Molecular cloning method using type IIS restriction enzymes

Abstract

Molecular cloning method using Type IIS restriction enzymes The invention is addressed to a cloning method for obtaining a destination nucleic acid vector comprising "n" inserts, wherein "n" is an integer of at least 1, and each insert comprises a sequence of interest "S" and, wherein the method comprises contacting in a reaction mixture: (i) a receiving backbone, wherein: (a) one end of the receiving backbone comprises a palindromic overhang and the other end comprises a non-palindromic overhang, or (b) each end of the receiving backbone comprises a different palindromic overhang; (ii) the n inserts, wherein each insert comprises two different overhangs, wherein the overhangs comprised in the n inserts are different overhangs, and each one is complementary only to another overhang comprised in another insert or to an overhang of the receiving backbone, and is non-palindromic, except the insert's overhang(s) complementary to the palindromic overhang(s) of the receiving backbone, and (iii) a DNA ligase; wherein the molar ratio of the receiving backbone and the insert(s) comprising a palindromic overhang is from 1:0.1 to 1:1.9 (receiving backbone:insert), and the molar ratio of the receiving backbone and the remaining inserts is from 1:1.1 to 1:3 (receiving backbone:insert). In a particular embodiment, at least one of the n inserts and the receiving backbone are obtained upon digestion of one or more polynucleotides and a receiving vector with Type IIS restriction enzyme(s).

Description

[0001] Molecular cloning method using Type IIS restriction enzymes.

[0002] This application claims the benefit of European Patent Application EP25382166.4 filed on February 24th2025.

[0003] Technical Field

[0004] The present invention relates to the field of molecular cloning methods for the obtention of nucleic acid vectors, and to the products thus obtained.

[0005] Background Art

[0006] Protein engineering through domain combination is a cutting-edge area in biotechnology and bioengineering, focusing on protein modularity to create new proteins with unique properties. This method involves reorganizing functional domains, akin to building with blocks, to achieve various biological activities. A significant challenge is generating cloning libraries, which are collections of protein variants designed to explore potential domain combinations. Creating these libraries requires overcoming technical challenges in cloning and assembly, necessitating high-efficiency methods like homologous recombination cloning. Different methodologies for designing modular cloning libraries include Golden Gate, Gibson assembly, and Gateway, each with its pros and cons. Gateway is simple and efficient but limited in cloning multiple coding sequences in frame, while Gibson assembly allows in-frame, scarless cloning, mainly used for mutagenesis libraries. Golden Gate cloning is a technique that utilizes type IIS restriction enzymes, which cut DNA outside their recognition sites, allowing for flexible selection of overhangs. This method enables efficient cloning of multiple inserts in "one-pot reactions" with minimal sample manipulation. The process involves cycles of restriction-ligation, enriching the desired ligation product. Commonly used type IIS enzymes include Bsal, Bbsl, and Esp3l. Golden Gate and its derivatives are employed for creating plasmid libraries and cloning complex polypeptides.

[0007] VersaTile, a type of Golden Gate assembly, describes a method for preparing a "tile vector” that contains “a coding polynucleotide sequence immediately preceded and followed by a type IIS recognition sequence, wherein said preceding and following recognition sequences are recognized by a same type IIS restriction enzyme, but have an opposite orientation" (similar to any conventional Golden Gate assembly reaction). The assembly takes place in a "one-pot” reaction, in the presence of the "tile plasmids”, a receiver plasmid containing sites for the same type IIS restriction enzyme, the corresponding Type IIS enzyme and DNA ligase, and following cycles of restriction-ligation. Thus, Versatile strategy replicates the conventional Golden Gate assembly method.

[0008] However, it is well known in the art that efficiency of the Versatile strategy - as well as other known Golden Gate based cloning methods - is significantly reduced when palindromic overhangs (such as those formed by Type IIP enzyme digestion) are generated by Type IIS restriction enzyme digestion duringrestriction / ligation cycles, due to self-religation of the digested fragments at the palindromic overhangs. Thus, not only the characteristics of the end products that can be efficiently obtained is limited in said methods, but also the type of original cloning vectors and insert sequences that can be used.

[0009] Therefore, there is a need in the art for a method that combines the advantages of Golden Gate based methods, and that is compatible with cloning vectors forming palindromic overhangs after restriction enzyme digestion, and / or that allow forming end products that retain the palindromic sequences, including Type IIP restriction enzyme target sites.

[0010] Summary of the Invention

[0011] The inventors have developed the TelClo cloning method which is characterized in that it comprises digestion of original nucleic acid material by Type IIS restriction enzyme and ligation of digested products in a same reaction mixture (i.e. a Golden Gate based method), wherein the vector backbone used comprises at least one palindromic overhang. As well known by an expert in the field, and as indicated in the background section, in Golden Gate based methods, it is contraindicated to work with palindromic overhangs, as self-religation of the digested products with said palindromic overhangs during restriction / ligation cycles can take place, which considerably reduces the method's efficiency and ligation fidelity. Surprisingly, however, the method developed by the inventors maintains a high performance and ligation fidelity. Importantly, the method comprises providing a molar ratio of around 1:0.1-1: 1.9 (vector backbone: insert ) for inserts comprising a palindromic overhang, and a molar ratio of around 1 :1.1-1 :3.5 (vector backbone:insert) for inserts with non-palindromic overhangs.

[0012] Indeed, as shown in example 5 (see Fig. 8 and 4), despite the predicted 50% efficiency of the method due to the presence of palindromic overhangs in the vector backbone and corresponding insert, 100% of successful clones were obtained with the TelClo method, when one insert was introduced in the vector backbone. When 6 inserts were introduced in the vector backbone, 93% successful clones were obtained, showing that the high efficiency and ligation fidelity of the method is maintained when several inserts are used.

[0013] The method thus provides the advantages associated to Golden Gate methods (such as performing the restriction / ligation reaction in a single pot and / or designing the position and orientation of one or several inserts within a destination vector in a versatile way by designing the sequence of the inserts' overhangs), and, in addition allows obtaining final products (i.e. a destination vector) with a sequence of ligated inserts, wherein the 5' or 3' end of the sequence of inserts can be and lead to a palindromic sequence, such as a Type IIP restriction enzyme target site, (re)generated during the cloning method. This is particularly useful for easy excision or substitution of the nucleic acid sequence immediately upstream or downstream the sequence of inserts in the destination vector by a simple Type IIP restriction enzyme digestion. In addition, the method also allows using "traditional” cloning vectors, i.e. comprising Type IIP based multicloning sites,or with minimal modifications for working with Type IIS enzymes (i.e. , for instance, no or only one Type IIS cloning site required instead of at least 2).

[0014] Thus, in a first aspect, the invention is addressed to a cloning method for obtaining a destination nucleic acid vector comprising “n” inserts, wherein “n” is an integer of at least 1, and each insert comprises a sequence of interest “S” and, wherein the method comprises contacting in a reaction mixture:

[0015] (I) a receiving backbone, wherein:

[0016] (a) one end of the receiving backbone comprises a palindromic overhang and the other end comprises a non-palindromic overhang, or

[0017] (b) each end of the receiving backbone comprises a different palindromic overhang;

[0018] (ii) the n inserts, wherein each insert comprises two different overhangs and

[0019] (a) when the receiving backbone is as defined in (i)-(a):

[0020] - when n is 1, an overhang comprised in the insert is a palindromic overhang complementary to the palindromic overhang of the receiving backbone such that, upon binding between said two complementary palindromic overhangs, a Type IIP restriction enzyme target site is formed and the other overhang comprised in the insert is non- palindromic and complementary to the non-palindromic overhang of the receiving backbone,

[0021] - when n is of 2 or more, an overhang comprised in a first insert is a palindromic overhang complementary to the palindromic overhang of the receiving backbone such that, upon binding between said two complementary palindromic overhangs, a Type IIP restriction enzyme target site is formed, and the other overhang comprised in said first insert and the overhangs comprised in the remaining (n-1) inserts are different non-palindromic overhangs, and each one of the said non-palindromic overhangs is complementary only to another non-palindromic overhang comprised in another insert, or to the non- palindromic overhang of the receiving backbone, provided the non-palindromic overhang comprised in said first insert is not complementary to the non-palindromic overhang of the receiving vector; and

[0022] (b) when the receiving backbone is as defined in (i)-(b):

[0023] - when n is 1, the insert comprises two palindromic overhangs, each one being complementary to a different palindromic overhang of the receiving backbone, such that upon binding between said two pairs of complementary palindromic overhangs, two different Type IIP restriction enzyme target sites are formed,

[0024] - when n is 2 or more, a first and a second insert comprise a palindromic overhang, each one being complementary to a different palindromic overhang of the receiving backbone, such that upon binding between said two pairs of complementary palindromic overhangs, two different Type IIP restriction enzyme target sites are formed, and the other overhang comprised in the first and the second inserts and the overhangs of the remaining (n-2)inserts are different non-palindromic overhangs, and each one of the said non-palindromic overhangs is complementary only to another non-palindromic overhang comprised in another insert;

[0025] and

[0026] (ill) a DNA ligase;

[0027] wherein the molar ratio of the receiving backbone and the insert(s) comprising a palindromic overhang is from 1:0.1 to 1:1.9 (receiving backbone:insert), and the molar ratio of the receiving backbone and the remaining inserts is from 1:1.1 to 1:3 (receiving backbone: insert).

[0028] As well known by an expert in the field, a main advantage of Type IIS restriction enzymes, is that the cleavage site is outside their recognition site, and the overhang formed by the Type IIS restriction enzyme digestion is determined by the number of nucleotides between the Type IIS recognition site and its cleavage site, independently of the specific nucleotides surrounding the cleavage site. Such a technical characteristic implies that any overhang sequence can be generated by Type IIS restriction enzyme digestion. Therefore, the method allows designing the overhangs formed, so that not only the position and orientation of the inserts is predetermined, but also the nucleotides comprised between the sequences S of the inserts in the destination vector. Thus, the method can be specifically designed to obtain a destination vector comprising several inserts in a predetermined position and orientation, more specifically, encoding a protein sequence resulting from the predetermined concatenation of peptides encoded by the n inserts or sequences S, that are in frame within the destination vector. As well understood by a skilled person, the method of the invention can be performed with any sequence S, as the formation of the end product does not depend on said sequences.

[0029] In a second aspect, the invention is addressed to a nucleic acid vector obtained by the cloning method according to the first aspect.

[0030] Additionally, as indicated above, an advantage of the method of the first aspect is that, in addition to provide the advantages of most Golden Gate based methods, it allows maintaining Type IIP target sites in the final product, despite the use of Type IIS restriction enzymes in the cloning method (whose recognition site is lost in the end product). The method thus allows an easy excision or substitution of nucleic acid sequences flanked by Type IIP target sites, conserved or regenerated with the method of the first aspect. When said nucleic acid sequence is a protein tag encoding sequence, substitution of the tag encoding sequence can thus be easily performed by traditional cloning methods.

[0031] Thus, in a third aspect, the invention is addressed to a method for the synthesis of a library of different nucleic acid vectors, wherein: each vector from the library is as a destination vector obtained by the first aspect of the invention wherein the n inserts comprise or consist of peptide encoding sequence; each vector from the library comprises a protein tag encoding sequence; and those vectors from the library that differ inthe combination and / or orientation of the n inserts, differ in the protein tag encoding sequence of interest, and those vectors from the library having the same combination and / or orientation of n inserts, have the same protein tag encoding sequence of interest; the method comprising:

[0032] (i) performing the method of the first aspect of the invention for obtaining different destination vectors that differ in the combination and / or orientation of the n inserts, each one comprising a protein tag encoding sequence flanked by two different type IIP target sites, and

[0033] (ii) substituting the protein tag from the destination vectors obtained from step (i) by another protein tag of interest that is the same for all destination vectors having the same combination and / or orientation of n inserts, and different between destination vectors having a different combination and / or orientation of n inserts.

[0034] In a fourth aspect, the invention is addressed to the library of vectors obtained by the method of the third aspect.

[0035] In a fifth aspect, the invention is addressed to a protein encoded by the nucleic acid vector according to the second aspect, wherein the nucleic acid sequence of the vector encoding the protein comprises the n inserts.

[0036] In a sixth aspect, the invention is addressed to a method for the synthesis of a protein, comprising (i) providing to a protein expression system the nucleic acid vector obtained by the cloning method according to the first aspect, wherein the n inserts comprise or consist of peptide encoding sequences and (ii) subjecting the protein expression system to conditions appropriate for the expression of the protein.

[0037] In a seventh aspect, the invention is addressed to a method for the synthesis of library of proteins, wherein the library of proteins comprises several proteins according to the fifth aspect, particularly of protein obtained by the method as defined by the sixth aspect as defined above, wherein proteins that differ in the sequence, number and / or position of the protein sequences encoded by the n inserts, also differ in a protein tag sequence, the method comprising:

[0038] (i) performing the method for the synthesis of a library of different nucleic acid vectors of the third aspect,

[0039] (ii) providing to a protein expression system the nucleic acid vectors of the library of vectors obtained from step (i), and

[0040] (ill) subjecting the protein expression system to conditions appropriate for the expression of the proteins.

[0041] In an eighth aspect, the invention is addressed to the protein library obtained by the method of the seventh aspect.In a ninth aspect, the invention is addressed to a kit comprising means for performing the method as defined by the first, third, sixth or seventh aspect.

[0042] In a tenth aspect, the invention is addressed to a reaction mixture comprising the receiving backbone, the n inserts, and the DNA ligase as defined in the first aspect.

[0043] Brief description of the drawings

[0044] Figure 1. Schematic Representation of the three receiving plasmids with or without signal peptides and no tag in the TelClo system. The figure illustrates three receiving plasmids in the TelClo system, each with different signal peptide configurations. (A) Plasmid 1 (SEQ ID NO. 26) includes an OmpA signal peptide for extracellular secretion. (B) Plasmid 2 (SEQ ID NO. 27) has a PelB signal peptide for secretion targeting. (C) Plasmid 3 (SEQ ID NO: 28) lacks a signal peptide, allowing protein expression without secretion. This setup offers flexibility for tailored plasmid configurations.

[0045] Figure 2. Schematic Representation of TelClo receiving vectors incorporating different Tags. Three plasmid designs in the TelClo system: (A) Plasmid 1 (SEQ ID NO. 29) has a His-tag and EcoRI site for tag exchange and screening; (B) Plasmid 2 (SEQ ID NO. 30) lacks a tag but has an Ndel site for similar purposes; (C) Plasmid 3 (SEQ ID NO. 31) includes a Streptavidin II tag and Acc65l site for tag exchange and screening.

[0046] Figure 3. Schematic Representation of Final Ligation Products. The figure illustrates the final ligation products (SEQ ID NO. 32-37) based on the number of DNA blocks concatenated, with a maximum of six blocks. Each ligation position is annotated with its corresponding overhangs (OH) and the "scar” introduced for each position during the assembly process. At the 3' end of the final construct, the BamHI site (GGATCC) for Tag exchange is included.

[0047] Figure 4. Colony check PCR showing the results for the screening of 30 colonies for 1 block (A) and 6 blocks (B) TelClo ligation.

[0048] Figure 5. Schematic Representation of the Receiving Plasmid pTT-SP-PelB-TagEx-Term in the TelClo System. The figure illustrates the structure of the receiving plasmid used in the TelClo system (SEQ ID NO.

[0049] 38). The expression of the final protein is inducible and regulated by the T7 promoter (not shown). The plasmid features two Bsal type-IIS restriction sites, which facilitate plasmid linearization and removal of the multiple cloning site (MCS). The 3' Bsal site generates a palindromic overhang that is part of a BamHI (type-IIP) restriction site. The C-terminal tag-shuffling sequence is flanked by this BamHI site and an Xhol restriction site, and it includes a tag-specific restriction site (in this case, Ndel) to enable both negative selection and diagnostic screening. The combination of BamHI and Xhol restriction sites allows the creation of receiving vectors with different tags or facilitates the exchange of an existing tag in plasmids alreadycontaining cloned proteins. Hindi II and Sall are negative selection enzymes used to digest the final ligation product, eliminating background from undigested plasmids. Key features include the ribosome binding site (RBS) upstream of the MCS, ensuring efficient translation of the expressed protein. This versatile design supports streamlined cloning and tag exchange processes within the TelClo system.

[0050] Figure 6. Generation of ready-to-clone duplexes via hybridization of ssDNA oligos. This figure illustrates the process of generating ready-to-clone DNA duplexes through the hybridization of singlestranded DNA (ssDNA) oligonucleotides. The hybridization step creates double-stranded DNA fragments with specific overhangs, facilitating seamless ligation into the cloning vector.

[0051] Figure 7. Generation of Ready-to-Clone Domain Coding Sequences by PCR Amplification. This figure depicts the process of producing ready-to-clone domain coding sequences through PCR amplification. The diagram highlights the key steps, including the generation of specific overhangs essential for seamless ligation. Small arrows indicate the cleavage sites and direction of the type IIS restriction enzyme, emphasizing the precision and compatibility achieved during the cloning process.

[0052] Figure 8. Estimated ligation fidelity for the proposed cloning system. The ligation fidelity significantly decreases in the presence of a palindromic overhang. But using a plasmid to insert molar ratio 1:1 for the insert with the palindromic overhang and 1 :2 for the rest of inserts, significantly overcomes this problem.

[0053] Detailed description of the invention

[0054] All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly through-out the specification and claims unless an otherwise expressly set out definition provides a broader definition.

[0055] As used herein, the indefinite articles "a” and "an” are synonymous with "at least one” or "one or more.” Unless indicated otherwise, definite articles used herein, such as "the” also include the plural of the noun.

[0056] For the purposes of the present invention, any ranges given include both the lower and the upper end-points of the range.

[0057] I- Cloning method of the first aspect of the invention

[0058] In a first aspect, the invention is addressed to a cloning method for obtaining a destination nucleic acid vector comprising “n” inserts, wherein “n” is an integer of at least 1, and each insert comprises a sequence of interest “S” and, wherein the method comprises contacting in a reaction mixture:(i) a receiving backbone, wherein:

[0059] (a) one end of the receiving backbone comprises a palindromic overhang and the other end comprises a non-palindromic overhang, or

[0060] (b) each end of the receiving backbone comprises a different palindromic overhang;

[0061] (ii) the n inserts, wherein each insert comprises two different overhangs and

[0062] (a) when the receiving backbone is as defined in (i)-(a):

[0063] - when n is 1, an overhang comprised in the insert is a palindromic overhang complementary to the palindromic overhang of the receiving backbone such that, upon binding between said two complementary palindromic overhangs, a Type IIP restriction enzyme target site is formed and the other overhang comprised in the insert is non- palindromic and complementary to the non-palindromic overhang of the receiving backbone,

[0064] - when n is of 2 or more, an overhang comprised in a first insert is a palindromic overhang complementary to the palindromic overhang of the receiving backbone such that, upon binding between said two complementary palindromic overhangs, a Type IIP restriction enzyme target site is formed, and

[0065] - the other overhang comprised in said first insert and the overhangs comprised in the remaining (n-1) inserts are different non-palindromic overhangs, and each one of the said non-palindromic overhangs is complementary only to another non-palindromic overhang comprised in another insert, or to the non-palindromic overhang of the receiving backbone, provided the non-palindromic overhang comprised in said first insert is not complementary to the non-palindromic overhang of the receiving backbone; and

[0066] (b) when the receiving backbone is as defined in (i)-(b):

[0067] - when n is 1, the insert comprises two palindromic overhangs, each one being complementary to a different palindromic overhang of the receiving backbone, such that upon binding between said two pairs of complementary palindromic overhangs, two different Type IIP restriction enzyme target sites are formed,

[0068] - when n is 2 or more, a first and a second insert comprise a palindromic overhang, each one being complementary to a different palindromic overhang of the receiving backbone, such that upon binding between said two pairs of complementary palindromic overhangs, two different Type IIP restriction enzyme target sites are formed, and

[0069] - the other overhang comprised in the first and the second inserts and the overhangs of the remaining (n-2) inserts are different non-palindromic overhangs, and each one of the said non-palindromic overhangs is complementary only to another non-palindromic overhang comprised in another insert;

[0070] and

[0071] (ill) a DNA ligase;wherein the molar ratio of the receiving backbone and the insert(s) comprising a palindromic overhang is from 1:0.1 to 1:1.9 (receiving backbone:insert), and the molar ratio of the receiving backbone and the remaining inserts is from 1:1.1 to 1:3 (receiving backbone:insert).

[0072] 1-1. Definitions of the first aspect of the invention

[0073] The term "cloning method” or "molecular cloning”, as used herein, refers to the term well-known by an expert in the field, comprising isolating nucleic acid fragment(s) of interest, and inserting said fragment(s) (also referred herein as "inserts”) into a vector (also referred herein as a "receiving vector”). Several copies of the obtained product (also referred herein as a "destination vector”) are then produced or replicated, by well known techniques, such as by transformation of bacteria with the obtained product and multiplication of the transformed bacteria. As well understood by a skilled person, the method may also comprise a step of isolation of the destination vector from the obtained bacteria, by well-known techniques by an expert in the field, such as the use of mini-prep or maxi-prep kits.

[0074] The term "nucleic acid molecule”, as used herein, refers to the term well-known by an expert in the field, particularly refers to biomolecules, formed by at least one polymer of nucleotides or sequence, of nucleotides. The term "polymer of nucleotides”, as used herein, is composed of monomer corresponding to nucleotides, joined to one another by covalent bonds and are thus single chains or sequences of nucleotides. Nucleotide polymers in the context of the present disclosure are of any length, as long as they comprise 2 nucleotides. The term "nucleotide”, as used herein, refers to the molecule composed of one of four nitrogen-containing nucleobases (cytosine [C], guanine [G], adenine [A], or a thymine [T] - or uracile [U] in case of an RNA molecule -), a sugar, which is a deoxyribose in a DNA molecule (a ribose in an RNA molecule), and a phosphate group. A nucleotide comprising a deoxyribose as sugar is also referred to as a deoxyribonucleotide. A nucleotide comprising a ribose as sugar is also referred to as a ribonucleotide. The term nucleotides, as used herein, also refers to non-standard nucleotides or nucleotide analogues. Nonlimiting examples of nucleotide analogues include 2'-deoxy guanosine, 2'-deoxy adenosine, 2'-0-methylguanosine, 2'-0-methyl (e.g., 2'-O-methylcytidine, 2'-0-methylpseudouridine, 2'-0-methyluridine, 2'-0-methyladenosine (2prime-O-methyladenosine as referred in the sequence listing), 2'-0-methylguanosine) ribonucleotide, 2'- amino, 2'-thio and 2'-fluoro modified ribonucleotide, 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'- fluoro-guanosine, 2'-fluoro-adenosine, 2'-amino-cytidine, 2'-amino-uridine, 2'-amino- adenosine, 2'-amino-guanosine, 2'-amino-butyryl-pyrene-uridine, 2'-amino-adenosine, 5-iodo-uridine, ribo- thymidine, 5-bromo-uridine, 2-aminopurine, 5-methyl-cytidine , 5-fluoro-cytidine, and 5- fluoro-uridine, 2,6-diaminopurine, 4-thio-uridine, 5-amino-ally l-uridine, and combinations thereof.

[0075] Nucleic acid molecules can also comprise or consist of at least one polymer modification selected from the group consisting of: peptide nucleic acid (PNA), Morpholino nucleic acid, glycol nucleic acid (GNA), threose nucleic acid (TNA), and hexitol nucleic acids (HNA), locked nucleic acids (LNA) (an nucleic acid molecule or region comprising at least one 2'-C,4'-C-oxy- methylene-linked bicyclic ribonucleotide monomer), 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxycarboxymethyluraci 1, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid, wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, 5-methyl-2-thiouracil, 3-(3-amino- 3-N-2-carboxypropyl) uracil and / or 2,6-diaminopurine.

[0076] In a nucleotide polymer, nucleotides are joined to one another by covalent bonds (known as the phosphodiester bond or linkage) between the sugar of one nucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate backbone. More particularly, a nucleotide polymer consists of a repeating structure wherein the phosphate of a nucleotide covalently bonds to the 3' carbon of the (deoxy)ribose of another nucleotide, and forms an alternating, unbranched polymer. Polymers are thus generally defined by a nucleotide sequence. In the context of this disclosure, nucleotide sequences defining a nucleotide polymer are represented in the 5' to 3' direction from left to right, and the first nucleotide presented in the sequence is the residue at position number 1. Thus, in the context of the present disclosure, the positions of nucleotides in a nucleotide polymer or nucleotide sequence are provided from the 5'-end to the 3'-end of said nucleotide polymer or nucleotide sequence (i.e. in a 5'- to 3'-end direction), being the residue at the 5'-end of the nucleotide polymer or nucleotide sequence at position 1. Concomitantly, nucleotide sequences or regions within a nucleotide polymer are provided from the 5'-end to the 3'-end direction of said polymer or nucleotide sequence, being the sequence or region at the 5' end of the nucleotide polymer or nucleotides sequence, at position 1.

[0077] As well known by an expert in the field, a nucleic acid molecule can be single stranded or double stranded, depending on whether it comprises one nucleotide polymer (single stranded) or two complementary nucleotide polymers bound to each other (double stranded). As well known by an expert in the field, in a double stranded nucleic acid molecule, the two nucleotide polymers are each one the reverse complement of the other. In a particularly preferred embodiment, the nucleic acid molecules referred in the present disclosure are double-stranded.

[0078] The term "complementary”, "binding to”, or "base pair”, as used herein in the context of two nucleotide polymers or nucleotide sequences, refers to the interaction between two nucleotides within said two nucleotide polymers or sequences by the formation of hydrogen bonds between the nitrogen basis of said nucleotides following base-pairing rules. Most common base pairing rule corresponds to Watson-crick base pairing (A with T and C with G). When several nucleotides in a nucleotide sequence base pair with several nucleotides of another sequence, said sequences are commonly understood to be complementary to eachother, bind to each other, or base pair with each other. Base pairing between two sequences can be complete or perfect, i.e. all the nucleotides within a sequence base pair with all the nucleotides of another sequence, or partial or imperfect, wherein several but not all the nucleotides of one sequence base pair with nucleotides in the other sequence. Thus, two nucleotide polymers can be considered complementary when they are at least at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% complementary to each other.

[0079] Methods to determine the percentage of complementarity between a first nucleotide sequence and a second nucleotide sequence are well-known by an expert in the field an include determining the percentage of sequence identity of the first nucleotide sequence with the reverse complementary of the second nucleotide sequence. Thus, a nucleotide sequence complementary to a target sequence can be alternatively defined as comprising or consisting of a nucleotide sequence that has identity with respect to the reverse complementary of its target sequence. Identity is of at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, or 100%, particularly of 100%, with respect to the reverse complementary of the nucleotide target sequence. Sequence identity, including determination of sequence complementarity for nucleic acid sequences, may be determined by sequence comparison and alignment algorithms known in the field. To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions / total # of positions* 100), optionally penalizing the score for the number of gaps introduced and / or length of gaps introduced.

