the group consisting of at least SnaB I , Not I, and Sal I.
16. 16. The coupling vector MC of PE3 according to claim 13, characterized in that the nucleic acid sequence is the multiple cloning module, and wherein the second gene pivot is selected from the group that cons SnaB I, Not I, and Sal I, and the third gene pivot is selected from the group consisting of at least Swa I, Rsr II, and BSiW I.
17. 17. Coupling vector C of PE3 in accordance with the claim 13, characterized in that the first gene pivot consists of, in order, AsiS I, Pac I, and Sbf I, the second gene pivot consists of, in order, Fse I, Ase I, and Mlu 1, the third gene pivot consists of , in order, SnaB I, Not I, and Sal I; and the fourth gene pivot consists of, in order, Swa I, Rsr II, and BSiW I.
18. 18. The coupling vector MC of PE3 according to claim 13, characterized in that it further comprises a means for releasing the PE3 module from the vector PE3, for insertion into a multigene coupling vector.