[0080] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity (i.e., a local alignment). A preferred, non-limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the length of the aligned sequences (i.e., a gapped alignment). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402. In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the entire length of the sequences aligned (i.e., aglobal alignment). A preferred, non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GOG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0081] In a particular embodiment, the "nucleic acid molecule” referred herein, is a DNA molecule, i.e. a nucleic acid molecule wherein all nucleotides are deoxyribonucleotides, more particularly a double stranded DNA molecules. In a particular embodiment, double stranded nucleic acid molecules referred herein, particularly double stranded DNA molecules, show 100% complementarity. In another particular embodiment, double stranded nucleic acid molecules referred herein, particularly double stranded DNA molecules, comprise or consist of a central region with 100% complementarity and short single stranded regions located at one or both ends of the molecules. More particularly, said short single stranded regions are 5' or 3' end overhangs. Thus, in a particularly preferred embodiment, the nucleic acid molecule referred herein, is a double stranded DNA molecule, comprising or consisting of a central region with 100% complementarity and a single stranded region at one or both ends of the molecule, more particularly wherein said short single stranded region is an overhang. More particularly the nucleic acid moleucles referred herein are double stranded DNA molecules showing 100% complementarity, or double stranded DNA molecules comprising or consistings of , particularly consisting of, a central region with 100% complementarity and a 5' and / or 3' overhang. In a particular embodiment, the vectors of the invention are double stranded nucleic acid molecules, particularly double stranded DNA molecules, showing 100% complementarity. In another particular embodiment, the polynucleotides of the first aspect are double stranded nucleic acid molecules, particularly double stranded DNA molecules, showing 100% complementarity. In another particular embodiment, the inserts of the first aspect, consist of double stranded nucleic acid molecules, particularly double stranded DNA molecules, comprising or consisting of, particularly consisting of, a central region with 100% complementarity and a 5' and / or 3' overhang. In another particular embodiment, the receiving backbone of the first aspect consists of a double stranded nucleic acid molecule, particularly a double stranded DNA molecule, comprising or consisting of, particularly consisting of, a central region with 100% complementarity and a 5' and / or 3' overhang. In another particular embodiment, the hybridized oligos of the first aspect, consist of a double stranded nucleic acid molecule, particularly double stranded DNA molecule, comprising or consisting of, particularly consisting of, a central region with 100% complementarity and a 5' and / or 3' overhang. In another particular embodiment, the linearized receiving backbone as defined in the first aspect, consists of double stranded nucleic acid molecules, particularly double stranded DNA molecules, comprising or consisting of, particularly consisting of, a central region with 100% complementarity and a 5' and / or 3' overhang.

[0082] In another particular embodiment, the inserts of the first aspect, the hybridized oligos of the first aspect, the receiving backbone of the first aspect, and the linearized receiving vectors of the first aspect, consist ofdouble stranded nucleic acid molecules, particularly double stranded DNA molecules, comprising or consisting of, particularly consisting of, a central region with 100% complementarity and a 5' and / or 3' overhang.

[0083] The term "vector”, or "nucleic acid vector” as used herein, refers to a nucleic acid molecule that is used as a vehicle to carry a particular nucleic acid segment or fragment into a host cell (such as a bacteria), where it can be expressed or replicated. As well known by an expert in the field, nucleic acid vectors are generally closed nucleic acid molecules, or circular nucleic acid molecules. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. As well understood by a skilled person, the vector referred in the present disclosure is doublestranded, is a double-stranded nucleic acid molecule, more particularly a double-stranded DNA vector. Thus, in a particular embodiment, the embodiments referred to double stranded DNA molecules are equally applicable to the vectors referred herein. In an embodiment, the vectors referred in the disclosure comprise at least one nucleotide analogue, particularly selected from the list provided in the definition of "nucleic acid” above. In another embodiment, the vectors referred in the disclosure comprise at least one polymer modification selected from the list provided in the definition of "nucleic acid” above.

[0084] Vectors, particularly artificially-constructed vectors, contain essential elements or regions found in all vectors, and in some cases, other optional features found only in some vectors. Non-limiting examples of such elements or regions, well-known by an expert in the field, are:

[0085] Origin of replication: Necessary for the replication and maintenance of the vector in the host cell. Promoter: Promoters are used to drive the transcription of a vector sequence, particularly a transgene or an inserted region, as well as the other genes in the vector such as the antibiotic resistance gene. Some cloning vectors need not have a promoter for the cloned insert but it is an essential component of expression vectors so that the cloned product may be expressed.

[0086] Cloning site: This may be a multiple cloning site or comprise features that allow for the insertion of foreign nucleic acid sequences into the vector through ligation. They comprise several restriction enzyme recognition sites or target sites, within and optionally also at the 5' and / or 3' end of the multicloning site.

[0087] Ribosome binding site (RBS) encoding sequence: A Ribosome Binding Site (RBS) is an RNA sequence found in mRNA to which ribosomescan bind and initiate translation. Translation initiation in bacteria almost always requires both an RBS sequence and a start codon.

[0088] Genetic markers: Genetic markers for viral vectors allow for confirmation that the vector has integrated with the host genomic DNA.

[0089] Antibiotic resistance: Vectors with antibiotic-resistance open reading frames allow for survival of cells, especially bacterial cells, that have taken up the vector in growth media containing antibiotics through antibiotic selection.Epitope: Some vectors may contain a sequence for a specific epitope that can be incorporated into the expressed protein. It allows for antibody identification and / or isolation of cells expressing the target protein.

[0090] Reporter genes: Some vectors may contain a reporter gene that allow for identification of plasmid that contains inserted DNA sequence. An example is lacZ-o which codes for the N-terminus fragment of p-galactosidase, an enzyme that digests galactose. A multiple cloning site is located within lacZ- ci, and an insert successfully ligated into the vector will disrupt the gene sequence, resulting in an inactive p-galactosidase. Cells containing vector with an insert may be identified using blue / white selection by growing cells in media containing an analogue of galactose (X-gal). Cells expressing p- galactosidase (therefore does not contain an insert) appear as blue colonies. White colonies would be selected as those that may contain an insert. Other commonly used reporters include green fluorescent protein and luciferase.

[0091] Targeting sequence: Expression vectors may include encoding sequences for a targeting sequence in the final protein that directs the expressed protein to a specific organelle in the cell or specific location such as the periplasmic space of bacteria. An example of targeting sequence is a signal peptide.

[0092] Protein purification tags: Some expression vectors include proteins or peptide encoding sequences that allows for easier identification, isolation and / or purification of the expressed protein. Examples include polyhistidine-tag, glutathione-S-transferase, and maltose binding protein. Some of these tags may also allow for increased solubility of the target protein. The target protein is fused to the protein tag, but a protease cleavage site positioned in the polypeptide linker region between the protein and the tag may be included, which allows the tag to be removed later if needed.

[0093] The term "destination nucleic acid vector”, or "destination vector” or "final vector”, as used herein, refers to the nucleic acid vector obtained by the method of the first aspect. Thus, the destination vector comprises or consists of, particularly consists of the n inserts of the first aspect of the invention and a larger sequence that serves as the backbone of the vector. In a particular embodiment, the backbone of the destination vector corresponds to the receiving backbone of the first aspect of the invention.

[0094] The term "insert”, as used herein, refers to a nucleic acid fragment, comprising a sequence of interest S, and the 5' and 3' overhangs required to be inserted in its corresponding position in the destination vector. The overhangs of the inserts are as defined in the first aspect of the invention. In a particular embodiment, the inserts as defined herein are double stranded DNA molecules, more particularly, comprising or consisting of, particularly consisting of, a central region showing 100% complementarity and comprising the sequence of interest S, and 2 overhangs. In another particular embodiment, the inserts of the first aspect are double stranded DNA molecules, consisting of a central region and two overhangs, wherein the central region shows 100% complementarity and consists of sequence S and its reverse complement.The term "sequence of interest "S'”' as used herein, refers to a nucleic acid sequence comprising at least 2 nucleotides, particularly at least 3, and can be the same or different between two of the n inserts, more particularly, between all the n inserts. In an embodiment, the sequence S is the same between at least 2 of the n inserts, particularly, between all the n inserts. In a particular embodiment, the sequence S is different between at least 2 of the n inserts, more particularly, between all the n inserts.

[0095] The term "reaction mixture”; as used herein, refers to the term well known by an expert in the field, particularly, the term refers to a composition comprising or consisting of a mix of reactants. As well understood by a skilled person, a reaction mixture can also be referred to as a combination of reactants that are in contact together, or comprised in a same volume, which can be a tube, also referred by a skilled person as a pot. The reaction mixture particularly comprises a buffer or reaction buffer, in addition to the reactants. In a particular embodiment, the reaction mixture of the first aspect comprises the n inserts, the receiving backbone, and the DNA ligase, more particularly also a reaction buffer. In a more particular embodiment, the reaction mixture of the first aspect comprises the n inserts, the receiving backbone, the DNA ligase, and Type IIS restriction enzymes, more particularly the Type IIS enzyme of the first aspect. In a yet more particular embodiment, the reaction mixture of the first aspect comprises the n inserts, the receiving backbone, the DNA ligase, and Type IIS restriction enzymes, more particularly the Type IIS enzyme of the first aspect and a reaction buffer. In a particular embodiment, the reaction buffer is that defined in "conditions appropriate for ligation by the DNA liagse” below.

[0096] The term "receiving backbone”, "vector backbone”, or "plasmid backbone”, as used herein, refers to a linear nucleic acid molecule in which the n inserts of the first aspect are inserted or ligated. In an embodiment, the receiving backbone corresponds to the nucleic acid molecule of the larger size from those used to form the final vector in the method of the first aspect. Thus, in a particular embodiment, the receiving backbone comprises the minimal elements required for the activity of the final vector, not including the n inserts, such as any of the essential or optional elements indicated above in the definition of the term "vector”, particularly at least an origin of replication, a promoter or both, more particularly, wherein the receiving backbone corresponds to the nucleic acid molecule of the larger size from those used to form the final vector in the method of the first aspect. In a more particular embodiment, the receiving backbone corresponds to the nucleic acid region of the destination vector not including the n inserts. Particularly, the receiving backbone referred herein is a double stranded DNA molecule, more particularly, comprising or consisting of, particularly consisting of, a central region showing 100% complementarity and 2 overhangs.

[0097] As defined in the first aspect, the receiving backbone comprises at each end an overhang, each overhang being complementary to one overhang of one of the n inserts (or each overhang being complementary to one overhang of the insert, when n is 1).The term "overhang”, as used herein, refers to a stretch of unpaired nucleotides at the end of a doublestranded nucleic acid molecule. These unpaired nucleotides can be in either strand of the double stranded nucleic acid molecule, creating either 3' or 5' overhangs. The simplest case of an overhang consists of a single nucleotide unpaired at the end of a strand. Particularly, an overhang as used herein refers to a stretch of unpaired nucleotides resulting from digestion of a double stranded DNA molecule with a restriction enzyme, particularly with a Type IIS restriction enzyme, more particularly with the Type IIS restriction enzyme of the first aspect. In an embodiment, the overhang referred in the present disclosure is of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides, more particularly, the length of the overhang is determined by the restriction enzyme used. In a particular embodiment, the overhangs of the insert referred herein are of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides, more particularly, the length of the overhangs is determined by the restriction enzyme used in each case. More particularly the restriction enzyme used to generate the overhangs of the n inserts is the Type IIS restriction enzyme of the first aspect. In another particular embodiment, the overhangs of the receiving backbone referred herein are of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides, more particularly, the length of the overhangs is determined by the restriction enzyme used in each case. More particularly the restriction enzyme used to generate the overhangs of the receiving backbone is the Type IIS restriction enzyme of the first aspect. In another particular embodiment, the restriction enzymes used to generate each overhang of the receiving backbone are those indicated below in connection with the restriction enzymes used to digest the receiving vector.

[0098] The term "palindromic overhang”, as used herein, refers to an overhang as just defined having a nucleotide sequence that is equal to its reverse complement. For example, the DNA sequence 5'-ACCTAGGT-3' is palindromic because its complement would be 5'-TGGATCCA-3', and its reverse complement, 5'-ACCTAGGT-3'. As well understood by a skilled person, a palindromic sequence can thus base pair "to itself'. In other words, a nucleic acid molecule with a palindromic overhang, can bind to an identical nucleic acid molecule that also comprises said identical palindromic overhang. In a particular embodiment, the palindromic overhangs referred herein result from digestion of a DNA double stranded molecule with a Type IIS or Type IIP restriction enzyme, particularly with the Type IIS restriction enzyme of the first aspect. In a more particular embodiment, the Type IIP restriction enzyme referred herein corresponds to a Type IIP restriction enzyme that digests the receiving vector as indicated below. In another particular embodiment, the palindromic overhangs referred herein can also result from the annealing of two complementary oligos, for instance in the case of inserts formed upon hybridization of two complementary oligos, wherein at least one of said oligos, particularly both of them, comprises in addition to the complementarity region, a 5' and / or 3' end single stranded region, i.e. a 5' and / or 3' end overhang, that is palindromic.

[0099] The term "non-palindromic overhang”, as well understood by a skilled person, refers to any overhang, as defined above, provided it is not palindromic. As well understood by a skilled person, a non-palindromic overhang cannot base pair to itself. In a particular embodiment, the non-palindromic overhangs referredherein result from digestion of a DNA double stranded molecule with a Type IIS restriction enzyme, particularly with the Type IIS restriction enzyme of the first aspect. In another particular embodiment, the non-palindromic overhangs referred herein can also result from the annealing of two complementary oligos, for instance in the case of inserts formed upon hybridization of two complementary oligos, wherein at least one of said oligos, particularly both of them, comprises in addition to the complementarity region, a 5' and / or 3' end single stranded region, i.e. a 5' and / or 3' end overhang, that is non-palindromic.

[0100] The term "DNA ligase”, as used herein, refers to the term well-known by an expert in the field, in particular to a type of enzyme that facilitates the joining of DNA strands together by catalysing the formation of a phosphodiester bond. The mechanism of DNA ligase comprises forming two covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide ("acceptor"), with the 5' phosphate end of another ("donor"). Two ATP molecules are consumed for each phosphodiester bond formed. Non-limiting examples of DNA ligases include E. Coll DNA ligase, or T4 DNA ligase. E. coll DNA ligases is encoded by the tig gene in E. coll. It uses energy gained by cleaving nicotinamide adenine dinucleotide (NAD) to create the phosphodiester bond. The T4 DNA ligase is the most-commonly used in cloning methods and is the DNA ligase from bacteriophage T4 (a bacteriophage that infects Escherichia coli bacteria). It can ligate either cohesive or blunt ends of DNA, oligonucleotides, as well as RNA and RNA-DNA hybrids, but not singlestranded nucleic acids. Unlike E. coli DNA ligase, T4 DNA ligase cannot utilize NAD and it has an absolute requirement for ATP as a cofactor. A typical reaction for inserting a fragment into a plasmid vector would use about 0.01 (sticky ends) to 1 (blunt ends) units of ligase. The optimal incubation temperature for T4 DNA ligase is 16 °C. The DNA ligase "T4 DNA ligase” is thus very well-known by an expert in the field and corresponds to any of those supplied by a broad number of commercial providers. In a particularly preferred embodiment, the DNA ligase is the T4 DNA ligase.

[0101] The term "Type IIP restriction enzyme”, "Type IIP enzyme” or "Type IIP”, as used herein, refers to a subtype of Type II restriction enzyme. Type II restriction enzymes are traditionally used for everyday molecular biology applications such as gene cloning and DNA fragmentation and analysis. These enzymes cleave DNA at fixed positions with respect to their recognition sequence, creating reproducible fragments and distinct gel electrophoresis patterns. Type IIP is the most important subtype, accounting for over 90% of the enzymes used in molecular biology. Type IIP enzymes recognize symmetric (or ‘palindromic’) DNA sequences of 4 to 8 base pairs in length and generally cleave within that sequence. Some Type IIP enzymes generate 5'-overhangs (‘staggered ends') of four bases (e.g., Hindlll: A'AGCTT) or of two bases (Ndel: CA'TATG). Others generate 3'-overhangs of four (Sacl: GAGCT'C) or two bases (Pvul: CGAT'CG). And yet others produce 'flush' (or 'blunt') ends (e.g., EcoRV: GAT'ATC). They are the simplest and smallest of all restriction enzymes, typically 250-350 amino acids in length.

[0102] In a particular embodiment, the Type IIP restriction enzyme referred herein is selected from the list consisting of BamHI, Xhol, EcoRI, Hindlll, Ncol, Notl, Sall, Spel, Xbal, Xmal, Kpnl, Ndel, Agel, and Smal. In a moreparticular embodiment, the type IIP restriction enzyme is selected form BamHI, EcoRI, and Xhol, particularly is BamHI. Thus, the Type IIP target sites referred herein are selected from the target sites of said Type IIP enzymes, particularly are selected from BamHI, EcoRI, and Xhol target sites, particularly is the BamHI target site.

[0103] The term "Type IIS restriction enzyme”, "Type IIS enzyme” or "Type IIS” as used herein, refers to a subtype of Type II restriction enzyme different from Type IIP. In Type IIP restriction enzymes, the amino acids that catalyze cleavage and those that recognize the DNA are integrated into a single protein domain that cannot be effectively subdivided. In Type IIS enzymes, in contrast, they are partitioned into separate domains linked by a short polypeptide connector. As a result, Type IIS proteins are larger than Type IIP proteins, typically 400-600 amino acids in length. When Type IIS enzymes bind to DNA, the catalytic domain is positioned to one side of, and several bases away from, the sequence bound by the recognition domain, and so cleavage is 'shifted' to one side of the sequence. Type IIS enzymes generally bind to DNA as monomers and recognize asymmetric DNA sequences. They cleave outside of this sequence, within one to two turns of the DNA. By convention, the recognition sequence is written in the orientation in which cleavage occurs downstream, to the right of the sequence, in a 5' to 3' end direction. In the present disclosure, the "forward strand of a type IIS recognition site”, corresponds to the strand of said Type IIS recognition site that leaves downstream (or that is upstream) the cleavage site of said Type IIS enzyme, in a 5' to 3' end direction. Concomitantly, the "reverse strand of a type IIS recognition site”, is the reverse complement strand of the "forward strand of type IIS recognition site” as just defined.

[0104] The exact positions of cleavage are indicated by the number of bases away from the recognition sequence in each strand. For example, the Type IIS enzyme Fokl recognizes the asymmetric sequence GGATG in duplex DNA and cleaves this ('top' or "forward”) strand 9 bases to the right, or downstream in a 5' to 3' end direction, and the complementary ('bottom' or "reverse”) strand four bases further down, producing 4-base 5'-overhanging ends. The specificity of Fokl is written: GGATG 9 / 13 or GGATG(9 / 13).

[0105] Type IIS cleavage domains have no inherent sequence-specificity, and so the sequence of the overhang they generate varies from one recognition site to another. Fragments produced by Type IlS-digestion of natural DNA molecules generally have different overhangs, therefore, and will not anneal to one another. However, if the sequence of the overhang is predetermined, by designing it into a PGR primer, for example, then it can be made to complement another and to be directional. As previously indicated, this feature is used in ‘Golden Gate' methods where multiple fragments can be stitched together in the correct order and orientation in a single ligation. Often used Type IIS enzymes in Golden Date methods are Bsal (GGTCTC 1 / 5), and BsmBI (GGTCTC 1 / 5). In a particular embodiment, the Type IIS restriction enzyme referred in the present disclosure is selected from the list consisting of: Bsal, Bbsl, Esp3l, PaqCI, Sapl, BspQI, BtgZI, and combinations thereof. More particularly, the Type IIS restriction enzyme is selected from the list consisting of: Bsal, Bbsl, Esp3l and combinations thereof. Yet more particularly, it is selected from the list consistingof Bsal, Bbsl and the combination thereof, more particularly from Bsal or Bbsl. In particularly preferred embodiment, the Type IIS restriction enzyme is Bsal.

[0106] The term "restriction enzyme recognition site”, as used herein, refers to the DNAsite specifically recognized by a restriction enzymeAs indicated in the definition of Type IIP and Type IIS enzymes, the recognition site is defined by a DNA sequence specific for each restriction enzyme. In the case of the Type IIP restriction enzymes, the recognitions site includes the cleavage site and is thus also referred to as the target site or Type IIP target site. In the case of Type IIS restriction enzymes, the recognition site of the enzyme does not include the enzyme's cleavage site, which is some nucleotides away from the recognition site. The term "restriction enzyme recognition sequence”, as used herein, refers to the DNA sequence specifically recognized by a restriction enzyme. As well understood by a skilled person, the restriction enzyme recognition sequence of a Type IIP enzyme includes also the cleavage site. Also, as well understood by a skilled person, when a restriction enzyme recognition sequence is herein described, it is implied that a restriction enzyme recognition site is present which is defined by said restriction enzyme recognition sequence.

[0107] The term "directly attach to each other”; or "directly linked to each other”, as used herein in the context of 2 nucleotide regions, or nucleotide sequences, refers to the fact that the nucleotide at the 3' end of a nucleotide region or sequence is linked to the 5' end nucleotide of another nucleotide region or sequence by a covalent bond, particularly by a phosphodiester bond. More specifically, the phosphodiester bond is formed between the 3'-OH of the 3' end nucleotide of one nucleotide region or sequence, and the 5' phosphate group of the 5' end nucleotide of another nucleotide region or sequence.

[0108] The term "disposable sequence” as used herein, refers to the term well-known by an expert in the field. Particularly, the term refers to a sequence without any associated activity, and that can be deleted from a longer sequence, such as the receiving vector, without affecting the function of the active elements comprised in said longer sequence, such as an origin of replication, promoters, genetic markers, antibiotic resistant genes, epitopes, reporter genes, targeting sequence, the protein purification tags etc in a receiving vector. A disposable sequence in a cloning vector can for instance refer to a region of a cloning or multicloning site, or the whole cloning site or multicloning site, comprised in the cloning vector. In a particular embodiment, the disposable sequence referred in the first aspect of the invention is comprised or consists of, particularly consists of a multicloning site.

[0109] The term "5' region”, as used herein in the context of a nucleotide sequence, refers to the region within the nucleotide sequence consisting of 49%, 45%, 40%, 35%, 30%, 25%, 22%, 20%, 15%, 10%, 8%, 7%, 5%, 3%, 2%, 1% of consecutive nucleotides of the nucleotide sequence and comprising the 5' end nucleotide of the sequence (i.e. consisting of the said consecutive nucleotides starting from the 5' end nucleotide of the sequence) wherein 100% represents all nucleotides of the sequence. As well understood by a skilled person, a short nucleotide sequence comprised at the 5' end region of a longer sequence, refers to a short nucleotidesequence that is within said 5' region but that not necessarily comprises the 5' end nucleotide of said longer sequence. Concomitantly, a short nucleotide sequence at the 5' end of a longer sequence, is a short sequence within the 5' region of said long nucleotides sequence and that comprises the 5' end (nucleotide) of said long sequence.

[0110] The term "3' region”, as used herein, refers to the region within a nucleotide sequence consisting of 49%, 45%, 40%, 35%, 30%, 25%, 22%, 20%, 15%, 10%, 8%, 7%, 5%, 3%, 2%, 1% of consecutive nucleotides of the nucleotide sequence and comprising the 3' end nucleotide of the nucleotide sequence (i.e. consisting of the said consecutive nucleotides starting from the 3' end nucleotide of the sequence) , wherein 100% represents all nucleotides of the sequence. As well understood by a skilled person, a nucleotide sequence comprised a the 3' end region of a longer sequence, refers to a region that is within said 3' region but that not necessarily comprises the 3' end nucleotide of said longer sequence. Concomitantly, a short nucleotide sequence at the 3' end of a longer sequence, is a short sequence within the 3' region of said long nucleotides sequence that comprises the 3' end (nucleotide) of said long sequence.

[0111] 1.2-Embodiments of the first aspect of the invention

[0112] In an embodiment, “n” is an integer of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, particularly of at least 1, more particularly of at least 2. In a particular embodiment, “n” is of no more than 50, 40, 30, 25, 20, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, particularly of no more than 10, yet more particularly of no more than 6.

[0113] In a particular embodiment, “n” is of at least 1 and of no more than 50, 40, 30, 25, 20, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, particularly of no more than 10, yet more particularly of no more than 6. In a particular embodiment, “n” is of at least 2 and of no more than 50, 40, 30, 25, 20, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, particularly of no more than 10, yet more particularly of no more than 6. In a particular embodiment, “n” is of at least 3 and of no more than 50, 40, 30, 25, 20, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, particularly of no more than 10, yet more particularly of no more than 6. In a particular embodiment, “n” is of at least 6 and of no more than 50, 40, 30, 25, 20, 18, 15, 12, 10, 9, 8, 7, particularly of no more than 10.

[0114] In another particular embodiment, n is of 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, more particularly of 1-10. In a yet more particular embodiment, n is of 1-6. In another particular embodiment, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 22, 25, 27, 30, 35, 40, 45, or 50. In another particular embodiment, n is 1. In another particular embodiment, n is 6. In another particular embodiment, n is 10.

[0115] As well understood by a skilled person, the term "a first insert” as used in the first aspect of the invention, refers to an insert of interest firstly mentioned, however it is not necessarily located at the 5' end of the sequence of n inserts in the destination vector, this is, it is not necessarily the insert located in the first position in the sequence of n inserts in the destination vector (in a 5' to 3' end direction). Thus, the term "a first insert”, as used in the first aspect, is interchangeable with "a first insert of interest”, "an insert n(a)”, or"an insert n' ”, and can be located at any end of the sequence of n inserts in the destination vector.

[0116] In a particular embodiment, the above-mentioned "first insert”, or insert n(a), is located at the 3' end of the sequence of n inserts in the destination vector, or in other words, it is located in the last position in the sequence of n inserts in the destination vector (in a 5' to 3' end direction). In an embodiment, the above-mentioned "first insert”, or insert n(a), is located at the 5' end of the sequence of n inserts in the destination vector, or in other words, it is located in the first position in the sequence of n inserts in the destination vector (in a 5' to 3' end direction).

[0117] Concomitantly, as well understood by a skilled person, the term "a second insert” as used in the first aspect of the invention, refers to an insert of interest secondly mentioned, however it is not necessarily located immediately downstream the insert at the 5' end of the sequence of n inserts in the destination vector, this is, it is not necessarily the insert located in the second position in the sequence of n inserts in the destination vector (in a 5' to 3' end direction). Thus, the term "a second insert”, as used in the first aspect, is interchangeable with "a second insert of interest”, "an insert n(b)”, or "an insert n” ”, and can be located at any end of the sequence of n inserts in the destination vector, provided it is located at the opposite end in said sequence of n inserts than the above-mentioned "a first insert”. As well understood by a skilled person when "a first insert” is substituted by "a first insert of interest” according to the embodiment above, "a second insert” is substituted by "a second insert of interest”, and so on. As also well understood by a skilled person, the above mentioned "a first insert” and "a second insert” are located each one at an opposite end of the sequence of n inserts in the destination vector.

[0118] In a particular embodiment, said "second insert”, or insert n(b), is located at the 5' end end of the sequence of n inserts in the destination vector and the above-mentioned "first insert”, or insert n(a), is located at the 3' end of the said sequence of n inserts, or in other words, the above mentioned "second insert” is located in the first position in the sequence of n inserts in the destination vector (in a 5' to 3' end direction) and the above-mentioned "first insert” is located in the last position in the sequence of n inserts in the destination vector (in a 5' to 3' end direction). In an embodiment, the above-mentioned "second insert” is located at the 3' end of the sequence of n inserts in the destination vector and the above-mentioned "first insert” is located at the 5' end of the said sequence of n inserts, or in other words, the above-mentioned "second insert” is located in the last position in the sequence of n inserts in the destination vector (in a 5' to 3' end direction) and the above-mentioned "first insert” is located in the first position in the sequence of n inserts in the destination vector (in a 5' to 3' end direction).

[0119] In a particular embodiment, when n is 1, the receiving backbone is as defined in (i)-a).

[0120] In another particular embodiment of the first aspect, the overhangs of at least one of the n insert(s), more particularly the n inserts, are obtained upon digestion of a DNA double stranded molecule by Type IISrestriction enzyme(s), particularly, by a same Type IIS restriction enzyme, more particularly by the same Type IIS restriction enzyme for all the n inserts, yet more particularly by the Type IIS restriction enzyme of the first aspect as defined below. In a particular embodiment, the term "at least one”; as used herein, refers to 1 , 2, 3, 4, 5, 6, particularly to 1. More particularly, the at least one of the n inserts obtained upon digestion of one or more polynucleotides by Type IIS enzyme(s), as defined herein, consist of the inserts with a double stranded region, or complementarity region, particularly a sequence S, comprising more than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, particularly more than 150 nucleotides.

[0121] In another particular embodiment, n is any of the values indicated above higher than 1, particularly of at least 2. In this case, as well understood by a skilled person, the first aspect of the invention can be formulated as follows (wherein the value n can be substituted by any of those indicated above higher than 1).

[0122] A cloning method for obtaining a destination nucleic acid vector comprising “n” inserts, wherein “n” is an integer of at least 2, and each insert comprises a sequence of interest “S” and, wherein the method comprises contacting in a reaction mixture:

[0123] (I) a receiving backbone, wherein:

[0124] (a) one end of the receiving backbone comprises a palindromic overhang and the other end comprises a non-palindromic overhang, or

[0125] (b) each end of the receiving backbone comprises a different palindromic overhang;

[0126] (II) the n inserts, wherein each insert comprises two different overhangs and

[0127] (a) when the receiving backbone is as defined in (i)-(a):

[0128] - an overhang comprised in a first insert is a palindromic overhang complementary to the palindromic overhang of the receiving backbone such that, upon binding between said two complementary palindromic overhangs, a Type IIP restriction enzyme target site is formed,

[0129] - the other overhang comprised in said first insert and the overhangs comprised in the remaining (n-1) inserts are different non-palindromic overhangs, and each one of the said non-palindromic overhangs is complementary only to another non-palindromic overhang comprised in another insert, or to the non-palindromic overhang of the receiving backbone, provided the non-palindromic overhang comprised in said first insert is not complementary to the non-palindromic overhang of the receiving backbone; and

[0130] (b) when the receiving backbone is as defined in (i)-(b):

[0131] - a first and a second insert comprise a palindromic overhang, each one being complementary to a different palindromic overhang of the receiving backbone, such that upon binding between said two pairs of complementary palindromic overhangs, two different Type IIP restriction enzyme target sites are formed, - the other overhang comprised in the first and the second inserts and the overhangs of the remaining (n-2) inserts are different non-palindromic overhangs, and each one of the said non-palindromic overhangs is complementary only to another non-palindromic overhang comprised in another insert;

[0132] and

[0133] (ill) a DNA ligase;wherein the molar ratio of the receiving backbone and the insert(s) comprising a palindromic overhang is from 1:0.1 to 1:1.9 (receiving backbone:insert), and the molar ratio of the receiving backbone and the remaining inserts is from 1:1.1 to 1:3 (receiving backbone: insert).

[0134] As well understood by a skilled person, the destination vector, receiving backbone and the n inserts of the first aspect of the invention are double stranded nucleic acid molecules as defined herein, particularly are double stranded DNA molecules (double stranded nucleic acid molecules wherein all nucleotides are deoxyribonucleotides).

[0135] In another particular embodiment, the destination vector is double stranded and comprises at least one nucleotide analogue, more particularly, comprises one nucleotide analogue, wherein the rest are (classical) deoxyribonucleotides. In another particular embodiment, the n inserts are double stranded and at least one of the n inserts of the first aspect, particularly each of the n inserts, comprises at least one nucleotide analogue, more particularly comprises one nucleotide analogue, wherein the rest are (classical) deoxyribonucleotides. In another particular embodiment, the receiving backbone is double stranded and comprises at least one nucleotide analogue, particularly one nucleotide analogue, wherein the rest are (classical) deoxyribonucleotides. In another particular embodiment, the n inserts are double stranded and at least one of the n inserts of the first aspect, particularly each of the n inserts, comprises at least one nucleotide analogue, particularly one nucleotide analogue, wherein the rest of nucleotides in said insert(s) are (classical) deoxyribonucleotides, and the receiving backbone is double stranded and comprises at least one nucleotide analogue, more particularly comprises one nucleotide analogue, wherein the rest of nucleotides in the receiving backbone are (classical) deoxyribonucleotides. In a particular embodiment, the nucleotide analogue referred in the embodiments above is selected from those disclosed in the definition of "nucleic acid” above. More particularly, the nucleotide analogue selected is the same for the destination vector, the insert(s) and the receiving backbone. In another particular embodiment, the term "at least one of the n inserts” referred in the embodiments of the paragraph herein consists of 1 of the n inserts.

[0136] In another embodiment, the destination vector is double stranded and comprises at least one nucleotide polymer modification selected from those provided in the definition of "nucleic acid” above, particularly comprises one of said modifications, wherein the rest of the vector consists of (classical) deoxyribonucleotide polymer structures. In a particular embodiment, the n inserts are double stranded and at least one of the n inserts of the first aspect, particularly each of the n inserts, comprise at least one nucleotide polymer modification selected from those indicated in the definition of "nucleic acid” above, particularly comprises one of said modifications, wherein the rest of the corresponding insert(s) and of the n inserts consists of (classical) deoxyribonucleotide polymer structures. In another particular embodiment, the receiving backbone is double stranded and comprises at least one nucleotide polymer modification selected from those indicated in the definition of "nucleic acid” above, particularly comprises one of said modifications, wherein the rest of the receiving backbone consists of (classical)deoxy ribonucleotide polymerstructures. In another particular embodiment, the n inserts are double stranded and at least one of the n inserts of the first aspect, particularly each of the n inserts, comprises at least one polymer modification selected from those indicated in the definition of "nucleic acid” above, particularly comprises one of said modifications, wherein the rest of said the corresponding insert(s) consists of (classical) deoxyribonucleotide polymer(s), and the receiving backbone is double stranded and comprises at least one modification selected from those indicated in the definition of "nucleic acid” above, particularly comprises one of said modifications, wherein the rest of the receiving backbone consists of (classical) deoxyribonucleotide(s). In a particular embodiment, the modification selected and referred in the embodiments just above is the same for the destination vector, the insert(s) and the receiving backbone. In another particular embodiment, the term "at least one of the n inserts” referred in the embodiments of the paragraph herein consists of 1 of the n inserts.

[0137] In a particular embodiment, the receiving backbone is as defined in (i)-(a) of the first aspect, particularly, wherein the palindromic overhang is at the 5' end of the forward strand or 3' end of the reverse strand of the receiving backbone. More particularly, the palindromic overhang is a 5' overhang at the 5' end of the forward strand or a 3' overhang a the 3' end of the reverse strand of the receiving backbone. In another particular embodiment, the receiving backbone is as defined in (i)-(a) of the first aspect, particularly, wherein the palindromic overhang is at the 3' end of the forward strand or 5' end of the reverse strand of the receiving backbone. More particularly, the palindromic overhang is a 3' overhang at the 3' end of the forward strand, o a 5' overhang a the 5' end of the reverse strand of the receiving backbone.

[0138] As well understood by a skilled person, the term "forward strand of a double-stranded nucleic acid molecule” refers to the strand comprising the sequences of interest, oriented in a 5' to 3' end direction, and not the reverse complement of said sequences. For instance, in case the sequence of interest is a peptide encoding sequence, the forward strand of a double stranded DNA molecule is that comprising the nucleic acid sequence encoding said peptide (not its reverse complement), i.e. the nucleic acid sequence that is transcribed into the RNA that gives rise to the protein of interest once translated. Concomitantly, the reverse strand is that comprising the sequences complementary to said sequences of interest.

[0139] In the context of the present disclosure, as well understood by a skilled person, the sequences S of interest of the n inserts are all comprised in the same strand of the double stranded destination vector, and said strand of the destination vector corresponds to the "forward strand of the destination vector”. Concomitantly, the strand of the sequence of n inserts in the destination vector comprising the sequences S of the inserts, corresponds to the "forward strand of the sequence of n inserts” (and is comprised in the destination vector). Accordingly, the forward strand of the receiving backbone is the strand ligated to the forward strand of the n inserts in the method of the first aspect and comprising the sequences of interest S. Similarly the forward strand of an insert is that comprising the sequence S, whereas the reverse strand of an insert, as used herein, refers to the reverse complement.

[0140] As well understood by a skilled person, the forward strand of the receiving backbone is part of the forwardstrand of the destination vector. Similarly, the forward strand of the sequence of n inserts in the destination vector is comprised in, or has been ligated to, the forward strand of the receiving vector as herein defined.

[0141] In a particular embodiment, the forward strand of the destination vector comprises the sequences of interest S of the n inserts. More particularly, the forward strand of the destination vector of the first aspect comprises an expression promoter upstream the sequence of n inserts, particularly, oriented to induce transcription of the sequences of interest S comprised in the n inserts within the destination vector. In a particular embodiment, the forward strand of the receiving backbone comprises said expression promoter comprised in the forward strand of the destination vector. In a particular embodiment, the expression promoter is the commonly known T7 expression promoter.

[0142] In another particular embodiment, the sequence of n inserts within the destination vector is under the control of an expression promoter (upstream said sequence of n inserts in the destination vector).

[0143] As well understood by a skilled person the term "sequence of n inserts” as used herein, refers to the n inserts as defined in the first aspect, placed one after the other in a certain order within the destination vector, i.e. once their complementary overhangs have based paired to each other and / or to the complementary overhangs of the receiving backbone, or once the n inserts and the receiving backbone have been ligated to each other

[0144] As also well understood by a skilled person, the directionality of double stranded nucleic acid molecules, is herein provided with respect to the forward strand of the nucleic acid molecule. For instance, the directionality of the sequence of n inserts is provided with respect to the forward strand of the sequence of n inserts as defined above. Thus, the 5' to 3' end direction of the sequence of n inserts corresponds to that wherein its forward strand is also in a 5' to 3' end direction. As an example, an insert at the 5' end of the sequence of n inserts is located at the 5' end of the forward strand of the sequence of n inserts, particularly, is located such that the strand comprising the sequence S of said insert, is at the 5' end of the forward strand of the sequence of n inserts. As another example, an expression promoter upstream the sequence of n inserts in the forward strand of the destination vector, or even simply in the destination vector, is located in the forward strand of the destination vector upstream the sequence of n inserts.

[0145] Similarly, and as well understood by a skilled person, the sequences or formulas provided to define a nucleic acid molecule or a region within a nucleic acid molecule, when the nucleic acid molecule or region is double stranded, are provided herein with respect to the forward strand of the corresponding nucleic acid molecule, unless otherwise indicated. The term "expression promoter”, as used herein, refers to nucleotide sequences comprised in a vector that drives transcription of a sequence, particularly a transgene or an inserted region, as well as other genes in the vector such as the antibiotic resistance gene, the protein tag encoding sequences etc. In a particular embodiment, the expression promoter as referred herein is the commonlyknown T7 expression promoter. Thus, the term "a sequence under the control of an expression promoter”, as used herein, refers to a sequence whose transcription is driven by the expression promoter. A sequence under the control of an expression promoter can also be referred herein as a sequence "operatively linked to the expression promoter”. As well understood by a skilled person, the nucleotide sequence operatively linked to the expression promoter does not need to be directly linked to the corresponding promoter sequence and nucleotides can be comprised between the 3' end of the corresponding sequence of the expression promoter and the 5' end of the nucleotide sequence operatively linked to the expression promoter.

[0146] As indicated in the method of the first aspect, the overhangs of the n inserts are either palindromic and complementary to a palindromic overhang of the receiving backbone, or are non-palindromic, different between each other, and each one is complementary only to another non-palindromic overhang of another insert or of the receiving backbone. Therefore, each insert's overhang can only bind to only another overhang of another insert, or of the receiving backbone. Thus, in a particular embodiment, the overhangs of the n inserts and of the receiving backbone predetermine the position and / or orientation of each insert within the destination vector. Therefore, the order in which the inserts are placed in the sequence of n inserts as defined above can be predetermined by the overhangs of the n inserts and of the receiving backbone. In a particular embodiment, the order and orientation in which the inserts are placed in the sequence of n inserts within the destination vector is predetermined by the overhangs of the n inserts and of the receiving backbone.

[0147] In another particular embodiment, the palindromic overhang of the receiving backbone defined in (i)-(a) of the method of the first aspect consists of the overhang formed upon cleavage of the Type IIP target site referred in (ii)-(a) by the corresponding Type IIP enzyme. In another particular embodiment, the palindromic overhangs of the receiving backbone defined in (i)-(b) of the method of the first aspect consist of the overhangs formed upon cleavage of the Type IIP target sites referred in (ii)-(b) by the corresponding Type IIP enzymes.

[0148] In a particular embodiment, the Type IIP target sites referred in the first aspect of the invention, particularly in (ii)-a) and (ii)-b) of the first aspect, are target sites for any of the Type IIP enzymes selected from those provided in the definition of Type IIP enzyme above. Thus, in a particular embodiment, the Type IIP enzymes referred in any of the embodiments of the first aspect are selected from the Type IIP enzymes provided in the definition of Type IIP enzyme above.

[0149] In a particular embodiment of the first aspect, the DNA ligase is any well-known DNA ligase, particularly is selected from any of the DNA ligases indicated in the definition of "DNA ligase”, more particularly is a T4 DNA ligase. In another particular embodiment, the reaction mixture further comprises a DNA ligase buffer, particularly a T4 DNA ligase buffer. In another particular embodiment, the reaction mixture comprises a Type1

[0150] IIS enzyme buffer, particularly a Type IIS enzyme buffer indicated in the definition of "conditions appropriate for digestion” below.

[0151] In an embodiment, the molar ratio (receiving backbone:insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of 1 :0.1-1 :1.9, 1 :0.2-1 :1.8, 1:0.3-1:17, 1 :0.4-1 :1.6, 1 :0.5-1 :1.5, 1:0.6-1:1.4, 1:07-1:1.3, 1 :0.8-1 :1.2, 1 :0.9-1 :1.1 more particularly of 1 :0.9-1 :1.1. Yet more particularly the molar ratio (receiving backbone: insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of around 1:1. In a particular embodiment, the molar ratio of the receiving backbone and the insert(s) comprising a palindromic overhang (receiving backbone: insert) is 1:0.5-1:1.5. In another embodiment, the molar ratio (receiving backbone: insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of 1:0.1-1:1.2, 1:0.1-1:1.2, 1:0.3-1:1.2, 1:0.4-1:1.2, 1:0.5-1:1.2, 1:0.6-1:1.2, 1:07-1:1.2, 1:0.8-1:1.2, 1:0.9-1:1.2, more particularly is of 1:0.5-1:1.2. In another embodiment, the molar ratio (receiving backbone:insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of 1 :0.1-1 :1.1, 1 :0.1-1 :1.1, 1 :0.3-1 :1.1, 1 :0.4-1 :1.1 , 1 :0.5-1 :1.1, 1 :0.6-1 :1.1, 1:07-1:1.1, 1:0.8-1:1.1, 1:0.9-1:1.1, more particularly is of 1:0.5-1:1.1. In another embodiment, the molar ratio (receiving backbone:insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of 1:0.1 -1:1, 1:0.1-1:1, 1:0.3-1:1, 1:0.4-1:1, 1:0.5-1:1, 1:0.6-1:1, 1:07-1:1, 1:0.8-1:1, 1:0.9-1:1, more particularly is of 1:0.5-1:1. In another embodiment, the molar ratio (receiving backbone:insert) of the receiving backbone and the remaining inserts, i.e. not comprising a palindromic overhang, is of 1:1.1-1:3, 1:1.2-1:2.9, 1:1.3-1:2.8, 1:1.4-1:2.6, 1:1.5-1:2.5, 1:1.6-1:2.4, 1:17-1:2.3, 1:1.8-1:2.2, 1:1.9-1:2.1, more particularly of around 1:1.9-1:2.1. Yet more particularly the molar ratio (receiving backbone:insert) of the receiving backbone and the insert(s) comprising a non-palindromic overhang is of around 1:2. In an embodiment, the molar ratio (receiving backbone:insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of 1:0.1-1:1.9 and the molar ratio (receiving backbone:insert) of the receiving backbone and the remaining inserts, is of 1:1.1-1:3, 1:1.2-1:2.9, 1:1.3-1:2.8, 1:1.4-1:2.6, 1:1.5, 1:2.5, 1:1.6-1:2.4, 1:17-1:2.3, 1:1.8-1:2.2, 1:1.9-1:2.1, particularly of around 1:1.9-1:2, yet more particularly of around 1:2.. In an embodiment, the molar ratio (receiving backbone:insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of 1:0.2-1: 1.8 and the molar ratio (receiving backbone:insert) of the receiving backbone and the remaining inserts, is of 1:1.1-1:3, 1:1.2-1:2.9, 1:1.3-1:2.8, 1:1.4-1:2.6, 1 :1.5, 1:2.5, 1 :1.6-1:2.4, 1:17-1:2.3, 1:1.8-1:2.2, 1:1.9-1:2.1, particularly of around 1:1.9-1:2, yet more particularly of around 1:2. more particularly of around 1:2. In an embodiment, the molar ratio (receiving backbone: insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of 1:0.3-1:17 and the molar ratio (receiving backbone: insert) of the receiving backbone and the remaining inserts, is of 1:1.1-1:3, 1:1.2-1:2.9, 1:1.3-1:2.8, 1:1.4-1:2.6, 1:1.5, 1:2.5, 1:1.6-1:2.4, 1:17-1:2.3, 1:1.8-1:2.2, 1:1.9-1:2.1, particularly of around 1:1.9-1 :2, yet more particularly of around 1 :2. In an embodiment, the molar ratio (receiving backbone: insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of 1:0.4-1: 1.6 and the molar ratio (receiving backbone:insert) of the receiving backbone and the remaining inserts, is of 1:1.1-1:3, 1:1.2-1:2.9, 1:1.3-1:2.8, 1:1.4-1:2.6, 1:1.5, 1:2.5, 1:1.6-1:2.4, 1:17-1:2.3, 1:1.8-1:2.2, 1:1.9-1:2.1, particularly of around1:1.9-1:2, yet more particularly of around 1:2.. In an embodiment, the molar ratio (receiving backbone: insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of 1:0.5-1: 1.5 and the molar ratio (receiving backbone:insert) of the receiving backbone and the remaining inserts, is of 1:1.1-1:3, 1 :1.2-1 :2.9, 1 :1.3-1 :2.8, 1 :1.4-1 :2.6, 1:1.5, 1:2.5, 1 :1.6-1 :2.4, 1:17-1:2.3, 1 :1.8-1 :2.2, 1 :1.9-1 :2.1, particularly of around 1:1.9-1:2, yet more particularly of around 1:2. In an embodiment, the molar ratio (receiving backbone:insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of 1 :0.6-1:1.4and the molar ratio (receiving backbone:insert) of the receiving backbone and the remaining inserts, is of 1:1.1-1:3, 1:1.2-1:2.9, 1:1.3-1:2.8, 1:1.4-1:2.6, 1:1.5, 1:2.5, 1:1.6-1:2.4, 1:17-1:2.3, 1:1.8-1:2.2, 1:1.9-1:2.1, particularly of around 1:1.9-1:2, yet more particularly of around 1:2. In an embodiment, the molar ratio (receiving backbone:insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of 1:07-1 :1.3and the molar ratio (receiving backbone:insert) of the receiving backbone and the remaining inserts, is of 1:1.1-1:3, 1:1.2-1:2.9, 1:1.3-1:2.8, 1:1.4-1:2.6, 1:1.5, 1:2.5, 1:1.6-1:2.4, 1:17-1:2.3, 1:1.8-1:2.2, 1 :1.9-1 :2.1, particularly of around 1:1.9-1:2, yet more particularly of around 1:2. In an embodiment, the molar ratio (receiving backbone: insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of 1:0.8-1:1.2 and the molar ratio (receiving backbone: insert) of the receiving backbone and the remaining inserts, is of 1:1.1-1:3, 1:1.2-1:2.9, 1:1.3-1:2.8, 1:1.4-1:2.6, 1 :1.5, 1:2.5, 1 :1.6-1:2.4, 1:17-1:2.3, 1:1.8-1:2.2, 1:1.9-1:2.1, particularly of around 1:1.9-1:2, yet more particularly of around 1:2. In an embodiment, the molar ratio (receiving backbone: insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of 1 :0.9-1: 1.1 and the molar ratio (receiving backbone: insert) of the receiving backbone and the remaining inserts, is of 1:1.1-1:3, 1:1.2-1:2.9, 1:1.3-1:2.8, 1:1.4-1:2.6, 1:1.5, 1:2.5, 1:1.6-1:2.4, 1:17-1:2.3, 1:1.8-1:2.2, 1:1.9-1:2.1, particularly of around 1:1.9-1:2, yet more particularly of around 1:2. In an embodiment, the molar ratio (receiving backbone: insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of around 1:1 and the molar ratio (receiving backbone: insert) of the receiving backbone and the remaining inserts, is of 1:1.1-1:3, 1:1.2-1:2.9, 1:1.3-1:2.8, 1:1.4-1:2.6, 1:1.5, 1:2.5, 1:1.6-1:2.4, 1:17-1:2.3, 1:1.8-1:2.2, 1:1.9-1:2.1, particularly of around 1:1.9-1:2, yet more particularly of around 1:2. In an embodiment, the molar ratio (receiving backbone: insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of around 1 :0.1-1 :1 and the molar ratio (receiving backbone:insert) of the receiving backbone and the remaining inserts, is of 1:1.1-1:3, 1:1.2-1:2.9, 1:1.3-1:2.8, 1:1.4-1:2.6, 1:1.5, 1:2.5, 1:1.6-1:2.4, 1:17-1:2.3, 1:1.8-1:2.2, 1:1.9-1:2.1, particularly of around 1:1.9-1:2, yet more particularly of around 1:2. In an embodiment, the molar ratio (receiving backbone:insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of around 1:0.2-1:1 and the molar ratio (receiving backbone:insert) of the receiving backbone and the remaining inserts, is of 1:1.1-1:3, 1:1.2-1:2.9, 1:1.3-1:2.8, 1:1.4-1:2.6, 1:1.5, 1:2.5, 1:1.6-1:2.4, 1:17-1:2.3, 1:1.8-1:2.2, 1 :1.9-1 :2.1, particularly of around 1:1.9-1:2, yet more particularly of around 1:2. In an embodiment, the molar ratio (receiving backbone: insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of around 1:0.3-1:1 and the molar ratio (receiving backbone:insert) of the receiving backbone and the remaining inserts, is of 1:1.1-1:3, 1:1.2-1:2.9, 1:1.3-1:2.8, 1:1.4-1:2.6, 1:1.5, 1:2.5, 1:1.6-1:2.4, 1:17-1:2.3, 1:1.8-1:2.2, 1 :1.9-1 :2.1, particularly of around 1:1.9-1:2, yet more particularly of around 1:2. In anembodiment, the molar ratio (receiving backbone: insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of around 1:0.4-1:1 and the molar ratio (receiving backbone:insert) of the receiving backbone and the remaining inserts, is of 1:1.1-1:3, 1:1.2-1:2.9, 1:1.3-1:2.8, 1:1.4-1:2.6, 1:1.5, 1:2.5, 1:1.6-1:2.4, 1:17-1:2.3, 1:1.8-1:2.2, 1:1.9-1:2.1, particularly of around 1:1.9-1:2, yet more particularly of around 1:2. In an embodiment, the molar ratio (receiving backbone:insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of around 1:0.5-1:1 and the molar ratio (receiving backbone: insert) of the receiving backbone and the remaining inserts, is of 1:1.1-1:3, 1:1.2-1:2.9, 1:1.3-1:2.8, 1:1.4-1:2.6, 1:1.5, 1:2.5, 1:1.6-1:2.4, 1:17-1:2.3, 1:1.8-1:2.2, 1:1.9-1:2.1, particularly of around 1:1.9-1:2, yet more particularly of around 1:2. In an embodiment, the molar ratio (receiving backbone: insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of around 1 : 0.6- 1 : 1 and the molar ratio (receiving backbone:insert) of the receiving backbone and the remaining inserts, is of 1:1.1-1:3, 1:1.2-1:2.9, 1:1.3-1:2.8, 1:1.4-1:2.6, 1:1.5, 1:2.5, 1:1.6-1:2.4, 1:17-1:2.3, 1:1.8-1:2.2, 1:1.9-1:2.1, particularly of around 1:1.9-1:2, yet more particularly of around 1:2. In an embodiment, the molar ratio (receiving backbone:insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of around 1:07-1:1 and the molar ratio (receiving backbone:insert) of the receiving backbone and the remaining inserts, is of 1:1.1-1:3, 1:1.2-1:2.9, 1:1.3-1:2.8, 1:1.4-1:2.6, 1:1.5, 1:2.5, 1:1.6-1:2.4, 1:17-1:2.3, 1:1.8-1:2.2, 1 :1.9-1 :2.1, particularly of around 1:1.9-1:2, yet more particularly of around 1:2. In an embodiment, the molar ratio (receiving backbone: insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of around 1:0.8-1:1 and the molar ratio (receiving backbone:insert) of the receiving backbone and the remaining inserts, is of 1:1.1-1:3, 1:1.2-1:2.9, 1:1.3-1:2.8, 1:1.4-1:2.6, 1:1.5, 1:2.5, 1:1.6-1:2.4, 1:17-1:2.3, 1:1.8-1:2.2, 1 :1.9-1 :2.1, particularly of around 1:1.9-1:2, yet more particularly of around 1:2. In an embodiment, the molar ratio (receiving backbone: insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is of around 1:0.9-1:1 and the molar ratio (receiving backbone:insert) of the receiving backbone and the remaining inserts, is of 1:1.1-1:3, 1:1.2-1:2.9, 1:1.3-1:2.8, 1:1.4-1:2.6, 1:1.5, 1:2.5, 1:1.6-1:2.4, 1:17-1:2.3, 1:1.8-1:2.2, 1:1.9-1:2.1, particularly of around 1:1.9-1:2, yet more particularly of around 1:2.

[0152] In a preferred embodiment, the molar ratio (receiving backbone:insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is higher than the molar ratio (receiving backbone:insert) of the receiving backbone and the remaining inserts. In other words, in a preferred embodiment, the molar concentration for inserts comprising a palindromic overhang (with respect to the molar concentration of the receiving backbone) is lower than the molar concentration for the rest of inserts (i.e. not comprising a palindromic overhang; with respect to the molar concentration of the receiving backbone). In a more preferred embodiment, the molar ratio (receiving backbone:insert) of the receiving backbone and the insert(s) comprising a palindromic overhang is as defined in any of the embodiments of the previous paragraph, provided the molar concentration for inserts comprising a palindromic overhangs (with respect to the molar concentration of the receiving backbone) is lower than the molar concentration for the rest of inserts (i.e. not comprising a palindromic overhang; with respect to the molar concentration of the receivingbackbone). In another particularly preferred embodiment, the molar ratio (receiving backbone:insert) of the receiving backbone and the insert(s) not comprising a palindromic overhang is as defined in any of the embodiments of the previous paragraph, provided the molar concentration for inserts comprising a palindromic overhangs (with respect to the molar concentration of the receiving backbone) is lower than the molar concentration for the rest of inserts (i.e. not comprising a palindromic overhang; with respect to the molar concentration of the receiving backbone). In another particularly preferred embodiment, the molar ratio (receiving backbone: insert) of the receiving backbone and the insert(s) comprising a palindromic overhang and the molar ratio of the receiving backbone and the remaining inserts (not comprising a palindromic overhang), is as defined in any of the embodiments of the previous paragraph, provided the molar concentration for inserts comprising a palindromic overhangs (with respect to the molar concentration of the receiving backbone) is lower than the molar concentration for the rest of inserts (i.e. not comprising a palindromic overhang). In another particular embodiment, the molar ratio (receiving backbone:insert) of the receiving backbone and the insert(s) comprising a palindromic overhang and the molar ratio of the receiving backbone and the remaining inserts (not comprising a palindromic overhang), is as defined in any of the embodiments of the first aspect, particularly in the embodiments of the paragraph above, provided the molar concentration of inserts comprising a palindromic overhang is equal or lower, particularly lower, than that of the receiving backbone. Thus, in a particular embodiment of the first aspect, the molar concentration of inserts comprising a palindromic overhang is equal or lower, particularly lower, than that of the receiving backbone in the reaction mixture of the first aspect. In another particular embodiment of the first aspect, the molar concentration of inserts comprising a palindromic overhang is equal to that of the receiving backbone in the reaction mixture of the first aspect.

[0153] In an embodiment, the cloning method of the first aspect further comprises subjecting the reaction mixture to conditions appropriate for ligation of the receiving backbone with the n inserts.

[0154] The term "conditions appropriate for ligation”, or "conditions suitable for ligation”, refers to the conditions that allow a particular DNA ligase to form a phosphodiester bond between two DNA strands. A skilled person knows the appropriate conditions required for ligation when using a specific DNA ligase, such as T4 DNA ligase or E. coli ligase. As well known by an expert in the filed, said conditions comprise in general terms incubating the corresponding nucleic acid molecules to be ligated together with the corresponding DNA ligase, in a specific buffer for a certain time and at a certain temperature. These conditions are well known by an expert in the field for each DNA ligase to be used and can be applied for instance by following the provider's instructions for the corresponding enzyme.

[0155] In a particular embodiment, subjecting the reaction mixture to conditions appropriate for ligation comprises adding to the reaction mixture a buffer suitable for the activity of the DNA ligase, particularly of the DNA ligase of the first aspect. A buffer suitable for the corresponding DNA ligase is well known by an expert in the field and particularly corresponds to a buffer specified in the DNA ligase provider's instructions used inthe method of the first aspect. In a particular embodiment, the buffer suitable for the activity of the DNA ligase is a ligase buffer, particularly the DNA ligase buffer specified in the provider's instructions of the DNA ligase of the first aspect. In a particular embodiment, the ligation buffer is the T4 DNA ligase buffer, particularly wherein the DNA ligase is a T4 DNA ligase. In a particular embodiment, the ligase buffer, particularly the T4 DNA ligase buffer, comprises or consist of: Tris-HCI, particularly at around 50mM; MgCI2, particularly at around 10 mM; ATP, particularly at around 1 mM; and DTT, particularly at around 10 mM. More particularly the ligase buffer is as defined above and is at a pH of 7-8, 7.2-7.8, 7.4-7.6, particularly of around 7.5.

[0156] In a further particular embodiment, the DNA ligase buffer is an E. coll ligase buffer, particularly wherein the DNA ligase of the first aspect is an E. Coll ligase. In another particular embodiment, the ligase buffer, particularly the E. coll ligase buffer, comprises or consists of Tris-HCI, particularly at around 50 mM; MgCI2, particularly at around 4mM; NAD, particularly at around 4 piM; DTT, and optionally also albumin, particularly at around 50pig / ml. More particularly the ligation buffer is as just defined and is at a pH of around 7.5-8.5, 7.7-8.3, 7.9- 8.1, particularly at around 8.

[0157] As well known by an expert in the field, some DNA ligases, such as the T4 DNA ligase, are also active in certain restriction enzyme buffers, particularly in Type IIS restriction enzyme buffers, such as the particularly preferred Type IIS enzyme buffer with the components specified in the embodiments below addressed to the "conditions appropriate for digestion”. Thus, in another embodiment, the buffer suitable for ligation by the DNA ligase, particularly that of the first aspect, comprises or consists of a digestion buffer ( i.e. a restriction enzyme buffer), particularly a digestion buffer specified in the embodiments below addressed to the Type IIS digestion buffer, more particularly wherein the DNA ligase is a T4 DNA ligase. In a particular embodiment, the buffer suitable for ligation by the DNA ligase comprises ATP, particularly wherein the DNA ligase is a T4 DNA ligase. Thus, in a particular embodiment, when the buffer suitable for ligation by the DNA ligase comprises a restriction enzyme digestion buffer, particularly a restriction enzyme digestion buffer as defined below, ATP is added to the digestion buffer, particularly wherein the DNA ligase is a T4 DNA ligase. In another particular embodiment, when the buffer suitable for ligation by the DNA ligase consists of a digestion buffer, particularly as defined in below in "conditions appropriate for digestion”, the digestion buffer comprises ATP, particularly wherein the DNA ligase is a T4 DNA ligase. More particularly, the ATP added to or comprised in the digestion buffer is added at or comprised in a concentration of 0.1 -2mM; , 0.3-1.7mM, 0.7-1.5mM. 0.8-1.2mM, 0.9-1.1mM, more particularly of around 1mM.

[0158] In a particular embodiment, conditions appropriate for ligation, comprise, subjecting the reaction mixture of the first aspect, particularly the reaction mixture of the previous paragraph, to 4 °C - 37 °C, 9 °C -32 °C, 12 °C-30 °C, 14 °C-27 °C. In a more particular embodiment, the temperature is of 10 °C -21 °C, 12 °C -19 °C, 14 °C -18 °C, 15 °C -17 °C, particularly of 15 °C -17 °C. More particularly, the temperature is of around 16 °C.In a particular embodiment, subjecting the reaction mixture to conditions appropriate for ligation comprises incubating the reaction mixture of the first aspect, particularly under the conditions defined in any of the embodiments of previous paragraph below the definition of "conditions appropriate for ligation”, for at least 1, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 45, at least 60 minutes, at least 70, at least 80, at least 90, at least 120 minutes, more particularly for at least 5 minutes. More particularly, for around 1, 3, 4, 5, 10, 15, 20, 25, 30, 45, 60, 70, 80, 90, 120 minutes, yet more particularly for around 5 minutes.

[0159] In an embodiment of the first aspect, at least one of the n inserts, particularly the n inserts are obtained by digestion of one or more polynucleotides with Type IIS restriction enzyme(s). In another embodiment, the receiving backbone is obtained by digestion of the receiving vector with Type IIS restriction enzyme(s). More particularly, at least one of the n inserts, particularly the n inserts, are obtained by digestion of one or more polynucleotides with Type IIS restriction enzyme(s), and the receiving backbone is obtained by digestion of the receiving vector with Type IIS restriction enzyme(s), particularly with the same Type IIS restriction enzyme(s) as the one or more polynucleotides.

[0160] In another embodiment of the first aspect, at least one of the n inserts, particularly the n inserts are obtained by digestion of one or more polynucleotides with Type IIS restriction enzyme(s), particularly with the Type IIS enzyme of the first aspect, each polynucleotide comprising one or more of the n inserts, wherein each insert is comprised within the corresponding polynucleotide within a region F of formula 5'-R1-E1-S-E2-R2-3', wherein:

[0161] - each one of R1 and R2 is a sequence consisting of a recognition sequence for a Type IIS restriction enzyme, particularly for the Type IIS enzyme of the first aspect, oriented in opposite directions within F, - E1 is an extension sequence comprising the cleavage site C1 of the Type IIS restriction enzyme that recognizes R1,

[0162] - S consists of the sequence of interest of the corresponding insert,

[0163] - E2 consists of an extension sequence comprising the cleavage site C2 of the Type IIS restriction enzyme that recognizes R2,

[0164] - R1, E1, S, E2 and R2 are directly attached to each other, and

[0165] wherein upon cleavage by the corresponding Type IIS restriction enzyme, particularly by the Type IIS enzyme of the first aspect, C1 and C2 form the overhangs comprised in the inserts as defined in (ii)-a) or (ii)-b) of claim 1.

[0166] In a particular embodiment, the one or more polynucleotides referred above are digested by a sole Type IIS enzyme that is the same for all said polynucleotides, also herein referred as the Type IIS restriction enzyme of the first aspect. Thus, in another particular embodiment, R1 and R2 are recognized by the same Type IISenzyme, also corresponding to the Type IIS restriction enzyme of the first aspect. In a particularly preferred embodiment, the Type IIS enzyme of the first aspect is selected from any of those provided in the definition of "Type IIS enzyme” above, particularly is selected from Bsal, Bbsl, or Esp3l, more particularly from Bsal or Bbsl. In an even yet more preferred embodiment, the Type IIS enzyme of the first aspect is Bsal.

[0167] The one or more polynucleotides as just defined are also referred herein as the one or more polynucleotides of the first aspect, the polynucleotide of the first aspect, or, when referring to one polynucleotide as just defined, to the polynucleotide of the first aspect.

[0168] The term "polynucleotide”; as used herein, refers to a nucleic acid molecule, as defined herein, of any length, including at least 2 nucleotides, particularly of at least 3, 4, 5, 6, 9, 12, 15, 18 or 21 nucleotides, more particularly at least 6 nucleotides. In a particular embodiment, the polynucleotide, comprises no more than 700, 600, 500, 400, 350, 300, 250, 220, 200, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 12, 9 nucleotides. In another particular embodiment, the polynucleotide of the first aspect of the invention comprises at least 9 nucleotides, and no more than 700, 600, 500, 400, 350, 300, 250, 220, 200, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 12 nucleotides. In another particular embodiment, the polynucleotide of the first aspect of the invention comprises at least 12 nucleotides and no more than 700, 600,500, 400, 350, 300, 250, 220, 200, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15. In another particular embodiment, the polynucleotide of the first aspect of the invention comprises at least 15 nucleotide and no more than 700, 600, 500, 400, 350, 300, 250, 220, 200, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15. In another particular embodiment, the polynucleotide of the first aspect of the invention comprises at least 18 nucleotide and no more than 700, 600, 500, 400, 350, 300, 250, 220, 200, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 21. In another particular embodiment, the polynucleotide of the first aspect of the invention comprises at least 21 nucleotides and no more than 700, 600, 500, 400, 350, 300, 250, 220, 200, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30. In another particular embodiment, the polynucleotide of the first aspect of the invention comprises around 700, 600, 500, 400, 350, 300, 250, 220, 200, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 12, 9, 6 or 3 nucleotides. In another particular embodiment, the polynucleotide of the first aspect of the invention comprises at least 100 nucleotide and no more than 700, 600, 500, 400, 350, 300, 250, 220, 200, 180, 170, 160, 150, 140, 130, 120, 110. In another particular embodiment, the polynucleotide of the first aspect of the invention comprises at least 150 nucleotide and no more than 700, 600, 500, 400, 350, 300, 250, 220, 200, 180, 170, 160. In another particular embodiment, the polynucleotide of the first aspect of the invention comprises at least 200 nucleotide and no more than 700, 600, 500, 400, 350, 300, 250, 220. In another embodiment, the polynucleotide is of more than 100, 110, 120, 130, 140, 150, 170, 200, 210, particularly of more than 150 nucleotides.As well understood by a skilled person, the one or more polynucleotides of the first aspect of the invention are double-stranded, particularly double stranded DNA polynucleotides. Similarly, regions F are also doublestranded, particularly, double stranded DNA regions. Thus, in the context of the polynucleotides of the first aspect, when referring to the number of nucleotides of the polynucleotide, the term "nucleotide” refers to or can be substituted by "base-pair”. As well understood by a skilled person, when an insert comprises a nucleotide analogue, or a nucleic acid molecule modification, as indicated in any of the embodiments above, the polynucleotide comprising said insert(s) also comprises said analogue or nucleic acid molecule modification.

[0169] In a particular embodiment, the polynucleotides of the first aspect comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, particularly, at least 2 of the n inserts. In another particular embodiment, the polynucleotides of the first aspect comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the n inserts. In a particular embodiment, each of polynucleotide of the first aspect comprises as many regions F as inserts. In another particular embodiment, the polynucleotides comprise only one insert within a region F as defined above. As well understood by a skilled person, each insert referred herein is comprised in a region F, as defined herein, which is itself comprised in a polynucleotide of the first aspect. Thus, in a more particular embodiment, at least one of the n inserts, particularly the n inserts, are obtained by digestion of at least one polynucleotide, particularly n polynucleotides, with Type IIS restriction enzymes, particularly by the Type IIS enzyme of the first aspect, each polynucleotide comprising one of the n inserts and wherein each insert is comprised within the corresponding polynucleotide within a region F as defined herein.

[0170] In a particular embodiment, at least one of the n inserts are obtained by providing to the reaction mixture, the one or more polynucleotides of the first aspect, together with the Type IIS restriction enzyme(s) that recognize R1 and R2 in each of said polynculeotides, particularly the Type IIS restriction enzyme of the first asecpt, and subjecting the reaction mixture to conditions appropriate for digestion of the one or more polynucleotides by the Type IIS restriction enzyme(s).

[0171] In an embodiment, the one or more polynucleotide of the first aspect are Polymerase Chain Reaction (PCR) products, hybridized complementary oligos or combinations thereof. Thus, in an embodiment, the one or more polynucleotide of the first aspect are obtained by a PCR reaction, upon hybridization of 2 complementary oligos, or the combination thereof. Thus, in an embodiment, some polynucleotides are PCR products, and others a are hybridized complementary oligos. As well understood by a skilled person, two hybridized oligos as referred herein are two complementary oligos hybridized or bound to each other. The term "complementary” has been defined above and applies herein. Methods to generate polynucleotides by PCR or upon hybridization of two complementary oligos are well-known by an expert in the field. Non-limiting examples of such methods are provided in the examples below. In an embodiment, when the polynucleotide is of less than 200, 180, 170, 160, 150, 140, 130, 120, 110, 100 nucleotides in length, particularly of lessthan 150 nucleotides, it is obtained upon hybridization o two complementary oligos or corresponds to two hybridized complementary oligos.

[0172] In another particular embodiment, at least one of the n inserts, particularly the n inserts, is obtained upon hybridization of two complementary oligos, each oligo consisting of one strand of the corresponding insert. In another particular embodiment, the n inserts are each one obtained upon hybridization between two complementary oligos, each oligo consisting of one strand of the corresponding insert. Thus, in a particular embodiment, at least one of the n inserts is a hybridized oligo, more particularly the n inserts are hybridized oligos. Methods to obtain inserts upon hybridization between two complementary oligos are well known by an expert in the field. Non-limiting examples of such methods include those provided in the examples below.

[0173] In a more particular embodiment, the two complementary oligos forming the at least one of the n inserts, particularly the n inserts, as described just above (i.e. inserts that are hybridized oligos), are phosphorylated at one or both ends, particularly at the 5' end. Phosphorylation of the oligos is preferably performed before hybridization between the complementary oligos. Methods to phosphorylate oligos, especially before hybridization between the oligos for subsequent ligation, are well known by an expert in the field. Nonlimiting examples include those provided below in the examples.

[0174] In another embodiment, when the base paired region, particularly the sequence S, of the insert is of less than 200, 180, 170, 160, 150, 140, 130, 120, 110, 100 nucleotides in length, particularly of less than 150 nucleotides, said insert is obtained upon hybridization between two complementary oligos, particularly wherein each of the two oligos corresponds to one strand of the corresponding insert. As well understood by a skilled person, the length of the base paired region is determined by the number of nucleotides of one strand of the insert comprised in said base paired region, in other words, it refers to the number of base pairs within the insert. In a particular embodiment, inserts corresponding to hybridized oligos or obtained upon hybridization between two complementary oligos, comprise or consist of a protein tag encoding sequence, or a signal peptide encoding sequence, particularly a protein tag encoding sequence.

[0175] In a particular embodiment, the inserts with a base paired region, particularly a sequence S, of less than 200, 180, 170, 160, 150, 140, 130, 120, 110, 100 nucleotides in length, particularly of less than 150 nucleotides in length, are hybridized oligos or are obtained upon hybridization between two complementary oligos, as defined in any of the embodiments above. In another particular embodiment, the at least one of the n inserts obtained upon digestion of one or more polynucleotides by type IIS enzyme(s), particularly by the type IIS restriction enzyme of the first aspect, as indicated in any of the embodiments herein, consists of those inserts (from the n inserts), comprising a base paired region, particularly a sequence S, of more than 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, particularly more than 150 nucleotides. Thus, in a particularly preferred embodiment, the inserts with a base paired region, particularly a sequence S, of less than 200, 180, 170, 160, 150, 140, 130, 120, 110, 100 nucleotides in length, particularly of less than150 nucleotides in length, are hybridized oligos or are obtained upon hybridization between two complementary oligos, as defined in any of the embodiments above, and the inserts of a higher length, particularly of more than 150 nucleotides, are obtained upon digestion of one or more polynucleotides with Type IIS enzyme(s), particularly by the type IIS enzyme of the first aspect, as defined in any of the embodiments above.

[0176] As well understood by a skilled person, when the insert is obtained upon hybridization between two complementary oligos, or corresponds to two hybridized oligos, each oligo forming the insert comprises a single stranded region at one of its ends, corresponding to an overhang of the insert, whereas the rest of the oligo is hybridized to the other oligo, also corresponding to the base-paired region of the insert or of the hybridized oligo as referred herein. Particularly, the regions of the two oligos that are hybridized to each other are 100% complementary, also corresponding to the base-paired region of the insert or of the hybridized oligo as referred herein. In a particularly preferred embodiment, when an insert is obtained upon hybridization of two complementary oligos as defined above, the overhangs of the corresponding insert are as those formed upon digestion of a polynucleotide of the first aspect by Type IIS restriction enzyme(s), particularly, by the Type IIS restriction enzyme of the first aspect. Thus, in a particularly preferred embodiment, when an insert is obtained upon hybridization of two complementary oligos as defined above, the overhangs of the corresponding insert are as those formed upon digestion of extension sequences E1 and E2 as defined in the present disclosure, comprised in the polynucleotide of the first aspect, by Type IIS restriction enzyme(s), particularly, by the Type IIS restriction enzyme of the first aspect.

[0177] In a particular embodiment, when at least one the n inserts, particularly the n inserts, are obtained upon hybridization of two complementary oligos as defined herein, the receiving backbone is obtained upon digestion of the receiving vector as defined below by Type IIS restriction enzyme(s), particularly by the Type IIS restriction enzyme of the first aspect, in the reaction mixture of the first aspect, as indicated in any of the embodiments below. More particularly, in the previous embodiment, the receiving vector is a receiving vector as defined below comprising V1 and V2 or comprising V3 and V4, particularly comprising V1 and V2. In a particularly preferred embodiment, when n is 1, and the insert is obtained upon hybridization between two complementary oligos, the receiving backbone is obtained upon digestion of the receiving vector as defined below by Type IIS restriction enzyme(s), particularly by the Type IIS restriction enzyme of the first aspect, in the reaction mixture of the first aspect, as indicated in any of the embodiments below. More particularly, in the previous embodiment, the receiving vector is a receiving vector as defined below comprising V1 and V2 or comprising V3 and V4, particularly comprising V1 and V2.

[0178] In an embodiment, R1 and R2 within a same region F have different nucleotide sequences, but are recognized by the same Type IIS enzyme, particularly by the Type IIS restriction enzyme of the first aspect. In a particular embodiment, all sequences R1 in the polynucleotides of the first aspect are equal, and allregions R2 in the polynucleotides of the first aspect are equal, particularly, wherein R1 and R2 are recognized by the same Type IIS enzyme, the Type IIS of the first aspect.

[0179] The term "oriented in opposite directions” as used herein, in the context of two Type IIS restriction enzyme recognition sequences comprised in a longer double stranded nucleic acid molecule, refers to the fact that the forward strand of the longer nucleic acid molecule, comprises the forward strand of one of the Type II recognition sites defined by one of said Type IIS recognition sequences, and the reverse strand of the other Type IIS recognition site defined by the other Type IIS recognition sequence. The term "forward strand of a type IIS enzyme recognition site” has been provided above in the definition of "Type IIS enzyme”. In other words, the term "oriented in opposite directions” in the context of regions R1 and R2, refers to the fact that the cleavage site of the restriction enzyme that recognizes R1 (i.e. C1) is downstream R1 (in the nucleotide sequences comprising R1, i.e. the forward strand of the polynucleotide comprising it), and the cleavage site of the restriction enzyme that recognizes R2 (i.e. C2), is upstream R2 (in the same nucleotide sequence comprising R1 and R2, i.e. the forward strand of the polynucleotide comprising it).

[0180] The term "extension sequence”, or "linker sequence”, as used herein, refers to a sequence with no function besides the fact it bridges or links two regions within a longer nucleotide sequence, or that it separates said two nucleotide regions, in a length equivalent to that of the extension sequence itself. In a particular embodiment, the extension sequence is an innocuous nucleic acid sequence, this is, it does not affect the activity of any of the nucleic acid sequences within the n inserts nor vectors of the first aspect, particularly within the destination vector, such as that of a promoter sequence, a protein tag encoding sequence, or an antibiotic resistance encoding sequence etc. In a particular embodiment, the extension sequence does not affect the structure nor activity of a peptide or protein encoded by the n inserts, or by the vectors of the first aspect, particularly by the destination vector. Particularly, when referring to the effect of the activity of the extension sequence in the context of the activity of a protein or peptide (such as the tag, or the peptide / protein encoded by the n inserts), the activity of the extension sequence corresponds to that of the amino acid(s) or peptide encoded by said extension sequence.

[0181] In a particular embodiment, the extension sequence E1 is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleotides long, particularly at least 3 nucleotides long. In another particular embodiment, the extension sequence E2 is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 nucleotides long, particularly at least 3 nucleotides long. In a particularly preferred embodiment, E1 is at least 6 nucleotides long. In another particularly preferred embodiment, E2 is at least 6 nucleotides long. As well understood by a skilled person, the length of a nucleotide sequence strand, or single stranded sequence, as used herein, corresponds to the number of nucleotides said sequence consists of.In a particular embodiment, the extension sequence E1 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 nucleotides long, particularly 3 nucleotides long. In another particular embodiments, In a particular embodiment, the extension sequences E2 is 3, 4, 5, 6, at 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 nucleotides long, particularly 3 nucleotides long. In a particularly preferred embodiment, E1 is 6 nucleotides long. In another particularly preferred embodiment, E2 is 6 nucleotides long.

[0182] More particularly, both E1 and E2 have the same length and is selected from any of those just provided. In a yet more particular embodiment, E1 and E2 are at least 6 nucleotides long. In a yet more particular embodiment, E1 and E2 are 6 nucleotides long.

[0183] As well understood by a skilled person, each of E1 and E2 comprises one of the overhangs of the insert comprised in the same F region as said E1 and E2. Thus, the length of E1 and E2 is at least the number of nucleotides that separate the Type IIS enzyme's recognition sequence and its cleavage site. In a particular embodiment, all the embodiments herein addressed to E1 and / or E2 apply to all the E1 and E2 of the one or more polynucleotides of the first aspect.

[0184] As indicated in the definition of the Type IIS enzyme, the distance between the Type IIS enzyme' s recognition site and its cleavage site is specific for each Type IIS enzyme, and is well known by an expert in the field. Said distance is also indicated for each Type IIS enzyme in the instructions of the Type IIS enzyme's supplier.

[0185] In a particular embodiment, as indicated above, the Type IIS enzyme is selected from the list consisting of Bsal, Bbsl, Esp3l, PaqCI, Sapl, BspQI, BtgZI, particularly is selected from Bsal, Bbsl, and Esp3l. The nucleotides that separate the Bsal recognition sequence and its cleavage site is 1 nucleotide in the strand comprising the forward strand of the recognition site, and 5 nucleotides in the other strand. The nucleotides that separate the Bbsl recognition sequence and its cleavage site is 2 nucleotides in the strand comprising the forward strand of the recognition site, and 6 nucleotides in the other strand. The nucleotides that separate the Esp3l recognition sequence and its cleavage site is 1 nucleotide in the strand comprising the forward strand of the recognition site, and 5 nucleotides in the other strand.

[0186] Thus, E1 and E2 are at least 5 nucleotides long when type IIS enzyme is Bsal or Bbsl, and at least 6 nucleotides long when type IIS enzyme is Esp3l.

[0187] As well understood by a skilled person, E1 and E2 within each region F comprise the overhangs of the corresponding insert, comprised in said region. Additionally, as indicated in the method of the first aspect, upon binding between the complementary palindromic overhangs of the one or two inserts comprising one, and the receiving backbone, a type IIP target site is formed.In a particular embodiment, E1 or E2 of the region(s) F comprising an insert's palindromic overhang, comprises or consists of a type IIP target sequence. In another particular embodiment, when n=1 , E1 and / or E2 comprising a palindromic overhang, comprise(s) or consist(s) of (a) type IIP target sequence(s). In a particular embodiment, the Type IIP target sequences(s) correspond(s) to those of the type IIP target site(s) referred to in (ii)-a) or (ii)-b) of the method of the first aspect.

[0188] In an embodiment of the first aspect, when n is 1, the insert is obtained by digestion of a polynucleotide with a Type IIS restriction enzyme, the polynucleotide comprising said insert within a region F of formula 5'-R1-E1-S-E2-R2-3', wherein F and each sequence therein, is as defined in any of the embodiments of the first aspect. In another embodiment, when n=1, the receiving backbone is obtained upon digestion of the receiving vector as defined below by Type IIS restriction enzyme(s), particularly by the Type IIS restriction enzyme of the first aspect, in the reaction mixture of the first aspect, as indicated in any of the embodiments below. More particularly, in the previous embodiment, the receiving vector is a receiving vector as defined below comprising V1 and V2, or V3 and V4, particularly V1 and V2.

[0189] In a particular embodiment, at least one of the n inserts, particularly the n inserts, are obtained upon digestion of the polynucleotides of the first aspect by the Type IIS restriction enzyme in the reaction mixture of the first aspect. Thus, in a particular embodiment of the method of the first aspect, at least one of the n inserts, particularly the n inserts, more particularly those inserts indicated above to have been obtained upon digestion of the polynucleotide of the first aspect by type IIS enzyme(s) (i.e. having a base paired region or sequence S of a length indicated above), are obtained by providing to the reaction mixture, the one or more polynucleotide of the first aspect, together with the Type IIS enzyme(s) that recognize R1 and R2 in the one or more polynucleotides, particularly the Type IIS restriction enzyme of the first aspect, and subjecting the reaction mixture to conditions appropriate for digestion of the one or more polynucleotides by the Type IIS restriction enzyme(s). In another embodiment, the reaction mixture of the first aspect further comprises the polynucleotides of the first aspect, the Type IIS restriction enzyme(s), particularly the Type IIS restriction enzyme of the first aspect, and the method further comprises subjecting the reaction mixture to conditions appropriate for digestion.

[0190] As also indicated above, some or all of the n inserts, particularly the inserts with a base paired region or sequence S of a length indicated above for insert consisting of hybridized oligos, are obtained upon hybridization between two complementary oligos. Thus, in another particular embodiment, the reaction mixture of the first aspect further comprises the polynucleotides of the first aspect, the inserts consisting of hybridized oligos as defined in the first aspect, the Type IIS restriction enzyme(s), particularly the Type IIS restriction enzyme of the first aspect, and the method further comprises subjecting the reaction mixture to conditions appropriate for digestion.The term "conditions appropriate for digestion” is defined below and applies herein, as well as the embodiments provided below defining said conditions.

[0191] In another particular embodiment of the first aspect, the receiving backbone is obtained by digestion of a receiving vector with Type IIS restriction enzymes(s), particularly with the Type IIS restriction enzyme IIS of the first aspect, the receiving vector comprising a disposable sequence comprising two recognition sequences V1 and V2 for said Type IIS restriction enzyme(s), wherein:

[0192] - V1 is at the 5' region of the disposable sequence and V2 is at the 3' region of the disposable sequence, and

[0193] - V1 and V2 are oriented in opposite directions within the disposable sequence, the orientation of V1 being such that a cleavage site K1 of the Type IIS enzyme recognizing V1 is upstream V1 and at the 5' end of the disposable sequence, and the orientation of V2 being such that a cleavage site K2 of the Type IIS enzyme recognizing V2 is downstream V2 and at the 3' end of the disposable sequence, and,

[0194] wherein upon cleavage of K1 and K2 with the Type IIS restriction enzyme, the overhangs in the receiving backbone defined in (i)-a) or (i)-b) in the method of the first aspect are formed.

[0195] For simplicity, the receiving vector as defined just above is also referred to as the receiving vector comprising V1 and V2.

[0196] The term "disposable sequence”, as used herein, refers to the term well-known by an expert in the field. Particularly, the term refers to a sequence without any associated activity, and that can be deleted from a longer sequence, i.e. the receiving vector, without affecting to the function of the active elements comprised in said longer sequence, such as an origin of replication, promoters, genetic markers, antibiotic resistant genes, epitopes, reporter genes, targeting sequence, or the protein purification tags etc. A disposable sequence in a cloning vector can for instance refer to a region or the whole cloning site or multicloning site of a vector. In a particular embodiment, the disposable sequence referred in the first aspect of the invention is comprised or consists of, particularly consists of a multicloning site from the receiving vector.

[0197] The term "oriented in opposite directions” in the context of two type IIS recognition sequences within a longer double stranded nucleic acid molecule has been defined above and applies herein wherein the double stranded nucleic acid molecule is the receiving vector.

[0198] The terms "5' region”, "3' region”, "5' end” and "'3' end” have been defined above and apply herein.

[0199] In a particular embodiment, V1 and V2 are as R2 and R1, respectively, defined in the first aspect. In a more particular embodiment, V1 and V2 are the same as or different than R2 and R1, respectively, particularly are the same, wherein all R1 and R2 of all the polynucleotides of the first aspect are the same. Particularly, V1 and V2 are recognized by the same Type IIS restriction enzyme, more particularly by the type IIS of thefirst aspect.

[0200] In an embodiment, upon cleavage of K1 and K2 by the corresponding Type IIS restriction enzyme, the overhangs formed correspond to those of the receiving backbone defined in (i)-a) of the method of the first aspect. In another embodiment, upon cleavage of K1 and K2 with the corresponding Type IIS restriction enzyme, the overhangs formed correspond to those of the receiving backbone defined in (i)-b) of the method of the first aspect.

[0201] The sequence immediately upstream V1 and / or the sequence immediately downstream V2 in the receiving vector, comprising the type IIS cleavage sites K1 and / or K2 respectively, are also referred herein to as the extension sequences E_V1 and / or E_V2 respectively. In a particular embodiment, the definitions and embodiments addressed to the length of E2 or E1 are equally applicable to E_V1 or E_V2 respectively. Thus, in a particular embodiment, the length of E_V1 is as defined for E2, particularly is selected from any of the lengths provided above for E2. In another particular embodiment, the length of E_V2 is as defined for E1, particularly is selected from any of the lengths provided above for E1.

[0202] As well understood by a skilled person, the term "immediately upstream”, or "immediately downstream”, in the context of a region or sequence with respect to another one used as reference, indicates that both are directly linked to each other, wherein the sequence immediately upstream the reference one is directly linked to the 5' end of a reference sequence, and the sequence that is immediately downstream a reference one is directly linked to the 3' end of the reference sequence. The term "directly linked to each other” has been provided above and applies herein.

[0203] In an embodiment, the receiving vector comprising V1 and V2, can comprise in addition to the Type IIS recognition sequences, at least one Type IIP target sequence comprised in or consisting of E_V1 and / or E-V2 extension sequences. In a particular embodiment, the receiving vector as defined herein comprising V1 and V2, comprises at least one Typ IIP target sequence comprised in or consisting of E_V1 and / or E-V2 extension sequences. In another particular embodiment, the receiving vector as defined herein comprising V1 and V2, comprises one Type IIP target sequence comprised in or consisting of E_V1 or E_V2 extension sequences. In another particular embodiment, the receiving vector as defined herein comprising V1 and V2, comprises two Type IIP target sequences comprised in or consisting of E_V1 and E-V2 extension sequences.

[0204] In a particular embodiment, the at least one type IIP target sites comprised in the receiving vector and defined by the Type IIP target sequences referred in the embodiments of the previous paragraph, corresponds to at least one of the type IIP target sites referred to in (ii)-a) or (ii)-b) of the method of the first aspect. In another particular embodiment, the one type IIP target sites comprised in the receiving vector and defined by the Type IIP target sequences referred in the embodiments of the present paragraphcorresponds to the type IIP target site referred in (ii)-a) or one of the two type IIP targets sites referred in (II)-b) of the method of the first aspect. In another particular embodiment, the two type IIP target sites comprised in the receiving vector defined by the Type IIP target sequences referred in the embodiments of the previous paragraph correspond to the type IIP target site referred in (ii)-b) of the method of the first aspect.

[0205] In another embodiment, the receiving backbone is obtained by digestion of a receiving vector with a Type IIP restriction enzyme and with a Type IIS restriction enzyme, particularly with the Type IIS enzyme of the first aspect, the receiving vector comprising a disposable sequence comprising two restriction enzyme recognition sequences V3 and V4, wherein:

[0206] a) V3 is at the 5' region of the disposable sequence and V4 is at the 3' region of the disposable sequence, b) V3 or V4 is a target sequence of the said Type IIP restriction enzyme, and:

[0207] - if V3 is a target sequence of said Type IIP restriction enzyme: (i) V3 is at the 5' end of the disposable sequence, (ii) V4 is a recognition sequence of the Type IIS restriction enzyme, and the orientation of V4 is such that a cleavage site K4 of the Type IIS enzyme recognizing V4 is downstream V4 and at the 3' end of the disposable sequence, and (iii) upon cleavage of V3 by the Type IIP restriction enzyme and of K4 by the Type IIS restriction enzyme, the overhangs in the receiving backbone defined in (i)-a) or (i)-b) of the first aspect are formed;

[0208] - if V4 is a target sequence of said specific Type 11 P restriction enzyme: (i) V4 is at the 3' end of the disposable sequence, (ii) V3 is a recognition sequence of the Type IIS restriction enzyme, and the orientation of V3 is such that a cleavage site K3 of the Type IIS enzyme recognizing V3 is upstream V3 and at the 5' end of the disposable sequence, and (iii) upon cleavage of K3 by the Type IIS restriction enzyme and of V4 by the Type IIP restriction enzyme, the overhangs in the receiving backbone defined in (i)-a) or (i)-b) of the first aspect are formed.

[0209] For simplicity, the receiving vector as defined just above is also referred to as the receiving vector comprising V3 and V4.

[0210] The term "disposable sequence”, "restriction enzyme recognition sequence”, "5' Region”, "3' region”, "5' end, "3' end”, "Type IIS enzyme”; "Type IIP enzyme” have been defined above. In a particular embodiment, the type IIP restriction enzyme is selected from any of those provided in the definition of the term "Type IIP enzyme” above.

[0211] For simplicity, the sequence immediately upstream V3 when V3 is the Type IIS recognition sequence, and comprising the cleavage site K3, is also referred herein as extension sequence E_V3. The sequence immediately downstream V4 when V4 is the Type IIS recognition sequence, and comprising the cleavage site K4, is also referred herein as extension sequence E_V4. In a particular embodiment, E_V3 or E_V4 comprise or consist of a type IIP restriction enzyme target sequence, similarly to E_V1 or E_V2, respectively. More particularly, the type IIP target sequences correspond to those forming the type IIP targetsite referred in (ii)-a) or one of the two type IIP targets sites referred in (ii)-b) of the method of the first aspect.

[0212] In a further particular embodiment, all the embodiments defining E_V1 are equally applicable to E_V3. In another particular embodiment, all the embodiments defining E_V2 are equally applicable to E_V4. Thus, in a particular embodiment, the length of E_V3 is as defined for E2, particularly is selected from any of the lengths provided above for E1. In another particular embodiment, the length of E_V4 is as defined for E1, particularly is selected from any of the lengths provided above for E1.

[0213] In a particular embodiment, the Type IIP target sequence V3 or V4, comprises or consists of one of the type IIP target sequences forming the Type IIP target site referred in (ii) -a) of the method of the first aspect, or one of the type IIP target sites referred in (ii)-b) of the method of the first aspect. In another particular embodiment, the Type IIP target sequence V3 or V4, comprises or consists of one of the type IIP target sequences forming the Type IIP target site referred in (II) -a) of the method of the first aspect. In another particular embodiment, the Type IIP target sequence V3 or V4, comprises or consists of one of the type IIP target sequences forming the Type IIP target sites referred in (ii)-b) of the method of the first aspect. In a further embodiment, E_V3 or E_V4 comprises or consists of one of the type IIP target sequence forming the Type IIP target site referred in (ii)-b) of the method of the first aspect, and the type IIP target sequence V4 or V3, respectively, is one of the Type IIP target sequences forming the other Type IIP target site referred in (ii)-b).

[0214] In another embodiment of the first aspect, the receiving backbone is obtained by digestion of a receiving vector with two different Type IIP restriction enzymes, the receiving vector comprising a disposable sequence comprising two target sequences V5 and V6 of said two different Type IIP restriction enzymes, wherein:

[0215] a) V5 is at the 5' end of the disposable sequence and V6 is at the 3' end of the disposable sequence, and b) upon cleavage of V5 and V6 with their corresponding Type IIP restriction enzyme, the overhangs in the receiving backbone defined in (i)-b) of claim 1 are formed.

[0216] For simplicity, the above defined receiving vector is also herein referred to as the receiving vector comprising V5 and V6.

[0217] The term” disposable sequence”, "restriction enzyme recognition sequence”, "5' end, "3' end”, and "Type IIP enzyme” have been defined above. In a particular embodiment, the type IIP restriction enzymes are selected from any of those provided in the definition of the term "Type IIP enzyme” above.

[0218] In a particular embodiment, at least one of Type IIP target sequences V5 or V6, comprises or consists of one of the sequences forming the type IIP target site referred in (II) -a) of the method of the first aspect, or one of the type IIP target sites referred in (ii)-b) of the method of the first aspect. In another particularembodiment, one of Type IIP target sequences V5 or V6, comprises or consists of one of the sequences forming the type IIP target site referred in (II) -a) of the method of the first aspect, or one of the type IIP target sites referred in (ii)-b) of the method of the first aspect. In another particular embodiment, the two Type IIP target sequences V5 and V6, each one comprises or consists of one of the sequences forming the two type IIP target sites referred in (ii)-b) of the method of the first aspect.

[0219] In a particular embodiment of the first aspect, the n inserts and the receiving backbone are obtained by providing to the reaction mixture: the one or more polynucleotides as defined in the first aspect; the receiving vector comprising V1 and V2, V3 and V4, or V5 and V6; and the Type IIS restriction enzyme(s), particularly the Type IIS restriction enzyme of the first aspect; and subjecting the reaction mixture to conditions appropriate for digestion of the one or more polynucleotides and optionally, when the receiving vector comprises V1 and V2 or V3 and V4, the receiving vector by said Type IIS restriction enzyme(s). In a particular embodiment of the first aspect, the n inserts and the receiving backbone are obtained by providing to the reaction mixture: the one or more polynucleotides as defined in the first aspect; the receiving vector comprising V1 and V2, or V3 and V4; the Type IIS restriction enzyme(s), particularly the Type IIS enzyme of the first aspect; and subjecting the reaction mixture to conditions appropriate for digestion of the one or more polynucleotides and the receiving vector by said Type IIS restriction enzyme(s). In another particular embodiment, the n inserts and the receiving backbone are obtained by providing to the reaction mixture: the one or more polynucleotides as defined in the first aspect; the receiving vector comprising V1 and V2; the Type IIS restriction enzyme(s), particularly the Type IIS of the first aspect; and subjecting the reaction mixture to conditions appropriate for digestion of the one or more polynucleotides and the receiving vector by said Type IIS restriction enzyme(s). In a particular embodiment of the first aspect, the n inserts and the receiving backbone are obtained by providing to the reaction mixture: the one or more polynucleotides as defined in the first aspect; the receiving vector V3 and V4; the Type IIS restriction enzyme(s), particularly the Type IIS of the first aspect; and subjecting the reaction mixture to conditions appropriate for digestion of the one or more polynucleotides and the receiving vector by said Type IIS restriction enzyme(s).

[0220] In another particular embodiment, the n inserts and the receiving backbone are obtained by providing to the reaction mixture: the one or more polynucleotides as defined in the first aspect; the hybridized oligos consisting of the inserts obtained upon hybridization of complementary oligos as defined in any of the embodiments above; the receiving vector comprising V1 and V2, V3 and V4, or V5 and V6; and the Type IIS restriction enzyme(s), particularly the Type IIS restriction enzyme of the first aspect; and subjecting the reaction mixture to conditions appropriate for digestion of the one or more polynucleotides and optionally, when the receiving vector comprises V1 and V2 or V3 and V4, the receiving vector by said Type IIS restriction enzyme(s). In a particular embodiment of the first aspect, the n inserts and the receiving backbone are obtained by providing to the reaction mixture: the one or more polynucleotides as defined in the first aspect; the hybridized oligos consisting of the inserts obtained upon hybridization of complementary oligos as defined in any of the embodiments above; the receiving vector comprising V1 and V2, or V3 and V4; theType IIS restriction enzyme(s), particularly the Type IIS enzyme of the first aspect; and subjecting the reaction mixture to conditions appropriate for digestion of the one or more polynucleotides and the receiving vector by said Type IIS restriction enzyme(s). In another particular embodiment, the n inserts and the receiving backbone are obtained by providing to the reaction mixture: the one or more polynucleotides as defined in the first aspect; the hybridized oligos consisting of the inserts obtained upon hybridization of complementary oligos as defined in any of the embodiments above; the receiving vector comprising V1 and V2; the Type IIS restriction enzyme(s), particularly the Type IIS of the first aspect; and subjecting the reaction mixture to conditions appropriate for digestion of the one or more polynucleotides and the receiving vector by said Type IIS restriction enzyme(s). In a particular embodiment of the first aspect, the n inserts and the receiving backbone are obtained by providing to the reaction mixture: the one or more polynucleotides as defined in the first aspect; the hybridized oligos consisting of the inserts obtained upon hybridization of complementary oligos as defined in any of the embodiments above; the receiving vector V3 and V4; the Type IIS restriction enzyme(s), particularly the Type IIS of the first aspect; and subjecting the reaction mixture to conditions appropriate for digestion of the one or more polynucleotides and the receiving vector by said Type IIS restriction enzyme(s).

[0221] In another particular embodiment, and as well understood by a skilled person, when the n inserts are hybridized oligos, or are obtained upon hybridization between two complementary oligos, the embodiments provided in the paragraph above apply by deleting reference to the one or more polynucleotides of the first aspect.

[0222] In another particular embodiment of the first aspect, the receiving backbone is obtained by providing to the reaction mixture receiving vector comprising V1 and V2, or V3 and V4; and the Type IIS restriction enzyme(s), particularly the Type IIS restriction enzyme of the first aspect; and subjecting the reaction mixture to conditions appropriate for digestion of the the receiving vector by said Type IIS restriction enzyme(s). In another particular embodiment of the first aspect, the receiving backbone is obtained by providing to the reaction mixture receiving vector comprising V1 and V2 and the Type IIS restriction enzyme(s), particularly the Type IIS restriction enzyme of the first aspect; and subjecting the reaction mixture to conditions appropriate for digestion of the the receiving vector by said Type IIS restriction enzyme(s). In another particular embodiment of the first aspect, the receiving backbone is obtained by providing to the reaction mixture receiving vector comprising V3 and V4 and the Type IIS restriction enzyme, particularly the Type IIS restriction enzyme of the first aspect; and subjecting the reaction mixture to conditions appropriate for digestion of the the receiving vector by said Type IIS restriction enzyme. More particularly, when at least one of the n inserts, more particularly the n inserts, are obtained upon hybridization of the two complementary oligos as indicated in any of the embodiments above, the receiving backbone is obtained as defined in the embodiments herein. Yet more particularly, when n=1 , the receiving backbone is obtained as defined in the embodiments herein.In a particular embodiment, and as well understood by a skilled person, those embodiments defining how the n inserts and / or the receiving backbone are obtained in the first aspect and comprising providing elements to the reaction mixture, can be re-worded to indicate that the reaction mixture of the first aspect comprises the further components provided to the reaction mixture corresponding to those indicated therein, instead than indicating that said components are provided to the reaction mixture.

[0223] The term "conditions appropriate for digestion”, or "conditions suitable for digestion”, as used herein, refers to conditions that fulfill the requirements for digestion of nucleic acid molecules of interest by the corresponding restriction enzyme, having a recognition site within said nucleic acid molecules of interest. As well known by an expert in the filed, said conditions comprise in general terms incubating the corresponding nucleic acid molecules to be digested with the corresponding restriction enzyme, in a specific buffer for a certain time and at a certain temperature. These conditions are well known by an expert in the field for each restriction enzyme and can be applied for instance by following the provider's instructions for the corresponding enzyme.

[0224] Thus, in a particularly preferred embodiment, the reaction mixture of the first aspect comprises Type IIS restriction enzyme(s), particularly, the type IIS restriction enzyme(s) referred in the first aspect, more particularly the Type IIS enzyme of the first aspect.

[0225] In a particular embodiment, the conditions appropriate for digestion comprise adding to the reaction mixture a buffer used for, or suitable for, digestion with a restriction enzyme, particularly the Type IIS enzyme(s) referred in the first aspect. As well understood by a skilled person, when several Type IIS enzymes are used, the same buffer is used for all of them.

[0226] In a particular embodiment, buffer used for, or suitable for, digestion with a restriction enzyme is a restriction enzyme digestion buffer, particularly specified in the provider's instructions for any of the corresponding enzyme(s), particularly for the Type IIS enzyme of the first aspect, which is a Type IIS digestion buffer. In a more particular embodiment, the restriction enzyme digestion buffer is a well-known digestion buffer by a skilled person, for digestion of nucleic acid molecules by several restriction enzymes at the same time, particularly by several Type IIS and Type IIP enzymes, more particularly by several Type IIS. In a particular embodiment, such buffer corresponds to the commonly known "universal” digestion buffer. In a particularly preferred embodiment, the Type IIS digestion buffer comprises: Potassium Acetate, particularly at around 50mM, Tris-acetate, particularly at around 20mM, Magnesium Acetate, particularly at around 10mM, optionally BSA, , particularly at around 100 pig / ml, wherein BSA is particularly recombinant BSA. Yet more particularly, the pH of the Type IIS enzyme buffer is of 7-9, 7.5-8.5, particularly of 7.6-8.2, more particularly of around 7.9. As well known by an expert in the field, several Type IIS enzymes (and the Type IIP enzymes such as EcoRI and Xhol) are active in a ligation buffer, particularly in the T4 DNA ligase buffer, and some DNA ligases (and the Type IIP enzymes such as EcoRI and Xhol) are also active in Type IIS digestionbuffers, especially in the Type IIS digestion buffer specified in the paragraph above. Indeed, as indicated in the definition of "conditions appropriate for ligation” the T4 DNA ligase is active in Type IIS enzyme digestion buffers, particularly wherein said buffer comprises or is supplemented with ATP, especially in the concentrations indicated therein, and also especially in the digestion buffer specified in the paragraph above.

[0227] Thus, in a particularly preferred embodiment, the buffer added to the reaction mixture for digestion, particularly by Type IIS enzyme(s) (i.e. added to the reaction mixture as part of conditions suitable for digestion), is selected from the buffers indicated above that are added to the reaction mixture for ligation by the DNA ligase (i.e. added to the reaction mixture as part of a condition suitable for digestion).

[0228] More particularly, the buffer added to the reaction mixture for digestion, particularly by Type IIS enzyme(s) (i.e. added to the reaction mixture as part of conditions suitable for digestion), consists of the buffer added to the reaction mixture for ligation by the DNA ligase (i.e. added to the reaction mixture as part of a condition suitable for digestion), particularly any of those indicated in "conditions appropriate for ligation”. Thus, in a particularly preferred embodiment, only one buffer is used to subject the reaction mixture to conditions appropriate for ligation and to conditions appropriate for digestion, particularly, said buffer is a buffer as defined in "conditions appropriate for ligation”. ,

[0229] Preferably, the buffer added to the reaction mixture for digestion and for ligation, is a ligase buffer for ligation by the DNA ligase of the first aspect, particularly specified in the DNA ligase provider's instructions. In a more particular embodiment, the buffer added to the reaction mixture for digestion and ligation is a T4 DNA ligase buffer, more particularly wherein the DNA ligase of the first aspect is the T4 DNA ligase.

[0230] In another particular embodiment, the reaction mixture comprises only one buffer selected from those indicated in the definition of "conditions appropriate for ligation”, particularly wherein the DNA ligase of the first aspect is a T4 DNA ligase, more particularly, whereinthe reaction mixture is subjected to conditions appropriate for ligation and to conditions appropriate for digestion, as defined herein. .

[0231] Therefore, in a particularly preferred embodiment, the reaction mixture of the first aspect comprises in addition to the receiving backbone, the n inserts and the DNA ligase, Type IIS restriction enzyme(s), particularly the Type IIS enzyme of the first aspect, a buffer, particularly wherein the buffer is selected from any of those provided herein for subjecting the reaction mixture to conditions appropriate for ligation, more particularly, a ligation buffer, yet more particularly a T4 DNA ligation buffer. Particularly, when the buffer is a ligation buffer, the DNA ligase is a T4 DNA ligase.

[0232] The volume of buffer added to the reaction mixture of the first aspect is that specified by the enzyme's supplier in a corresponding ligation reaction or restriction enzyme digestion reaction, particularly is 5%, 10%, 15%, 20%, 30%, 40%, 50%, 75%, 90%, 95% of the volume of the reaction mixture, more particularly is 10%of the volume of the reaction mixture.

[0233] In a further embodiment, conditions appropriate for digestion, particularly by a Type IIS enzyme, comprise subjecting the reaction mixture of the first aspect, particularly the reaction mixture of the previous paragraphs below the definition of "conditions appropriate for digestion”, at the temperature indicated by the restriction enzyme's provider. In a particular embodiment, said temperature is of about 30° C -45° C, 32° C -42° C, 34° C -40° C, 35° C-40° C, 35° C -39° C, 36° C-38° C, particularly 36° C-38° C. In a more particular embodiment, the temperature is of around 37° C.

[0234] In another embodiment, subjecting the reaction mixture to conditions appropriate for digestion of nucleic acid molecules by Type IIS enzyme(s), particularly by the type IIS enzyme of the first aspect, comprise incubating the reaction mixture of the first aspect, particularly to the conditions defined in any of the embodiments of the previous paragraph below the definition of "conditions appropriate for digestion”, for a certain amount of time, particularly for at least 1, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 45, at least 60 minutes, at least 70, at least 80, at least 90, at least 120 minutes, more particularly for at least 5 minutes. More particularly, for around 1, 3, 4, 5, 10, 15, 20, 25, 30, 45, 60, 70, 80, 90, 120 minutes, yet more particularly for around 5 minutes.

[0235] As well understood by a skilled person, the method of the first aspect of the invention, in a particularly preferred embodiment, comprises subjecting the reaction mixture to conditions appropriate for digestion (particularly by the type IIS enzyme(s), more particularly the type IIS enzyme of the first aspect) and to conditions appropriate for ligation, wherein the buffer suitable for digestion consists of the buffer suitable for ligation, more particularly wherein the n inserts are obtained upon digestion of the one or more polynucleotides of the first aspect by the Type IIS restriction enzyme(s), particularly by the Type IIS enzyme of the first aspect.

[0236] In a particular embodiment, the method of the first aspect of the invention comprises subjected the reaction mixture to at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 17, at least 20, at least 22, at least 25, at least 27, at least 30, at least 32, at least 35, restriction / ligation cycles, particularly to at least 30 restriction / ligation cycles. In another more particular embodiment, the method of the first aspect of the invention comprises subjected the reaction mixture to around 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 35, restriction / ligation cycles, particularly to around 30 restriction / ligation cycles.

[0237] As well known by an expert in the field, a restriction / ligation cycle comprises subjecting the reaction mixture to conditions appropriate for digestion by the Type IIS enzyme(s), such as those defined herein, and subsequently to conditions appropriate for ligation by the DNA ligase of the first aspect, such as those defined herein.In a particular embodiment, one restriction / ligation cycle comprises incubating the reaction mixture for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 30, at least 45, at least 60 min, particularly at least 5 min, under conditions suitable for digestion by the corresponding Type IIS enzyme(s), particularly the type IIS of the first aspect, as those defined herein, and subsequently, for the same amount of time, under conditions suitable for ligation by the DNA ligase of the first aspect, as those defined herein.

[0238] In a particular embodiment, one restriction / ligation cycle comprises incubating the reaction mixture for around 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 45, 60 min, particularly around 5 min, under conditions suitable for digestion by the corresponding Type IIS enzyme, as those defined herein, and subsequently, for the same amount of time, under conditions suitable for ligation by the DNA ligase of the first aspect, as those defined herein.

[0239] Thus, in a particular embodiment, one restriction / ligation cycle comprises incubating the reaction mixture for any of the amounts of time indicated above, under conditions suitable for digestion by the corresponding Type IIS enzyme, wherein the temperature is as indicated in any embodiment herein, particularly +- 2 ° C that indicated in the Type IIS enzyme's supplier instructions, and subsequently, for the same amount of time, under conditions suitable for ligation by the DNA ligase of the first aspect, wherein the temperature is as indicated in any embodiment herein, particularly, +-2 ° C that indicated in the DNA ligase enzyme's supplier instructions.

[0240] In another particular embodiment, one restriction / ligation cycle comprises incubating the reaction mixture for any of the amounts of time indicated above, under conditions suitable for digestion by the corresponding Type IIS enzyme, wherein the temperature is as indicated in any embodiment herein, particularly +- 1 ° C that indicated in the Type IIS enzyme's supplier instructions, and subsequently, for the same amount of time, under conditions suitable for ligation by the DNA ligase of the first aspect, wherein the temperature is as indicated in any embodiment herein, particularly, +- 1 ° C that indicated in the DNA ligase enzyme's supplier instructions.

[0241] In a particular embodiment, the receiving vector referred herein as comprising V3 and V4, is provided to the reaction mixture of the method of the first aspect, in a linearized form, after digestion with the Type IIP restriction enzyme (with target sequence V3 or V4) in another or different reaction mixture. Thus, in a particular embodiment, the receiving vector referred herein as comprising V3 and V4, is provided to the reaction mixture after digestion with the Type IIP restriction enzyme (with target sequence V3 or V4) in another or different reaction mixture, particularly also after isolation from said reaction mixture. In another particular embodiment, the receiving vector referred herein as comprising V5 and V6, is provided to the reaction mixture of the method of the first aspect, in a linearized form, after digestion with the Type IIPrestriction enzymes in another or different reaction mixture. Thus, in a particular embodiment, the receiving vector referred herein as comprising V5 and V6, is provided to the reaction mixture after digestion with the Type IIP restriction enzymes (with target sequences V5 and V6) in another or different reaction mixture, particularly also after isolation from said reaction mixture. Conditions appropriate for digestion of a vector by a Type IIP restriction enzyme are well-known by an expert in the field and include simply following the instructions from the Type IIP enzyme's provider. Methods for the isolation of a vector from a restriction enzyme reaction are also well-known by an expert in the field and are provided below in the examples section.

[0242] In another particular embodiment, the receiving vector referred herein as comprising V3 and V4, or the receiving vector comprising V5 and V6, is provided to the reaction mixture of the method of the first aspect in a circular form, i.e. previous to digestion by the corresponding Type IIP restriction enzyme(s). In this case, digestion with the type IIP restriction enzyme(s) (with target sequence V3 or V4; or V5 and V6) is performed in the reaction mixture of the first aspect, comprising the corresponding Type IIP restriction enzyme(s), and under the conditions appropriate for digestion by the Type IIS enzyme(s), particularly the type IIS of the first aspect. Indeed, as indicated above, some Type IIP restriction enzymes are active in Type IIS digestion buffers, particularly in that specified in the definition of "conditions appropriate for digestion”, as well as in DNA ligase buffers, particularly in T4 DNA ligase buffer. The rest of conditions appropriate for digestion by most Type IIS restriction enzymes are also appropriate for digestion of most Type IIP restriction enzymes.

[0243] Thus, in a particular embodiment, the receiving vector comprising V3 and V4, or the receiving vector comprising V5 and V6, is provided to the reaction mixture of the first aspect before digestion by the Type IIP restriction enzyme(s) having V3 or V4; or V5 and V6, as target sequences, together with the corresponding Type IIP restriction enzyme(s). The conditions appropriate for digestion and ligation are as indicated in any of the embodiments herein, particularly the method comprises subjecting the reaction mixture to a number of restriction / ligation cycles provided herein, particularly to one cycle of restriction / ligation. More particularly, in such a case, the incubation under condition appropriate for digestion in the cycle is for at least 20 minutes, more particularly for at least 45 min, yet more particularly for at least 60 minutes. Yet more particularly, subsequently, the reaction mixture is incubated for the same amount of time, under conditions suitable for ligation by the DNA ligase of the first aspect, as those defined herein.

[0244] In a particular embodiment of the first aspect of the invention, the receiving backbone comprised in the reaction mixture is not dephosphorylated. The term "dephosphoryl ated” in the context of nucleic acid molecules used in a cloning method, refers to the fact that a phosphate group is comprised at the 5' end of the corresponding nucleic acid molecule and has not been removed. Indeed, in standard cloning protocols, a five-prime phosphate is required to serve as the donor in the ligation reaction. At a minimum, either the fragment ends or vector ends must be phosphorylated. Thus, to reduce possible relegation of digested vectors, in some protocols, the digested vector is dephosphorylated, and the inserts phosphorylated, so toavoid relegation of the digested vector and promote ligation of the inserts with the digested vector.

[0245] In a particularly preferred embodiment, each one of the sequences of interest “S” comprised in the n inserts consists of a peptide encoding sequence, and the peptide encoding sequences within the n inserts are in frame within the destination vector. In a yet more particular embodiment of the first aspect, each one of the sequences of interest “S” comprised in the n inserts consists of a peptide encoding sequence, and the peptide encoding sequences within the n inserts are in frame within the destination vector, wherein: each one of the extension sequences E1 and E2 (within each region F) in each of the one or more polynucleotides consists of p codons, being “p” an integer of at least 1, particularly, equal to 2, particularly wherein: (I) each codon encodes a neutral amino acid and / or (ii) the extension sequence further comprises a translation start codon in frame with the corresponding peptide encoding sequence S (i.e. sequence S comprised in the same region F as that comprising the start codon) and / or with the rest of peptide encoding sequences S within the destination vector, wherein the translation start codon is retained within the corresponding insert upon cleavage of the corresponding polynucleotide by the Type IIS restriction enzyme.

[0246] In another particular embodiment, upon binding between complementary overhangs of the n inserts, the sequences linking sequences S to and within the destination vector (i.e. the linking sequences between two sequences S comprised in the destination vector), each one consists of p codons, being “p” an integer of at least 1, particularly, equal to 2, wherein: (I) each codon encodes a neutral amino acid and / or (ii) the linking sequence further comprises a translation start codon in frame with the corresponding peptide encoding sequence S. Thus, as well understood by a skilled person, the overhangs of the inserts are such that upon binding between complementary overhangs of the n inserts, the sequences linking sequences S to and within the destination vector are as just defined. In a particularly preferred embodiment, the definitions and embodiments addressed to the extension sequences E1 and E2 are equally applicable to the linking sequences between two sequences S in the destination vector of the first aspect. The embodiments of the paragraph herein particularly apply to inserts obtained upon hybridization between two complementary oligos as defined herein.

[0247] The term "Protein”, as used herein, refers to the term well-known by an expert in the field. In particular, the term refers to large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence encoding them, and which usually results in protein folding into a specific 3D structure that determines its activity. The individual amino acid residues are bonded together by peptide bonds and adjacent amino acid residues. The sequence of amino acid residues in a protein is defined by the sequence of a gene, which is encoded in the genetic code. In general, the genetic code specifies 20 standard amino acids; although in certain organisms the genetic code can include selenocysteine and— in certain archaea— pyrrolysine. Shortly after or even during synthesis, the residues in a protein are often chemically modified by post-translational modification, which alters the physical and chemical properties,folding, stability, activity, and ultimately, the function of the proteins. Proteins can work together to achieve a particular function, and they often associate to form stable protein complexes.

[0248] A linear chain of amino acid residues is called a polypeptide. A protein contains at least one long polypeptide. Short polypeptides, are commonly called peptides. In a particular embodiment, a peptide is considered to comprise less than 100, 80, 70, 60, 50, 40, 35, 30, 25, 20, 15, 12, or 10 amino acids, particularly is of less than 30 amino acids.

[0249] In a particular embodiment, the number of codons p comprised in each of extension sequence E1 and E2 as defined herein, is of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7, more particularly is of at least 1. In another particular embodiment, the number of codons p comprised in each of extension sequence E1 and E2, is of at least 2. In another particular embodiment, the number of codons p comprised in each of extension sequence E1 and E2, is of 1, 2, 3, 4, 5, 6, or 7, more particularly is of 2.

[0250] In a particular embodiment, at least 1, at least 2, at least 3, at least 4, or all of regions F comprising the n inserts within the one or more polynucleotides of the first aspect, particularly one region F, comprises an extension sequence E1 or E2, particularly E1, as defined in (II) above (see the paragraph appearing five paragraphs above the present paragraph), particularly wherein the rest of extension sequences, i.e. the other extension sequence from said region F, and the rest of E1 and E2 (within the one or more polynucleotides of the first aspect) are as defined in (I) (see the paragraph appearing five paragraphs above the present paragraph). In another particular embodiment, 1, 2, 3, 4, or all of regions F comprising the n inserts within the one or more polynucleotides of the first aspect, particularly one region F, comprises an extension sequence E1 or E2, particularly E1, as defined in (II) above (see the paragraph appearing five paragraphs above the present paragraph), particularly wherein the rest of extension sequences, i.e. the other extension sequence from said region F, and the rest of E1 and E2 (within the one or more polynucleotides of the first aspect) are as defined in (I) (see the paragraph appearing five paragraphs above the present paragraph). In a particular embodiment, only E1 or E2, particularly E1, within one region F, is as defined in (II) above (see the paragraph appearing five paragraphs above the present paragraph), particularly wherein the rest of extension sequences i.e. the other extension sequence from said region F, and the rest of E1 and E2 (within the one or more polynucleotides of the first aspect) are as defined in (I) (see the paragraph appearing five paragraphs above the present paragraph).

[0251] In a particular embodiment, when the extension sequence is as defined in (II) above (see the paragraph appearing six paragraphs above the present paragraph), the start codon is in frame +1, +2 or +3 of the extension sequence. As well understood by a skilled person, a start codon in frame +1 of the extension sequence, is comprised in the frame (or sequence of consecutive codons), starting at nucleotide at position 1 of the extension sequence, and the start codon in frame +2 of the extension sequence, is comprised in the frame starting at nucleotide at position 2 of the extension sequence and so on. In a particularembodiment, the start codon is at position 1, or at position 2, particularly at position 2, i.e. the first nucleotide of the start codon corresponds to the nucleotide at position 1 or at position 2, of the extension sequences as defined in (ii) above (see the paragraph appearing six paragraphs above the present paragraph). As well understood by a skilled person, the peptide encoding sequences S downstream said translation start codon are in the same frame as said start codon. In another particular embodiment, between the start codon and the downstream peptide encoding sequence S, the extension sequence comprises one or more neutral codons, i.e. codons encoding neutral amino acids, that are also in frame with the start codon. In a particular embodiment, the peptide encoding sequences comprised or consisting of inserts n, downstream said translation start codon are in the same frame as said start codon.

[0252] The term "codon” as well understood by a skilled person, refers to a triplet of nucleotides encoding an amino acid, or the start or end of translation. A start codon determines the start of translation and can be selected from the list consisting of ATG, GTG, and TTG. In a particular embodiment the start codon of the first aspect of the invention is ATG.

[0253] The term "neutral amino acid”, as used herein, refers to an amino acid with neutral physicochemical properties, i.e. with minimal impact on a protein's structure and activity. Examples of neutral amino acids include glycine, leucine, serine, alanine, valine and isoleucine. In a particular embodiment, the neutral amino acids referred herein are selected from the list consisting of glycine, leucine, serine, alanine, serine, and combinations thereof. In a particular embodiment, the neutral amino acids referred herein are selected from the list consisting of glycine, leucine, serine, alanine and combination thereof. In another particular embodiment, the neutral amino acids referred herein are selected from the list consisting of glycine, leucine, serine and combination thereof. In another particular embodiment, the neutral amino acids referred herein are selected from glycine, leucine, and the combination thereof. In another particular embodiment, the neutral amino acids referred herein are selected from glycine, serine and the combination thereof. In another particular embodiment, the neutral amino acids referred herein are selected from serine, leucine and the combination thereof.

[0254] As well understood by a skilled person, codons encoding a neutral amino acid are codons encoding the neutral amino acids as defined above. The codons that encode an amino acid are well known by an expert in the feild, and can be found in any well known molecular biology book. Thus, as well understood by a skilled person, once the neutral amino acids used in the method of the first aspect are selected from those provided in the definition of neutral amino acid above, the codons encoding them is clearly known by an expert in the field.

[0255] In a particular embodiment, the codons encoding a neutral amino acid are selected from those provided below in table 4 of the examples, wherein the nucleotides encoding the amino acids in the column"aminoacid” are provided in the column "nucleotide” (in a 5' to 3' end direction for the nucleotides, and from the N- to the C- terminus for the case where 2 amino acids are disclosed). In another particular embodiment, extension sequences E1 and E2 as defined herein, are selected from the sequences disclosed in table 4 in the column "nucleotide”.

[0256] In a more particular embodiment, at least one the n sequences S comprises or consists of a peptide encoding sequence, wherein the peptide has antibacterial activity. The term "antibacterial activity”, as used herein, refers to the term well-known by an expert in the field, and more particularly refers to the fact that the peptide inhibits growth of at least one bacterial species, or kills at least one bacterial species. In another particular embodiment, n-1 of the S sequences comprise or consist of peptide encoding sequence wherein the peptides have antibacterial activity. In another particular embodiment, the n sequences S comprise or consist of a peptide encoding sequence wherein the peptides have antibacterial activity. In a more particular embodiment, at least one of the n sequences S, particularly one of the n sequences S, comprises or consists of a signal peptide encoding sequence as defined below. Particularly, the signal peptide is selected from any of those provided in the definition and embodiments defining the signal peptide below. In another particular embodiment, at least one of the n sequences S, particularly one of the n sequences S, comprises or consists of a tag peptide encoding sequence as defined below. Particularly, the tag peptide is selected from any of those provided in the definition and embodiments defining the tag peptide below. In another particular embodiment, one of the n sequences S encodes a tag peptide, and the rest of n sequences (n-1) encode a peptide with antibacterial activity. In another particular embodiment, one of the n sequences S encodes a signal peptide, and the rest of n sequences (n-1) encode a peptide with antibacterial activity. In another particular embodiment, one of the n sequences S encodes a tag peptide, another one of the n sequences S encodes a signal peptide, and the rest of n sequences (n-2) encode a peptide with antibacterial activity.

[0257] In a particular embodiment, when a sequence S comprises or consists of a signal peptide encoding sequence, the insert comprising it is at the 5' end of the sequence of n inserts within the destination vector. In another particular embodiment, when a sequence S comprises or consists of a signal peptide, the insert comprising it is at the 3' end of the sequence of n inserts within the destination vector.

[0258] In another particular embodiment, when a sequence S comprises or consists of a tag peptide, the insert comprising it is at the 3' end of the sequence of n inserts within the destination vector. In another particular embodiment, when a sequence S comprises or consists of a tag peptide, the insert comprising it is at the 5' end of the sequence of n inserts within the destination vector. In another particular embodiment, when a sequence S comprises or consists of a tag peptide, the insert comprising it is comprised between the 5' and the 3' end positions, within the sequence of n inserts in the destination vector.

[0259] In a particular embodiment, each of the n inserts consists of a peptide encoding sequence wherein E1 andE2 are as defined above.

[0260] In another particular embodiment, each one of the n inserts consists of a peptide encoding sequence, and the peptide encoding sequences within the n inserts are in frame within the destination vector, wherein: each one of the extension sequences E1 and E2 (within each region F) in each of the one or more polynucleotides consists of p codons, being “p” an integer of at least 1, particularly, equal to 2, particularly wherein: (I) each codon encodes an amino acid part of the peptide encoded by insert n and / or (ii) the extension sequence further comprises a translation start codon in frame with the corresponding peptide encoding sequence S (i.e. sequence S comprised in the same region F as that comprising the start codon) and / or with the rest of peptide encoding sequences S, wherein the translation start codon is retained within the corresponding insert upon cleavage of the corresponding polynucleotide by the Type IIS restriction enzyme.

[0261] In a particular embodiment, the extension sequence defined in (ii) in the paragraph above, is as the extension sequence comprising a start codon defined in the previous embodiments.

[0262] In another particular embodiment, when n consists of the peptide encoding sequence, any of the embodiments above addressed to sequence S comprising or consisting of a specific peptide encoding sequence applies, wherein the term "sequence S” is substituted by "insert”. Particularly, in the context of the location of the insert comprising the corresponding peptide encoding sequence S, the "insert comprising it” is substituted by the "insert”.

[0263] In an embodiment, the receiving vector as defined above (i.e. comprising V1 and V2, V3 and V4 or V5 and V6) comprises a protein tag encoding sequence upstream or downstream, particularly downstream, the disposable sequence and under the control of a same expression promoter as the disposable sequence. Particularly, the protein tag encoding sequence is in frame with the peptide encoding sequences consisting of sequences S, or, comprised in or consisting of inserts n, within the destination vector.

[0264] The term "protein tag”, or "peptide tag”, as used herein, refers to the term well-known by an expert in the field. Particularly the term refers to any protein tag well-known by an expert in the field, such as, but not limited to, histidine or streptavidine tags. They can be added to either end of the target protein (N- or C-terminus). Some tags are also inserted at sites within the protein of interest (internal tags).

[0265] In an embodiment, the protein tag encoding sequence is flanked by two Type IIP restriction enzyme target sequences within the receiving vector as defined above. The term "flanked by”, as used herein, in the context of a nucleotide sequence of interest flanked by two additional nucleotide sequences, refers to the fact that the nucleotide sequence of interest is directly attached at its 5' end to one of said additional nucleotide sequences, and directly attached to its 3' end to the other additional nucleotide sequence. As wellunderstood by a skilled person, the presence of two Type IIP target sequences flanking the tag encoding sequences allow easy tag exchange by traditional cloning techniques (i.e. well-known techniques by an expert in the based on digestion by the two Type IIP enzymes). In a particular embodiment, the Type IIP restriction enzymes referred herein are selected from the list consisting of Agel, BamHI, EcoRI, Hindlll, Ncol, Notl, Sall, Spel, Xbal, Xhol, Xmal, Kpnl, Ndel and Smal. In a more particular embodiment, the type IIP restriction enzyme are selected form BamHI, EcoRI, and Xhol, particularly are BamHI and Xhol, more particularly wherein BamHI is at the 5' end of the protein tag encoding sequence and Xhol is at the 3' end of the peptide encoding sequence.

[0266] In a particular embodiment, upon cleavage one of the two Type IIP restriction enzyme target sequences flanking the protein tag encoding sequence forms the palindromic overhang of the receiving backbone as defined in (i)-a or (i)-b of the method of the first aspect, particularly wherein said Type IIP target sequence is upstream the protein tag encoding sequence, and the protein tag encoding sequence is downstream the disposable sequence in the receiving vector. Thus, in a further particular embodiment, the protein tag encoding sequence is downstream the disposable sequence within the receiving vector, and upon cleavage, one of the two Type IIP restriction enzyme target sequences flanking the protein tag encoding sequence forms the palindromic overhang of the receiving backbone as defined in (i)-a or (i)-b of the method of the first aspect, particularly the palindromic overhang that is at the 5' end of the forward strand, or 3' end of the reverse strand, of the receiving backbone. In a more particular embodiment, said Type IIP target sequence is a BamHI target sequence.

[0267] In another embodiment, the receiving vector as defined herein comprises a signal peptide upstream or downstream, particularly upstream, the disposable sequence and under the control of a same expression promoter as the disposable sequence. Particularly, the signal peptide encoding sequence is in frame with the peptide encoding sequences consisting of sequences S, or, comprised in or consisting of inserts n, within the destination vector. Thus, in a particular embodiment, the signal peptide is upstream or downstream the sequence of n inserts in the destination vector, particularly upstream.

[0268] In a particular embodiment, the receiving vector comprises a Ribosome Entry Site (RBS) as defined above in the definition of a vector. Particularly, the RBS is upstream the disposable sequence in the receiving vector, more particularly, upstream the signal peptide comprised in the receiving vector that is itself upstream the disposable sequence.

[0269] The term "signal peptide”, " signal sequence”, "targeting signal”, "localization signal”, "localization sequence”, "transit peptide”, "leader sequence” or "leader peptide”, as used herein, refers to a short peptide (usually 16-30 amino acids long) present at the N-terminus (or occasionally nonclassical at the C-terminus or internally) of most newly synthesized proteins that are destined toward the secretory pathway. Signal peptides function to prompt a cell to translocate the protein, usually to the cellular membrane. As indictedabove, signal peptides are usually located at the N-terminus of proteins, but some have C-terminal or internal signal peptides (examples: peroxisomal targeting signal and nuclear localization signal), which are considered as non-classical peptides, and significantly differ in structure from the N- terminal signal peptides. Non-limiting examples of signal peptide include PelB, or OmpA.

[0270] The term "PelB”, as used herein, refers to pectate lyase B of Erwinia carotovora CE. It corresponds to a sequence of amino acids which, when attached to a protein, directs the protein to the bacterial periplasm, where the sequence is removed by a signal peptidase. The leader sequence consists of the 22 N-terminal amino acid residues. This leader sequence can be attached to any other protein (on the DNA level) resulting in a transfer of such a fused protein to the periplasmic space of Gram-negative bacteria, such as Escherichia coli, often used in genetic engineering. Protein secretion can increase the stability of cloned gene products. The pelB leader sequence as used herein refers to the peptide consisting of SEQ ID NO. 1:

[0271] MKYLLPTAAAGLLLLAAQPAMA

[0272] The term "OmpA” as used herein, refers to the "Outer membrane protein A” protein in E. coli. Said protein is a signal peptide formed by a 21 -amino acid sequence which targets fused proteins to the Sec secretion pathway in E. coli. The fused protein is not folded until it is secreted to the periplasm. The signal peptide is cleaved after residue 21 by signal peptidase after secretion (recognition sequence: Ala-X-Ala). The OmpA leader sequence as used herein refers to the peptide consisting of SEQ ID NO: 2: MAKKTAIAIAVALAGFATVAQA

[0273] In a particular embodiment, the signal peptide referred herein is selected from a PelB signal peptide or an OmpA signal peptide. In a particular embodiment, when the signal peptide is PelB, it is comprised or consists of insert at the 5' end of the sequence of n inserts in the destination vector. In another particular embodiment, when the signal peptide is OmpA, it is comprised or consists of insert at the 5' end of the sequence of n inserts in the destination vector.

[0274] In a particular embodiment, the insert at the 5' end of the sequence of n inserts in the destination vector comprises a translation start codon and the extension sequence E1 of the F region comprising said insert is as the extension sequences comprising a start codon defined in any of the embodiments above, particularly wherein the signal peptide encoding sequence and / or the protein tag encoding sequence within the destination vector, is downstream said translation start codon. Particularly, the signal peptide encoding sequence is immediately downstream the start codon, and the protein tag encoding sequence is downstream the sequence of n inserts within the destination vector, more particularly, wherein the protein tag encoding sequence is flanked by two type IIP target sequences as indicated in any of the embodiments above. More particularly, the Type IIP target sequence at the 5' end of the protein tag encoding sequence is a BamHI target site.As well understood by a skilled person, recognition sites or sequences for the Type IIS enzyme(s) and / or Type IIP enzyme(s) defined in the first aspect of the invention as being comprised in the n inserts, the receiving backbone, the one or more polynucleotides, and / or the receiving vector of the first aspect, are unique in the corresponding insert, receiving backbone, polynucleotide and / or receiving vector of the first aspect. In other words, the n inserts, the receiving backbone, the one or more polynucleotides and / or the receiving vector of the first aspect, do not comprise do not comprise recognition sites for the Type IIS and / or Type IIP restriction enzymes defined in any one of the embodiments of the first aspect, in addition to the recognition sites for said enzymes indicated in any of the embodiments of the first aspect (defined by the recognition sequences defined in any one of the embodiments of the first aspect). Particularly, recognition sites specific for the Type IIS and / or Type IIP enzyme(s) referred in any one of the embodiments of the first aspect, in addition to the recognition sites for said enzyme(s) indicated in any of the embodiments of the first aspect (defined by the recognition sequences defined in any one of said embodiments), have been deleted in the corresponding inserts, receiving backbone, polynucleotides and / or receiving vector. As well understood by a skilled person, when a restriction enzyme recognition sequence is herein referred to be comprised in a nucleic acid molecule, it is also understood that a corresponding restriction enzyme recognition site, comprising said recognition sequence, is therein comprised in the nucleic acid molecule.

[0275] In another particular embodiment, the receiving vector comprises negative selection restriction enzyme recognition sites, particularly comprised in the disposable sequence, for negative selection of the final product. Commonly, negative selection restriction enzyme recognition sites are Type IIP target sites present in the nucleic acid molecules used in a cloning method, but absent in the final product if the cloning method has been succesfull. In a particular embodiment, the negative selection enzyme recognition sites are Type IIP target sites, particularly any Type IIP target site, provided they are not used in the method of the first aspect to obtain the receiving backbone, the inserts, particularly, in any step before ligation of the nucleic acid products, particularly, in any step previous to the obtention of the destination vector.

[0276] In another particular embodiment, the cloning method of the first aspect comprises a further step of substituting the protein tag encoding sequence referred in any of the embodiment above, by a different protein tag encoding sequence of interest. In a particular embodiment, substitution of the protein tag encoding sequence comprises digestion of the destination vector of the first aspect comprising a protein tag encoding sequence flanked by two type IIP target sites, at said two Type IIP target sites, by the corresponding Type IIP enzymes. More particularly, said substitution further comprises inserting at or between said sites of the destination vector (resulting from digestion by the corresponding Type IIP restriction enzymes), the protein tag encoding sequence of interest.

[0277] In an embodiment, the method comprising substitution of the tag peptide encoding sequence of the destination vectors as defined in the the first aspect, is performed for the synthesis of a library of destination vectors, wherein vectors that differ in the combination and / or orientation of the n inserts, differ in the proteintag encoding sequence of interest, and those vectors having the same combination and / or orientation of n inserts, have the same protein tag encoding sequence of interest.

[0278] II- Nucleic acids and methods of the second, third and fourth aspects of the invention

[0279] In a second aspect, the invention is addressed to a nucleic acid vector obtained by the cloning method according to the first aspect.

[0280] The term "nucleic acid vector”, has been defined in the first aspect and equally applies to the second aspect. As well understood by a skilled person, the nucleic acid vector of the second aspect is as the destination vector of the first aspect. Thus, all the definition and embodiments addressed to the destination vector of the first aspect are equally applicable to the nucleic acid vector of the second aspect.

[0281] In a particular embodiment, the definitions and embodiments of the first aspect are equally applicable to the second aspect.

[0282] As indicated above, in a particular embodiment the method of the first aspect is performed for the synthesis of a library of destination vectors. Thus, in a third aspect, the invention is addressed to a method for the synthesis of a library of different nucleic acid vectors, wherein: each vector from the library is as a destination vector obtained by the first aspect of the invention wherein the n inserts comprise or consist of peptide encoding sequence; each vector from the library comprises a protein tag encoding sequence; and those vectors from the library that differ in the combination and / or orientation of the n inserts, differ in the protein tag encoding sequence of interest, and those vectors from the library having the same combination and / or orientation of n inserts, have the same protein tag encoding sequence of interest; the method comprising: (I) performing the method of the first aspect of the invention for obtaining different destination vectors that differ in the combination and / or orientation of the n inserts, each one comprising a protein tag encoding sequence flanked by two different type IIP target sites, and

[0283] (ii) substituting the protein tag from the destination vectors obtained from step (I) by another protein tag of interest that is the same for all destination vectors having the same combination and / or orientation of n inserts, and different between destination vectors having a different combination and / or orientation of n inserts.

[0284] In a particular embodiment of the method of the third aspect, step (ii) comprises digestion of the destination vectors obtained from step (I) by the corresponding two type IIP restriction enzymes and inserting the corresponding protein tag of interest instead, at said digested sites in the corresponding destination vectors. In a particular embodiment, insertion of the protein tag encoding sequence of interest is performed by ligation of an insert comprising said protein tag encoding sequence of interest.In another particular embodiment, the method of the third aspect comprises an intermediate step (i') between step (i) and (ii) of isolating the destination vectors obtained from step (i).

[0285] Methods for performing digestion of a vector by two Type IIP restriction enzymes, as well as for ligation of an insert in a digested vector and for isolation of a digested product are well known by an expert in the field, and include those disclosed above and in the examples below.

[0286] The term "library of vectors”, "library of nucleic acid vectors”, or "library of molecular vectors”, as used herein refers to a pull of nucleic acid vectors of interest, wherein at least 2 different groups of vectors can be found. All the vectors within one group have the same nucleic acid sequence, and the vectors between groups differ in the sequence or position of at least one nucleic acid sequence or region.

[0287] In a particular embodiment, step (i) of the method of the second aspect can also be defined as comprising obtaining a library of destination vectors, wherein those vectors that are different between each other, differ in the combination and / or orientation of the n inserts, each one comprising a protein tag encoding sequence flanked by two different type IIP target sites.

[0288] In a particular embodiment, all the definitions and embodiments addressed to the vectors of the invention are equally applicable to the vectors of the library of vectors of the third aspect. In another embodiment, all the definitions and embodiments of the first and second aspects, are equally applicable to the third aspect.

[0289] In a fourth aspect, the invention is addressed to the library of vectors obtained by the method of the third aspect. Thus, all the definitions and embodiments of the first, second and third aspect are equally applicable to the fourth aspect.

[0290] Ill- Additional aspects of the invention

[0291] In a fifth aspect, the invention is addressed to a protein encoded by the nucleic acid vector according to the second aspect, wherein the nucleic acid sequence of the vector encoding the protein comprises the n inserts.

[0292] The term protein has been defined in the first aspect of the invention and applies to the fifth aspect of the invention. In a particular embodiment, the definitions and embodiments of the first and second aspects of the invention are equally applicable to the fifth aspect.

[0293] In a particular embodiment, the protein of the fifth aspect comprises the peptide encoded by the sequences comprised in or consisting of the n inserts of the first aspect and comprised in the destination vector of the first aspect and also comprises the signal peptide and the tag encoding peptide as defined in the first aspect.More particularly, the protein of the fifth aspect comprises in an N- to C-terminal direction, the signal peptide, the n peptides encoded by the sequence comprised in or consisting of the n inserts, and the tag protein.

[0294] In a sixth aspect, the invention is addressed to a method for the synthesis of a protein, comprising (i) providing to a protein expression system the nucleic acid vector obtained by the cloning method according to the first aspect, wherein the n inserts comprise or consist of peptide encoding sequences, particularly wherein the sequences of interest S comprise or consist of peptide encoding sequences, and (ii) subjecting the protein expression system to conditions appropriate for the expression of the protein.

[0295] The term "protein-expression system”, as used herein, refers to means of producing or synthesizing functional proteins of interest. Given the size and complexity of proteins, chemical synthesis is generally not performed, but living cells and their cellular machinery are usually harnessed as factories to build and construct proteins based on supplied genetic templates. Traditional strategies for recombinant protein expression involve transfecting cells with a DNA vector that contains the template and then culturing the cells so that they transcribe and translate the desired protein. Typically, the cells are then lysed to extract the expressed protein for subsequent purification. Both prokaryotic and eukaryotic in vivo protein expression systems are widely used. Living cells suitable for protein expression are well-known by an expert in the filed an include mammalian, insect, yeast, bacterial, algal and cell-free systems, generally comprising cell extracts obtained said cells. In a particular embodiment, the protein expression system as used herein, refers to the host cells in which the DNA encoding the protein of interest is transfected, which is then subjected to cell culture conditions promoting the growing of the cells which results in the expression of the protein of interest. In a particular embodiment the protein expression system is a bacterial cell, particularly an E. coli cell, more particularly especially suitable for protein expression such as E. coli DH5o competent cells.

[0296] The term "conditions appropriate for the expression of the protein”; as used herein in the context of the protein expression system, refers to the condition promoting the growth of the cells, or the expression of the protein in the corresponding cells extract. Said conditions are well known by an expert in the feild and include those specified in the cell or cell extract provider's instructions.

[0297] In a particular embodiment, the method of the sixth aspect comprises a step i-a) previous to step (i) as defined above, of performing the cloning method of the first aspect, so that the nucleic acid vector provided to the protein expression system in step (i) is the vector obtained from step i.a).

[0298] Thus, in a particular embodiment, the method of the sixth aspect comprises: i-a) performing the cloning method as defined in the first aspect of the invention, wherein the n inserts comprise or consist of peptide encoding sequences, particularly wherein sequences of interest S comprise of consist of peptide encoding sequences, i-b) providing to a protein expression system the nucleic acid vector obtained from step i-a) and(ii) subjecting the protein expression system to conditions appropriate for the expression of the protein.

[0299] As well understood by a skilled person, all the embodiments and definitions of the first aspect defining the method wherein the n inserts comprise or consist of peptide encoding sequences, particularly including those wherein sequences S are peptide encoding sequences, apply to the method of the sixth aspect, particularly to that defined in the paragraph above.

[0300] In a particular embodiment, the protein obtained by the method of the sixth aspect is as the protein defined in the fifth aspect of the invention. In a particular embodiment, the invention is addressed to a protein obtained by the method of the sixth aspect. All the embodiments and definitions provided for the protein of fifth aspect apply to the protein obtained by the method of the sixth aspect.

[0301] In a particular embodiment, the definitions and embodiments of the first, second, third, fourth and fifth aspects of the invention are equally applicable to the sixth aspect.

[0302] In a seventh aspect, the invention is addressed to a method for the synthesis of a library of proteins, wherein the library of proteins comprises several proteins according to the fifth aspect, particularly of proteins obtained by the method as defined by the sixth aspect as defined above, wherein those proteins from the library that differ in the sequence, number and / or position of the protein sequences encoded by the n inserts, also differ in a protein tag sequence, and those proteins from the library with the same sequence, number and / or position of the protein sequences encoded by the n inserts, also comprise the same protein tag sequence, the method comprising:

[0303] (i) performing the method for the synthesis of a library of different nucleic acid vectors of the third aspect,

[0304] (ii) providing to a protein expression system the nucleic acid vectors of the library of vectors obtained from step (i), and

[0305] (iii) subjecting the protein expression system to conditions appropriate for the expression of the proteins.

[0306] The term "library of protein”, as used herein refers to a pull of proteins of interest, wherein at least 2 different groups of proteins can be found. All the proteins within one group have the same amino acid sequence, or are encoded by the same DNA sequence, and the proteins between groups differ in the sequence or position of at least one amino acid sequence or region.

[0307] The terms "protein expression system”, and "subjecting the protein expression system to conditions appropriate for the expression of the proteins” have been defined above and apply herein.

[0308] In a particular embodiment, the method of the seventh aspect comprises isolating or purifying the proteinsfrom the protein expression system, particularly, using the protein tags referred in the seventh aspect. Methods for the isolation / purification of protein using protein tags are well-known by an expert in the filed and include, for instance, the use of magnetic beads or nanomagnetic resins comprising an antibody specific for the protein tag of interest.

[0309] In an eighth aspect, the invention is addressed to the protein library obtained by the method of the seventh aspect.

[0310] In a particular embodiment, the definition and embodiments of the first, second, third, fourth, fifth, sixth and aspects, also apply to the seventh and eighth aspect of the invention.

[0311] In ninth aspect, the invention is addressed to a kit comprising means for performing the method as defined by the first aspect, the third aspect, the sixth aspect, or the seventh aspect of the invention, particularly the method as defined by the first aspect.

[0312] In a particular embodiment, the kit of the ninth aspect further comprises a solid support. In the present invention, the term "solid support” includes a nitrocellulose membrane, glass or a polymer. The most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of strips, tubes, beads, discs or microplates, or any other surface suitable for conducting the cloning method.

[0313] In another embodiment of the ninth aspect, the kit comprises instructions on how to use it. In another particular embodiment, the kit comprises at least one element, particularly all elements, selected from the list consisting of vectors, inserts, DNA ligase, Restriction enzymes, particularly a Type IIS restriction enzyme, and buffer solution(s). In an embodiment, the kit comprises the receiving vectors and inserts of the first aspect of the invention, more particularly, further comprises the Type IIS restriction enzyme(s) of the first aspect.

[0314] In a particular embodiment, the definition, and embodiments of the first, second, third, fourth, fifth, sixth, seventh, and eighth aspects, are equally applicable to the eighth aspect of the invention.

[0315] In a tenth aspect, the invention is addressed to a reaction mixture comprising the receiving backbone, the n inserts, and the DNA ligase as defined in the first aspect.

[0316] In a particular embodiment, the reaction mixture of the tenth aspect is as the reaction mixture defined in the first aspect of the invention. In a particular embodiment, the definitions and embodiments of the first aspect are equally applicable to the reaction mixture of the tenth aspect.

[0317] For completeness, the present description is also disclosed in the following clauses:Clause 1- A cloning method for obtaining a destination nucleic acid vector comprising “n” inserts, wherein “n” is an integer of at least 1, and each insert comprises a sequence of interest “S” and, wherein the method comprises contacting in a reaction mixture:

[0318] (i) a receiving backbone, wherein:

[0319] (a) one end of the receiving backbone comprises a palindromic overhang and the other end comprises a non-palindromic overhang, or

[0320] (b) each end of the receiving backbone comprises a different palindromic overhang;

[0321] (ii) the n inserts, wherein each insert comprises two different overhangs and

[0322] (a) when the receiving backbone is as defined in (i)-(a):

[0323] - when n is 1, an overhang comprised in the insert is a palindromic overhang complementary to the palindromic overhang of the receiving backbone such that, upon binding between said two complementary palindromic overhangs, a Type IIP restriction enzyme target site is formed and the other overhang comprised in the insert is non- palindromic and complementary to the non-palindromic overhang of the receiving backbone,

[0324] - when n is of 2 or more, an overhang comprised in a first insert is a palindromic overhang complementary to the palindromic overhang of the receiving backbone such that, upon binding between said two complementary palindromic overhangs, a Type IIP restriction enzyme target site is formed, and the other overhang comprised in said first insert and the overhangs comprised in the remaining (n-1) inserts are different non-palindromic overhangs, and each one of the said non-palindromic overhangs is complementary only to another non-palindromic overhang comprised in another insert, or to the non- palindromic overhang of the receiving backbone, provided the non-palindromic overhang comprised in said first insert is not complementary to the non-palindromic overhang of the receiving backbone; and

[0325] (b) when the receiving backbone is as defined in (i)-(b):

[0326] - when n is 1, the insert comprises two palindromic overhangs, each one being complementary to a different palindromic overhang of the receiving backbone, such that upon binding between said two pairs of complementary palindromic overhangs, two different Type IIP restriction enzyme target sites are formed,

[0327] - when n is 2 or more, a first and a second insert comprise a palindromic overhang, each one being complementary to a different palindromic overhang of the receiving backbone, such that upon binding between said two pairs of complementary palindromic overhangs, two different Type IIP restriction enzyme target sites are formed,

[0328] - the other overhang comprised in the first and the second inserts and the overhangs of the remaining (n-2) inserts are different non-palindromic overhangs, and each one of thesaid non-palindromic overhangs is complementary only to another non-palindromic overhang comprised in another insert;

[0329] and

[0330] (ill) a DNA ligase;

[0331] wherein the molar ratio of the receiving backbone and the insert(s) comprising a palindromic overhang is from 1:0.1 to 1:1.9 (receiving backbone:insert), and the molar ratio of the receiving backbone and the remaining inserts is from 1:1.1 to 1:3 (receiving backbone:insert).

[0332] Clause 2. The cloning method according to clause 1, further comprising subjecting the reaction mixture to conditions appropriate for ligation of the receiving backbone with the n inserts.

[0333] Clause 3- The cloning method according to any one of clauses 1-2, wherein at least one of the n inserts, particularly the n inserts, are obtained by digestion of one or more polynucleotides with Type IIS restriction enzyme(s), particularly with the same Type IIS restriction enzyme, each polynucleotide comprising one or more of said (at least one of the n) inserts and wherein each insert is comprised within the corresponding polynucleotide within a region F of formula 5'-R1-E1-S-E2-R2-3', wherein:

[0334] - each one of R1 and R2 is a sequence consisting of a recognition sequence for a Type IIS restriction enzyme, particularly for the same Type IIS restriction enzyme, oriented in opposite directions within F, - E1 is an extension sequence comprising the cleavage site C1 of the Type IIS restriction enzyme that recognizes R1,

[0335] - S consists of the sequence of interest of the corresponding insert,

[0336] - E2 consists of an extension sequence comprising the cleavage site C2 of the Type IIS restriction enzyme that recognizes R2,

[0337] - R1, E1, S, E2 and R2 are directly attached to each other, and

[0338] wherein upon cleavage by the Type IIS restriction enzyme(s), C1 and C2 form the overhangs comprised in the inserts as defined in (ii)-a) or (ii)-b) of clause 1.

[0339] Clause 4- The cloning method according to clause 3, wherein the at least one of the n inserts are obtained by providing to the reaction mixture, the one or more polynucleotides as defined in clause 3 together with the Type IIS restriction enzyme(s) and subjecting the reaction mixture to conditions appropriate for digestion of the one or more polynucleotides by the Type IIS restriction enzyme(s).

[0340] Clause 5. The cloning method according to any one of clauses 1-4, wherein the receiving backbone is obtained by digestion of a receiving vector with one or two Type IIS restriction enzyme(s), particularly with the same Type IIS restriction enzyme that cleaves the one or more polynucleotides defined in clause 3, the receiving vector comprising a disposable sequence comprising two recognition sequences V1 and V2 for said one or two Type IIS restriction enzyme(s), wherein:

[0341] - V1 is at the 5' region of the disposable sequence and V2 is at the 3' region of the disposable sequence, and

[0342] - V1 and V2 are oriented in opposite directions within the disposable sequence, the orientation of V1 being such that a cleavage site K1 of the Type I IS enzyme recognizing V1 is upstream V1 and at the 5' end of thedisposable sequence, and the orientation of V2 being such that a cleavage site K2 of the Type IIS enzyme recognizing V2 is downstream V2 and at the 3' end of the disposable sequence, and,

[0343] wherein upon cleavage of K1 and K2 with the Type IIS restriction enzyme(s), the overhangs in the receiving backbone defined in (i)-a) or (i)-b) of clause 1 are formed.

[0344] Clause 6. The cloning method according to any one of clauses 1-4, wherein the receiving backbone is obtained by digestion of a receiving vector with a Type IIP restriction enzyme and with a Type IIS restriction enzyme, particularly with the same Type IIS restriction enzyme that cleaves the one or more polynucleotides defined in clause 3, the receiving vector comprising a disposable sequence comprising two restriction enzyme recognition sequences V3 and V4, wherein:

[0345] a) V3 is at the 5' region of the disposable sequence and V4 is at the 3' region of the disposable sequence, b) V3 or V4 is a target sequence of the said Type IIP restriction enzyme, and:

[0346] - if V3 is a target sequence of said Type IIP restriction enzyme: (i) V3 is at the 5' end of the disposable sequence, (ii) V4 is a recognition sequence of the Type IIS restriction enzyme, and the orientation of V4 is such that a cleavage site K4 of the Type IIS enzyme recognizing V4 is downstream V4 and at the 3' end of the disposable sequence, and (iii) upon cleavage of V3 by the Type IIP restriction enzyme and of K4 by the Type IIS restriction enzyme, the overhangs in the receiving backbone defined in (i)-a) or (i)-b) of clause 1 are formed;

[0347] - if V4 is a target sequence of said specific Type 11 P restriction enzyme: (i) V4 is at the 3' end of the disposable sequence, (ii) V3 is a recognition sequence of the Type IIS restriction enzyme, and the orientation of V3 is such that a cleavage site K3 of the Type IIS enzyme recognizing V3 is upstream V3 and at the 5' end of the disposable sequence, and (iii) upon cleavage of K3 by the Type IIS restriction enzyme and of V4 by the Type IIP restriction enzyme, the overhangs in the receiving backbone defined in (i)-a) or (i)-b) of clause 1 are formed.

[0348] Clause 7- The cloning method according to any one of clauses 1-6, wherein the the receiving backbone is obtained by providing to the reaction mixture, the receiving vector as defined in any one of clauses 5 or 6 together with the corresponding Type IIS restriction enzyme(s) defined in any one of clauses 5 or 6, and subjecting the reaction mixture to conditions appropriate for digestion of the receiving vector by the Type IIS restriction enzyme(s).

[0349] Clause 8- The cloning method according to any one of clauses 1-7, wherein at least one of the n inserts, particularly the n inserts, and the receiving backbone are obtained by providing to the reaction mixture, the one or more polynucleotides as defined in clause 3 and the receiving vector as defined in any one of clauses 5 or 6 together with the corresponding Type IIS restriction enzyme(s), particularly those defined in clause 3 and in clause 5 or 6, respectively, and subjecting the reaction mixture to conditions appropriate for digestion of the one or more polynucleotides and the receiving vector by the Type IIS restriction enzyme(s).

[0350] Clause 9. The cloning method according to any one of clauses 7 or 8, wherein the receiving vector is as defined in clause 6 and is provided to the reaction mixture in a linearized form, after digestion with the Type IIP restriction enzyme in another reaction mixture.Clause 10- The cloning method according to any one of clauses 1-4, wherein the receiving backbone is obtained by digestion of a receiving vector with two different Type IIP restriction enzymes, the receiving vector comprising a disposable sequence comprising two target sequences V5 and V6 of said two different Type IIP restriction enzymes, wherein:

[0351] a) V5 is at the 5' end of the disposable sequence and V6 is at the 3' end of the disposable sequence, and b) upon cleavage of V5 and V6 with their corresponding Type IIP restriction enzyme, the overhangs in the receiving backbone defined in (i)-b) of clause 1 are formed.

[0352] Clause 11- The cloning method according to any one of clauses 1-10, wherein at least on of the n inserts, particularly the n inserts, are obtained by providing the one or more polynucleotides as defined in clause 3 together with the Type IIS restriction enzyme(s) defined in clause 3, particularly with the same Type IIS restriction enzyme that cleaves the one or more polynucleotides defined in clause 3, to the reaction mixture and subjecting the reaction mixture to conditions appropriate for digestion of the one or more polynucleotides by the Type IIS restriction enzyme(s).

[0353] Clause 12. The cloning method according to anyone of clauses 10-11, wherein the receiving backbone is provided to the reaction mixture after digestion of the receiving vector with the corresponding Type IIP restriction enzymes that recognize V5 and V6 in another reaction mixture.

[0354] Clause 13. The cloning method according to any one of clauses 1-8 and 10-11, wherein at least one of the n inserts, particularly the n inserts, and the receiving backbone are obtained by providing to the reaction mixture, the one or more polynucleotides as defined in clause 3 and the receiving vector as defined in any one of clauses 6 or 10, together with the corresponding Type IIS restriction enzyme(s), particularly those defined in clause 3 and in clause 6, respectively, and the Type IIP restriction enzyme(s) defined in claim 6 or 10, and subjecting the reaction mixture to conditions appropriate for digestion by the Type IIS and Type lip restriction enzymes of the one or more polynucleotides and the corresponding receiving vector.

[0355] Clause 14- The cloning method according to any one of clauses 1-13, wherein the receiving backbone is not dephosphorylated.

[0356] Clause 15- The cloning method according to any one of clauses 4-14 wherein the method comprises performing at least two cycles of restriction / ligation.

[0357] Clause 16- The cloning method according to any one of clauses 3-15 wherein the one or more polynucleotides are PCR products, hybridized oligos or combinations thereof.

[0358] Clause 17- The cloning method according to any one of clauses 1-16, wherein at least one of the n inserts, particularly the n inserts, is / are obtained by hybridization of two complementary oligos, each oligo consisting of one strand of the corresponding insert.

[0359] Clause 18- The cloning method according to clause 17 wherein the at least one on the n inserts is of less than 200, 180, 170, 160, 150, 140, 130, 120 110 100, 90, 80 , 75, 60, 50 40, 30 , 20, or 10 nucleotides, particularly of less than 150 nucleotides.

[0360] Clause 19- The cloning method according to any one of clauses 17-18, wherein the receiving backbone is obtained by providing to the reaction mixture the receiving vector as defined in any one of clauses 5 or 6 together with the corresponding Type IIS restriction enzyme(s) defined in any one of clauses 5 or 6, andsubjecting the reaction mixture to conditions appropriate for digestion of the receiving vector by the Type IIS restriction enzyme(s), particularly wherein the Type IIS enzyme consists of the same Type IIS restriction enzyme that cleaves the one or more polynucleotides defined in clause 3.

[0361] Clause 20- The cloning method according to any one of clauses 17-19, wherein the rest of the n inserts are obtained upon digestion of one or more polynucleotides by type IIS restriction enzyme(s) as defined in clause 3, particularly, wherein the rest of n inserts correspond to the at least one of the n inserts defined in clause 3.

[0362] Clause 21- The cloning method according to clause 20 wherein the n inserts and the receiving backbone are obtained by providing to the reaction mixture, the hybridized oligos as defined in any on of clauses 17-18, the one or more polynucleotides as defined in clause 19 and the receiving vector as defined in any one of clauses 5 or 6 together with the corresponding Type IIS restriction enzyme(s), particularly those defined in clause 3 and in clause 5 or 6, respectively, more particularly the Type IIS restriction enzyme that cleaves the one or more polynucleotides defined in clause 3, and subjecting the reaction mixture to conditions appropriate for digestion of the one or more polynucleotides and the receiving vector by the Type IIS restriction enzyme(s).

[0363] Clause 22- The cloning method according to any one of the preceding clauses, wherein the overhangs of the n inserts and of the receiving backbone predetermine the position and / or orientation of each insert within the destination vector.

[0364] Clause 23- The cloning method according to any one of clauses 5-22, wherein the receiving vector comprises an expression promoter upstream the disposable sequence.

[0365] Clause 24- The cloning method according to any one of clauses 1-23, wherein each one of the sequences of interest “S” comprised in the n inserts consists of a peptide encoding sequence, and the peptide encoding sequences within the n inserts are in frame within the destination vector.

[0366] Clause 25- The cloning method according to clause 24, wherein:

[0367] each one of the extension sequences E1 and E2 in each of the one or more polynucleotides defined in clause 3 consists of p codons, being “p” an integer of at least 1, particularly, equal to 2, wherein: (I) each codon encodes a neutral amino acid and / or (ii) the extension sequence further comprises a translation start codon in frame with the corresponding peptide encoding sequence S, wherein the translation start codon is retained within the corresponding insert upon cleavage of the corresponding polynucleotide by the Type IIS restriction enzyme.

[0368] Clause 26- The cloning method according to any one of clauses 17-25, wherein the overhangs of the one or more inserts defined in clauses 17-18, are as the overhangs formed upon digestion of the extension sequences E1 and E2 as defined in clause 24 by the corresponding Type IIS enzyme.

[0369] Clause 27- The cloning method according to any one of clauses 1-26, wherein upon binding between complementary overhangs of the n inserts, the sequences linking sequences S to and within the destination vector, consists each one of p codons, being “p” an integer of at least 1, particularly, equal to 2, wherein: (I) each codon encodes a neutral amino acid and / or (ii) the linking sequence further comprises a translation start codon in frame with the corresponding peptide encoding sequence S.Clause 28- The cloning method according to any one of clauses 24-27, wherein the receiving vector comprises a protein tag encoding sequence upstream or downstream the disposable sequence and under the control of a same expression promoter as the disposable sequence.

[0370] Clause 29- The cloning method according to clause 28 wherein the protein tag encoding sequence is downstream the disposable sequence within the receiving vector.

[0371] Clause 30- The cloning method according to any one of clauses 28-29 wherein the protein tag encoding sequence is flanked by two Type IIP restriction enzyme target sites within the receiving vector, particularly, wherein the Type IIP target site upstream the protein tag encoding sequence is a BamHI target site and / or the the Type IIP target site downstream the protein tag encoding sequence is an Xhol target site.

[0372] Clause 31-The cloning method according to clause 30, wherein, upon cleavage, one of the two Type IIP restriction enzyme target sites flanking the protein tag encoding sequence forms the palindromic overhang of the receiving backbone as defined in (i)-a or (i)-b, particularly wherein said Type IIP target site is upstream the protein tag encoding sequence, and the protein tag encoding sequence is downstream the disposable sequence in the receiving vector.

[0373] Clause 32- The cloning method according to any one of clauses 28-31, wherein the protein tag encoding sequence is in frame with the peptide encoding sequences comprised or consisting of the n inserts within the destination vector, particularly with the peptide encoding sequences S of the n inserts

[0374] Clause 33- The cloning method according to any one of clauses 27-32 wherein (i) the peptide encoding sequence comprised or consisting of one of the n inserts, particularly the sequence S of one of the n inserts, encodes a signal peptide, or (ii) the receiving vector comprises a signal peptide upstream or downstream the disposable sequence and under the control of a same expression promoter as the disposable sequence. Clause 34- The cloning method according to clause 33 wherein (i) the signal peptide is at the 5' end or at the 3' end of the sequence of n inserts in the destination vector, particularly at the 5' end, or (ii) the signal peptide is upstream or downstream, particularly immediately upstream or downstream, the sequence of n inserts in the destination vector, particularly upstream, more particularly immediately upstream.

[0375] Clause 35- The cloning method according to any one of clauses 33-34, wherein the signal peptide encoding sequence is in frame with the peptide encoding sequences comprised or consisting of of the n inserts, particularly with the peptide encoding sequences S of the n inserts, within the destination vector.

[0376] Clause 36- The cloning method according to any one of clauses 27-35 wherein the insert at the 5' end of the sequence of n inserts in the destination vector comprises a translation start codon upstream the sequence S comprised in said insert, and in frame with said sequence S, particularly wherein the signal peptide encoding sequence and / or the protein tag encoding sequence within the destination vector, is downstream said translation start codon.

[0377] Clause 37- The cloning method according to clause 36, wherein the extension sequence E1 of the F region comprising the insert referred in clause 36, is as defined in (ii) of clause 25, particularly wherein the signal peptide encoding sequence and / or the protein tag encoding sequence within the destination vector, is downstream said translation start codon.Clause 38- The cloning method according to any one of clauses 27-37 wherein the signal peptide is selected from a PelB signal peptide or an OmpA signal peptide.

[0378] Clause 39- The cloning method according to any one of clauses 29-38, wherein the method further comprises a step of substituting the protein tag encoding sequence by a different protein tag encoding sequence of interest, particularly wherein said substitution comprises digestion of the destination vector at the Type IIP target sites flanking the protein tag encoding sequence by the corresponding Type IIP enzymes, and subsequently, insertion of the protein tag encoding sequence of interest at said Type IIP cleaved sites. Clause 40- The cloning method according to any one of clauses 1-39, wherein the n inserts, the receiving backbone, the one or more polynucleotides and / or the receiving vector as defined in any one of clauses 1-36 do not comprise recognition sites for the Type IIS and / or Type IIP restriction enzymes defined in any one of the previous clauses, in addition to the recognitions sites for said enzymes indicated in any of the previous clauses, (defined by the recognition ssequences defined in any one of the previous clauses), more particularly wherein recognition sites specific for the Type IIS enzyme(s) and / or Type IIP enzyme(s) referred in any one of the previous clauses, in addition to the recognition sites for said enzymes indicated in any of the previous clauses, (defined by the recognition sequences defined in any one of the previous clauses), have been deleted in the corresponding inserts, receiving backbone, polynucleotides and / or the receiving vector.

[0379] Clause 41- The cloning method according to clause 40 wherein:

[0380] - the n inserts, the receiving backbone, the one or more polynucleotides and / or the receiving vector as defined in any one of clauses 1-40 do not comprise recognition sites for the Type IIS restriction enzymes defined in any one of clauses 1-40, in addition to the recognition sites for said enzymes indicated in any of clauses 1-40 (defined by the recognition sequences defined in any one of clauses 1-40) particularly wherein recognition sites specific for the Type IIS enzyme(s) referred in any one of clauses 1-40, in addition to the recognition sites for said enzyme(s) indicated in any of clauses 1-40, (defined by the recognition sequences defined in any one of clauses 1-40), have been deleted in the corresponding inserts, receiving backbone, polynucleotides and / or receiving vector- the n inserts, the receiving backbone, the one or more polynucleotides and / or the receiving vector as defined in any one of clauses 1-40 do not comprise recognition sites for the Type IIP restriction enzymes defined in any one of clauses 1-40, in addition to the recognition sites for said enzymes indicated in any of clauses 1-40 ( defined by the recognition sequences defined in any one of clauses 1-40), particularly wherein recognition sites specific for the Type IIP enzyme(s) enzyme(s) referred in any one of clauses 1-40, in addition to the recognition sites for said enzyme(s) indicated in any of clauses 1-40 (defined by the recognition sequences defined in any one of clauses 1-40), have been deleted in the corresponding inserts, receiving backbone, polynucleotides and / or receiving vector- the n inserts, the receiving backbone, the one or more polynucleotides and / or the receiving vector as defined in any one of clauses 1-40 do not comprise recognition sites for the Type IIS and Type IIP restriction enzymes defined in any one of clauses 1-40, in addition to the recognition sites for said enzymes indicated in any of clauses 1-40, (defined by the recognition sequences defined in any one of clauses 1-40), particularly wherein recognition sites specific for the Type IIP enzyme(s) enzyme(s) referred in any one ofclauses 1-40, in addition to the recognition sites for said enzyme(s) indicated in any of clauses 1-40 (defined by the recognition sequences defined in any one of clauses 1-40), have been deleted in the corresponding inserts, receiving backbone, polynucleotides and / or receiving vector

[0381] Clause 42- A nucleic acid vector obtained by the cloning method defined by any one of clauses 1-41. Clause 43- A method for the synthesis of a library of different nucleic acid vectors, wherein: each vector from the library is as a destination vector obtained by the cloning method as defined by any one of clauses 28-41; each vector from the library comprises a protein tag encoding sequence; and those vectors from the library that differ in the combination and / or orientation of the n inserts, differ in the protein tag encoding sequence of interest, and those vectors from the library having the same combination and / or orientation of n inserts, have the same protein tag encoding sequence of interest; the method comprising:

[0382] (i) performing the cloning method as defined by any one of clauses 28-41, for the obtention of destination vectors that differ in the combination and / or orientation of the n inserts, each one comprising a protein tag encoding sequence flanked by two different type IIP target sites, and

[0383] (ii) substituting the protein tag from the destination vectors obtained from step (i) by another protein tag of interest that is the same for all destination vectors having the same combination and / or orientation of n inserts, and different between destination vectors having a different combination and / or orientation of n inserts.

[0384] Clause 44- A library of vectors obtained by the method as defined by clause 43.

[0385] Clause 45- A protein encoded by the nucleic acid vector defined by clause 42, wherein the nucleic acid sequence of the vector encoding the protein comprises the n inserts.

[0386] Clause 46- A method for the synthesis of a protein comprising (i) performing the cloning method as defined in any one of clauses 1-41, wherein the the n inserts comprise or consist of peptide encoding sequences, particularly the method as defined in any one of clauses 24-41 (ii) providing to a protein expression system the nucleic acid vector obtained from step (i) and (iii) subjecting the protein expression system to conditions appropriate for the expression of the protein.

[0387] Clause 47- A method for the synthesis of a library of proteins, wherein the library of proteins comprises several proteins as defined in clause 45, particularly of proteins obtained by the method as defined by clause 46, wherein those proteins from the library that differ in the sequence, number and / or position of the protein sequences encoded by the n inserts, also differ in a protein tag sequence, and those proteins from the library that have the same sequence, number and / or position of the protein sequences encoded by the n inserts, have the same protein tag sequence, the method comprising:

[0388] (i) performing the method for the synthesis of a library of different nucleic acid vectors as defined in clause 43,

[0389] (ii) providing to a protein expression system the nucleic acid vectors of the library of vectors obtained from step (i), and

[0390] (iii) subjecting the protein expression system to conditions appropriate for the expression of the proteins.

[0391] Clause 48- A library of proteins obtained by the method as defined by clause 47.Clause 49. A kit comprising means for performing the method as defined in any one of clauses 1-41, 43, 46 or 47.

[0392] Clause 50. A reaction mixture as defined in any one of clauses 1-41.

[0393] Clause 51- The reaction mixture according to clause 50 comprising the receiving backbone, the n inserts and the DNA ligase, and optionally the Type IIS restriction enzyme(s), as defined in any one of clauses 1-41.

[0394] Throughout the description and claims the word "comprise” and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise” encompasses the case of "consisting of'. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention.

[0395] The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

[0396] Examples

[0397] Example 1- Materials and methods

[0398] Bacterial strains and culture media

[0399] Escherichia coli DH5o (Fisher, Spain) was used for transformation. The E. coli cultures were routinely grown at 37°C in Lennox broth (LB; Condalab, Madrid, Spain) or on plates of LB supplemented with 2% (w / v) agar and with the antibiotic for selection of the clones, 100 pg / mL of ampicillin (Fisher, Spain) when the strain carries any of the vectors designed at this work. The media were sterilized by autoclave at 120°C for 15 min or by filtration using sterile 0.2 pm cellulose acetate filters.

[0400] In silico design of vectors, primers, DNA and their synthesis.

[0401] All the in silico design of vectors, primers and gene blocks was performed using the bioinformatic software Geneious Prime® 2022.1.1 and further versions up to Geneious Prime® 2025.0.2 (http: / / www.qeneious.com / ).

[0402] The plasmid pTT-SP-PelB-Tag Ex-Term vector was constructed by replacing the multiple cloning site (MGS) of the pT7.7 plasmid, which served as the backbone, with a new MGS and tag-shuffling sequence. The new vector was synthesized by Genscript (Genscript Inc., USA).

[0403] All primers used in this study were synthesized by Integrated DNA Technologies, Inc. (IDT, USA). The Tm of the primers was calculated with Geneious Prime and with the algorithms specific for Phusion polymerase.The genes encoding the domains of interest were optimized based on E. coli codon usage using Geneious Prime software and synthetized at Twist Bioscience (San Fracisco, CA, USA). To note, all restriction sites that could interfere with the cloning process were eliminated from the gene blocks when needed.

[0404] To enable the assembly of complex proteins composed of multiple domains, unique overhangs were designed for each DNA building block. The estimated ligation fidelity for the overhangs for the 6-block combination, was calculated using the NEBridge Ligase Fidelity Viewer® (https: / / liqasefidelity.neb.com / viewset / run.cqi).

[0405] PCR amplification of DNA blocks

[0406] To generate DNA fragments suitable for cloning, each domain sequence was amplified via PCR with specific primer pairs (Table 1). These primers featured a 4-nucleotide overhang at the 5' end, optimized for ligation compatibility, and included recognition sites for type IIS restriction enzymes. PCR amplification was performed for 1 to 6 fragments, with primers diluted to 10 pM in RNase / DNase-free water. Each 50 pl PCR reaction contained 0.4 pl DNA (25 ng / pl), 10 pl 5X buffer, 1 l dNTPs (10 mM), 2.5 pl of each primer (10 pM), and 0.5 pl Phusion High-Fidelity Polymerase (2 U / pl) (Fisher, Spain). The amplification protocol commenced with an initial denaturation at 98°C for 30 seconds, followed by 35 cycles of 98°C for 10 seconds, 62°C for 15 seconds, and 72°C for 15 seconds, concluding with a final extension at 72°C for 10 seconds and a hold at 4°C. Amplicon sizes (Table 1) were confirmed via electrophoresis on a 2% agarose gel (5 pl product + 1 pl 6X loading buffer) in 1X TAE at 100 V for 45 minutes. The products were purified using the DNA Clean & Concentrator-5 kit (Zymo Research Corp., Irvine, CA, USA), eluted in 15 pl, quantified with a NanoPhotometer® (Implen GmbH, Munich, Germany), and stored at -20°C.

[0407] Table 1. List of the primers used for the PCR of DNA blocks and amplicon expected size

[0408] >

[0409]

[0410] Duplex hybridization

[0411] For sequences under 150 base pairs, such as protein tags or domains, duplex DNA is synthesized through the hybridization of Forward and Reverse oligonucleotides (i.e. Table 2). These duplexes are engineered with ligation-compatible overhangs to enhance cloning efficiency. Oligonucleotide phosphorylation is performed prior to hybridization. Each oligonucleotide (100 piM) is phosphorylated by mixing 2 piL of the oligonucleotide with 10X ligase buffer and T4 Polynucleotide Kinase (Fisher, Spain) in a final volume of 20 piL. The reaction is incubated at 37°C for 1 hour to allow phosphorylation, and the enzyme is inactivated by heating at 65°C for 20 minutes. After phosphorylation, the oligos are mixed in a 1:1 ratio and diluted 1:10 using IDTE buffer (IDT, USA). The mixture undergoes thermal cycling in a PCR machine, starting with a 5-minute incubation at 95°C for denaturation, followed by a 1 -minute incubation at 95°C with a gradual temperature decrease of 1 °C per cycle over 72 cycles to promote annealing. The reaction is then maintained at 4°C. The integrity of the duplexes is verified using a 2% agarose gel electrophoresis (5 pil product + 1 pil 6X loading buffer) in 1X TAE at 100 V for 45 minutes. Prior to ligation, the duplexes are diluted 1:50 in DNAse / RNAse-free water.

[0412] Table 2. List of oligonucleotides to perform oligo hybridization.

[0413] >

[0414]

[0415] Restriction digestion

[0416] All restriction enzymes were sourced from New England Biolabs, located in Ipswich, MA, USA, and utilized following the supplier's specified concentrations and temperatures. In a standard procedure, one unit of enzyme is employed to digest 1 pig of DNA (vector or liner DNA) for 1 hour at 37°C, within a total reaction volume of 50 pil, and using rCutSmart Buffer. An inactivation step is performed at the specified temperatureand for 20 min, calculated using the NEBcloner online tool (https: / / nebcloner.neb.eom / #l / ). After enzymatic digestion, the products are analyzed using 1-2% agarose gel electrophoresis in TAE buffer at 100 V for 1 hour and visualized under the G:B0X Syngene GelDoc.

[0417] Restriction / ligation of DNA Blocks into the vector

[0418] The TelClo restriction / ligation procedure was executed in a 25 l reaction volume, maintaining a 1:1 molar ratio of vector (1 ,75nM) to insert with a palindromic overhang (1.75 nM), and a 1 :2 ratio for additional inserts (3.5 nM). Vector and inserts were diluted to the desired concentrations in RNase / DNase-free water, with molarity determined via NEBioCalculator (https: / / nebiocalculator.neb.eom / #l / ligation). The reaction mixture comprised 1 pl vector, 1 pl per insert, 2.5 pl of 10X T4 DNA ligase buffer, 0.5 pl T4 DNA ligase (New England Biolabs, USA), 1.5 pl Bsa-HF-v2, and DNAse / RNAse-free water to a total of 25 pl. The cycling conditions were set at 37°C for 5 minutes and 16°C for 5 minutes over 30 cycles, followed by 80°C for 5 minutes, and maintained at 4°C. All dilutions and reagents were stored at -20°C and cataloged under the appropriate sample type. A negative selection digestion was conducted by incorporating 3 pl of CutSmart Buffer, 1 pl of Hindi 11, and 1 pl of Sall into the reaction, incubating at 37°C for 1 hour, followed by enzyme inactivation at 80°C for 20 minutes, and stored at -20°C until transformation. All restriction enzymes were sourced from New England Biolabs.

[0419] Ligation transformation

[0420] The TelClo reaction mix was used to transform E. coli DH5o competent cells according to suppliers' instructions. A 5 pl aliquot of the reaction mixture, along with 10 pg of pUC-19 as a positive control for transformation, was added to 50 pl of competent cells, which were incubated on ice for 30 minutes, heat-shocked at 42°C for 45 seconds, and then cooled on ice for 2 minutes. Subsequently, 250 pl of SOC medium was added, and the cells were incubated at 37°C for 1 hour with shaking at 200 rpm to allow recovery and expression of the antibiotic resistance marker. A 20 pl and 200 pl aliquot of each cell suspension was then evenly spread onto LB agar plates supplemented with 100 pg / mL of ampicillin (Fisher, Spain) for selection and incubated at 37°C for 16-20 h. Colonies were screened for positive ligation products using diagnostic methods such as colony check PCR (described below) or restriction digestion (as described above).

[0421] Colony check PCR, plasmid extraction and sequencing

[0422] A portion of a single colony from a plate containing transformants was aseptically transferred using a sterile tip into 30 pl of DNAse / RNAse-free water. For the subsequent PCR assay, 1 pil of this colony suspension was combined with 0.5 piM of the primers T7_promoter_Fwd and GG-Rev (Table 3), along with RedTaq DNA polymerase (Fisher, Spain), culminating in a total reaction volume of 15 pil. The thermal cycling protocol commenced with an initial denaturation at 95°C for 10 minutes, followed by 20 cycles of denaturation at 95°C for 30 seconds, annealing at 50 °C for 20 seconds, and extension at 72 °C for 1 minute per kilobase pair. The process concluded with a final extension at 72°C for 2 minutes, after which the reaction was held at 4°C. The products are analyzed using 1-2% agarose gel electrophoresis in TAE buffer at 100 V for 1 hourand visualized under the G:BOX Syngene GelDoc. The expected sizes from 1 and 6 blocks are 701 bp and 2516 bp respectively.

[0423] Table 3. List of primers for colony check and sequencing reactions.

[0424] >

[0425]

[0426] Plasmid extraction from the transformants was conducted utilizing the NZYMiniprep kit (Lisboa, Portugal), followed by sequencing at LGC Genomics (Berlin, Germany) with primers T7_promoter forward, GG-Rev, and Test6-1_F, Test6-3_F as required. Sequence verification was performed using Geneious Prime software.

[0427] Example 2. Design of the receiving vector plasmid.

[0428] The TelClo system was developed using the plasmid pT7.7 as a backbone. However, sequences for signal peptides, multicloning sites, or tags can be incorporated into alternative plasmid backbones and thus alternative vector backbones can be used.

[0429] The initial cloning vector (or receiving vector) of the TelClo system (Fig. 1 and 5) includes:

[0430] 1. One multicloning site containing two binding sites (in opposite orientation) for the same type-IIS restriction enzyme (Bsal). The first restriction site contains the ATG sequence that signals the initiation of the translation process and will generate a non-palindromic overhang. In case of the presence of a signal peptide (see below), this overhang will not encode for an ATG, but for a compatible overhang for the cloning in frame of the signal peptide in N-terminal of the protein. On the other hand, the second Bsal restriction site will create a palindromic overhang that, after cloning of the desired proteins, will regenerate the BamHI (type-IIP) restriction site that is part of the "tag exchange” cassette.

[0431] 2. One "tag-shuffling” cloning sequence, that will allow for the creation of customized destination vectors for the cloning in-frame of proteins with specific tags in C-terminal (delimited by BamHI and Xholtype-1 IP restriction sites). Each different tag will contain a unique and specific restriction site, right before the Xhol restriction site, that will allow for negative selection when exchanging the tag for a different one, and for screening by diagnostic digestion for positive colonies when the tag is cloned in an existing plasmid. The tags will be part of the destination vector to increase the efficiency of the cloning reaction by elimination of one ligation block, but they can also be exchanged from a plasmid with an already cloned tagged or untagged protein.

[0432] 3. Negative Selection Restriction Enzyme Sites: Two additional type-IIP restriction sites (Hindlll and Sall) were incorporated within the MCS to enable negative selection. These sites allow the elimination of undigested or re-linearized plasmid background by digesting the final ligation product with both enzymes prior to transformation into competent cells.

[0433] 4. An optional signal peptide at 5' of the first Bsal restriction enzyme site, for the cloning in frame of a polypeptide sequence that targets the expressed proteins for secretion to the culture media. In this particular case, three different plasmids were contemplated: without signal peptide, with Pe / B signal peptide, and with OmpA signal peptide.

[0434] 5. A T7 promoter and IPTG inducible system for overexpression of the final ligation construct. 6. An antimicrobial resistance gene for proper antibiotic selection.

[0435] This TelClo design ensures a high level of flexibility, efficiency, and compatibility for downstream applications, including protein expression and tag exchange systems.

[0436] Exmple 3. Development of interchangeable tags facilitated by the palindromic overhang characteristic of the TelClo system.

[0437] The design facilitates the exchange of the N-terminal protein tag in the receiving vector, plasmid, or a library of plasmids, with pre-cloned proteins. Each Tag is followed by a proper negative selection restriction enzyme site (Fig. 2).

[0438] The tag exchange process involves two main steps:

[0439] 1. Linearization of the receiving plasmid (receiving vector, plasmid, or a (library of) plasmid(s), with precloned proteins) through digestion with BamHI and Xhol (type-IIP enzymes), followed by gel band extraction.

[0440] 2. Preparation of tags, which varies based on size:

[0441] - For small fragments (less than 150 bp), create a ready-to-clone DNA duplex via hybridization and phosphorylation of ssDNA oligos, eliminating the need for restriction digestion (Fig. 6).

[0442] - For larger tags, prepare as domain coding sequences using PCR, then digest and purify the PCR product.

[0443] Subsequently, ligate the linearized vector with the tag (either DNA duplex or digested PCR product) and inactivate the DNA ligase. Conduct negative selection by digestion with the restriction enzyme specific to the tag initially present in the plasmid, followed by transformation into competent cells.Example 4. Design of overhangs for DNA block assembly and cloning

[0444] To enable the assembly of complex proteins composed of multiple domains, unique overhangs were designed for each DNA building block. The overhangs ensured compatibility between DNA fragments during ligation and avoided palindromic sequences, which could compromise ligation fidelity. The only exception was the palindromic overhang for the end position (position E or PE ), generated after digestion of the receiving plasmid with Bsal (type-1 IS). In this case, a BamHI restriction site (type I l-P) was regenerated as a scar following the final polypeptide cloning (Fig. 3).

[0445] Universal overhangs for each ligation position were designed to maintain the reading frame and minimize structural or functional disruptions in the final protein. These overhangs encoded for two amino acids (6 nucleotides) with neutral physicochemical properties to ensure minimal impact on the protein's structure and activity (Table 4, Fig. 3). We show the design for the ligation of up to six DNA blocks, but the complexity of the final construct could be further expanded with the addition of supplementary compatible overhangs.

[0446] Table 4. List of Compatible Overhangs for Cloning Multiple DNA Blocks. The table provides details for the assembly of multiple DNA blocks, specifying: 1) Overhangs by Position: Overhangs created after digestion with the corresponding restriction enzyme for each position; 2) Six-Nucleotide Sequences: Nucleotide sequences that contain the designated overhangs, ensuring compatibility between DNA fragments and; 3) Extra "Scar” Amino Acids: The two additional amino acids encoded at each cloning position. Notably, for position 1, the sequence includes only a methionine residue to signal the initiation of the translation process. This design ensures accurate assembly and minimal interference with the functional or structural properties of the resulting protein.

[0447]

[0448] TelClo cloning proceeds in three steps:1. PCR amplification of the domain encoding sequences that, only after their amplification, will be flanked by compatible overhangs and type IIS restriction enzyme sites in opposite orientation (Fig 7). Alternatively, hybridization of ssDNA oligos to create ready-to-clone duplexes (Fig 6).

[0449] 2. Restriction / ligation cycling reaction, in the presence of the vector, the linear PCR fragments and / or DNA duplexes encoding the protein domains, the type IIS restriction enzyme (in this case Bsal), and the DNA ligase. The plasmid to insert molar ratio would be 1:1 for the insert with the palindromic overhang, and 1:2 for the rest of inserts.

[0450] 3. Inactivation of the DNA ligase followed by negative selection restriction reaction of the ligated product using both negative selection enzymes (Hindlll and Sall). After digestion with Hindlll and Sall, these enzymes will be inactivated by temperature and the cloning reaction mix will be used to transform into competent cells. Colonies growth under selection conditions will be screened for positive ligation products.

[0451] As well understood by a skilled person, steps 2 and 3 can be performed in a single pot.

[0452] Example 5. Validation of TelClo system by cloning 1-block or 6-blocks into the receiving vector. To produce DNA fragments ready for cloning, the DNA domain sequence is amplified via PCR, resulting in a linear double-stranded DNA sequence. This sequence encodes the domain of interest and is flanked by type IIS restriction enzyme recognition sites with compatible overhangs for ligation. The system's efficacy was evaluated by cloning either a single domain or a combination of six domains into the vector pTT-SP-PelB-Tag Ex-Term within the TelClo System. The correct assembly of domains was verified through colony check PCR and subsequent sequencing (Fig. 4). The results demonstrated a 100% assembly efficiency for a single block and 93% for six blocks, surpassing the predicted 50% efficiency as estimated by NEB online tools (Fig. 8).

Claims

CLAIMS1- A cloning method for obtaining a destination nucleic acid vector comprising “n” inserts, wherein “n” is an integer of at least 1, and each insert comprises a sequence of interest “S” and, wherein the method comprises contacting in a reaction mixture:(i) a receiving backbone, wherein:(a) one end of the receiving backbone comprises a palindromic overhang and the other end comprises a non-palindromic overhang, or(b) each end of the receiving backbone comprises a different palindromic overhang;(ii) the n inserts, wherein each insert comprises two different overhangs and(a) when the receiving backbone is as defined in (i)-(a):- when n is 1, an overhang comprised in the insert is a palindromic overhang complementary to the palindromic overhang of the receiving backbone such that, upon binding between said two complementary palindromic overhangs, a Type IIP restriction enzyme target site is formed and the other overhang comprised in the insert is non- palindromic and complementary to the non-palindromic overhang of the receiving backbone,- when n is of 2 or more, an overhang comprised in a first insert is a palindromic overhang complementary to the palindromic overhang of the receiving backbone such that, upon binding between said two complementary palindromic overhangs, a Type IIP restriction enzyme target site is formed, and the other overhang comprised in said first insert and the overhangs comprised in the remaining (n-1) inserts are different non-palindromic overhangs, and each one of the said non-palindromic overhangs is complementary only to another non-palindromic overhang comprised in another insert, or to the non- palindromic overhang of the receiving backbone, provided the non-palindromic overhang comprised in said first insert is not complementary to the non-palindromic overhang of the receiving backbone; and(b) when the receiving backbone is as defined in (i)-(b):- when n is 1, the insert comprises two palindromic overhangs, each one being complementary to a different palindromic overhang of the receiving backbone, such that upon binding between said two pairs of complementary palindromic overhangs, two different Type IIP restriction enzyme target sites are formed,- when n is 2 or more, a first and a second insert comprise a palindromic overhang, each one being complementary to a different palindromic overhang of the receiving backbone, such that upon binding between said two pairs of complementary palindromic overhangs, two different Type IIP restriction enzyme target sites are formed,- the other overhang comprised in the first and the second inserts and the overhangs of the remaining (n-2) inserts are different non-palindromic overhangs, and each one of thesaid non-palindromic overhangs is complementary only to another non-palindromic overhang comprised in another insert;and(ill) a DNA ligase;wherein the molar ratio of the receiving backbone and the insert(s) comprising a palindromic overhang is from 1:0.1 to 1:1.9 (receiving backbone:insert), and the molar ratio of the receiving backbone and the remaining inserts is from 1:1.1 to 1:3 (receiving backbone:insert),particularly, the method further comprising subjecting the reaction mixture to conditions appropriate for ligation of the receiving backbone with the n inserts.2- The cloning method according to claim 1, wherein at least one of the n inserts, particularly the n inserts, are obtained by digestion of one or more polynucleotides with Type IIS restriction enzyme(s), particularly with the same Type IIS restriction enzyme, each polynucleotide comprising one or more of said inserts and wherein each insert is comprised within the corresponding polynucleotide within a region F of formula 5'-R1-E1-S-E2-R2-3', wherein:- each one of R1 and R2 is a sequence consisting of a recognition sequence for a Type IIS restriction enzyme, particularly for the same Type IIS restriction enzyme consisting of the Type IIS enzyme that cleaves the one or more polynucleotides, oriented in opposite directions within F,- E1 is an extension sequence comprising the cleavage site C1 of the Type IIS restriction enzyme that recognizes R1,- S consists of the sequence of interest of the corresponding insert,- E2 consists of an extension sequence comprising the cleavage site C2 of the Type IIS restriction enzyme that recognizes R2,- R1, E1, S, E2 and R2 are directly attached to each other, andwherein upon cleavage by the Type IIS restriction enzyme(s), C1 and C2 form the overhangs comprised in the inserts as defined in (ii)-a) or (ii)-b) of claim 1.

3. The cloning method according to any one of claims 1-2, wherein the receiving backbone is obtained by digestion of a receiving vector with one or two Type IIS restriction enzyme(s), particularly with the same Type IIS restriction enzyme that cleaves the one or more polynucleotides defined in claim 2, the receiving vector comprising a disposable sequence comprising two recognition sequences V1 and V2 for said one or two Type IIS restriction enzyme(s), wherein:- V1 is at the 5' region of the disposable sequence and V2 is at the 3' region of the disposable sequence, and- V1 and V2 are oriented in opposite directions within the disposable sequence, the orientation of V1 being such that a cleavage site K1 of the Type I IS enzyme recognizing V1 is upstream V1 and at the 5' end of the disposable sequence, and the orientation of V2 being such that a cleavage site K2 of the Type IIS enzyme recognizing V2 is downstream V2 and at the 3' end of the disposable sequence, and,wherein upon cleavage of K1 and K2 with the Type IIS restriction enzyme(s), the overhangs in the receiving backbone defined in (i)-a) or (i)-b) of claim 1 are formed.

4. The cloning method according to any one of claims 1-3, wherein the receiving backbone is obtained by digestion of a receiving vector with a Type IIP restriction enzyme and with a Type IIS restriction enzyme, particularly with the same Type IIS enzyme that cleaves the one or more polynucleotides defined in claim 2, the receiving vector comprising a disposable sequence comprising two restriction enzyme recognition sequences V3 and V4, wherein:a) V3 is at the 5' region of the disposable sequence and V4 is at the 3' region of the disposable sequence, b) V3 or V4 is a target sequence of the said Type IIP restriction enzyme, and:- if V3 is a target sequence of said Type IIP restriction enzyme: (I) V3 is at the 5' end of the disposable sequence, (ii) V4 is a recognition sequence of the Type IIS restriction enzyme, and the orientation of V4 is such that a cleavage site K4 of the Type IIS enzyme recognizing V4 is downstream V4 and at the 3' end of the disposable sequence, and (ill) upon cleavage of V3 by the Type IIP restriction enzyme and of K4 by the Type IIS restriction enzyme, the overhangs in the receiving backbone defined in (i)-a) or (i)-b) of claim 1 are formed;- if V4 is a target sequence of said specific Type 11 P restriction enzyme: (I) V4 is at the 3' end of the disposable sequence, (ii) V3 is a recognition sequence of the Type IIS restriction enzyme, and the orientation of V3 is such that a cleavage site K3 of the Type IIS enzyme recognizing V3 is upstream V3 and at the 5' end of the disposable sequence, and (ill) upon cleavage of K3 by the Type IIS restriction enzyme and of V4 by the Type IIP restriction enzyme, the overhangs in the receiving backbone defined in (i)-a) or (i)-b) of claim 1 are formed.5- The cloning method according to any one of claims 1-4, wherein the receiving backbone is obtained by digestion of a receiving vector with two different Type IIP restriction enzymes, the receiving vector comprising a disposable sequence comprising two target sequences V5 and V6 of said two different Type IIP restriction enzymes, wherein:a) V5 is at the 5' end of the disposable sequence and V6 is at the 3' end of the disposable sequence, and b) upon cleavage of V5 and V6 with their corresponding Type IIP restriction enzyme, the overhangs in the receiving backbone defined in (i)-b) of claim 1 are formed.6- The cloning method according to any one of claims 1-5, wherein at least one of the n inserts, particularly the n inserts, and the receiving backbone are obtained by providing to the reaction mixture:- the one or more polynucleotides as defined in claim 2 and the receiving vector as defined in any one of claims 3 or 4 together with the corresponding Type IIS restriction enzyme(s) and subjecting the reaction mixture to conditions appropriate for digestion of the one or more polynucleotides and the receiving vector by the Type IIS restriction enzyme(s), or- the one or more polynucleotides as defined in claim 2 together with the corresponding Type IIS restriction enzyme(s), and the receiving backbone as defined in claim 5 after digestion of the receiving vector in another reaction mixture with the corresponding Type IIP restriction enzymes, and subjecting the reaction mixture to conditions appropriate for digestion of the one or more polynucleotides.7- The cloning method according to any one of claims 2-6, wherein each one of the sequences of interest “S” comprised in the n inserts consists of a peptide encoding sequence, and the peptide encoding sequences within the n inserts are in frame within the destination vector, particularly wherein:each one of the extension sequences E1 and E2 in each of the one or more polynucleotides consists of p codons, being “p” an integer of at least 1, particularly, equal to 2, wherein: (I) each codon encodes a neutral amino acid and / or (ii) the extension sequence further comprises a translation start codon in frame with the corresponding peptide encoding sequence S, wherein the translation start codon is retained within the corresponding insert upon cleavage of the corresponding polynucleotide by the Type IIS restriction enzyme.8- The cloning method according to claim 7, wherein the receiving vector comprises a protein tag encoding sequence upstream or downstream the disposable sequence and under the control of a same expression promoter as the disposable sequence, particularly, wherein the protein tag encoding sequence is flanked by two Type IIP restriction enzyme target sequences within the receiving vector9- A nucleic acid vector obtained by the cloning method defined by any one of claims 1-8.10- A method for the synthesis of a library of different nucleic acid vectors, wherein: each vector from the library is as a destination vector obtained by the cloning method as defined by any one of claims 7-8; each vector from the library comprises a protein tag encoding sequence; and those vectors from the library that differ in the combination and / or orientation of the n inserts, differ in the protein tag encoding sequence of interest, and those vectors from the library having the same combination and / or orientation of n inserts, have the same protein tag encoding sequence of interest; the method comprising:(I) performing the cloning method as defined by any one of claims 7-8, for the obtention of destination vectors that differ in the combination and / or orientation of the n inserts, each one comprising a protein tag encoding sequence flanked by two different type IIP target sites, and(ii) substituting the protein tag from the destination vectors obtained from step (I) by another protein tag of interest that is the same for all destination vectors having the same combination and / or orientation of n inserts, and different between destination vectors having a different combination and / or orientation of n inserts.11- A library of vectors obtained by the method as defined by claim 10.12- A protein encoded by the nucleic acid vector defined by claim 11, wherein the nucleic acid sequence of the vector encoding the protein comprises the n inserts.13- A method for the synthesis of a protein comprising (I) performing the cloning method as defined in any one of claims 1-8, wherein the the n inserts comprise or consist of peptide encoding sequences, particularly wherein the cloning method is as defined in any one of claims 7-8, (ii) providing to a protein expression system the nucleic acid vector obtained from step (I), and (ill) subjecting the protein expression system to conditions appropriate for the expression of the protein.14- A method for the synthesis of a library of proteins, wherein the library of proteins comprises several proteins as defined in claim 12, particularly of proteins obtained by the method as defined by claim 13, wherein those proteins from the library that differ in the sequence, number and / or position of the protein sequences encoded by the n inserts, also differ in a protein tag sequence, and those proteins from the librarythat have the same sequence, number and / or position of the protein sequences encoded by the n inserts, have the same protein tag sequence, the method comprising:(i) performing the method for the synthesis of a library of different nucleic acid vectors as defined in claim 10,(ii) providing to a protein expression system the nucleic acid vectors of the library of vectors obtained from step (I), and(ill) subjecting the protein expression system to conditions appropriate for the expression of the proteins.15- A library of proteins obtained by the method as defined by claim 14.

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