Recombinant DNA molecules and molecular cloning methods that allow for assembly of multiple DNA fragments into a single contiguous stretch of DNA are essential tools for molecular biology, biotechnology and medical research. The first recombinant DNA mole-cule was made in the late 1960s, shortly after the discovery of restriction enzymes and DNA ligases. Since then, various methods have been developed to speed-up and facilitate the generation of recombinant DNA molecules.

Protein engineering is typically done by manipulating the underlying coding DNA se-quences. Directional assembly of DNA modules coding for different protein domains is central to the development and optimization of novel biotherapeutic formats.

Molecular cloning has progressed from the cloning of a single DNA fragment to the as-sembly of multiple DNA components into a single contiguous stretch of DNA. However, there is still a need for efficient technologies that allow for the generation of complex con-structs (see Endy, Nature 2005 Nov 24;438(7067):449-53). In particular, a set of standard and reliable engineering mechanisms is desired in order to remove much of the tedium and surprise during assembly of genetic components into larger systems (see Knight, T. F. (2003). Idempotent Vector Design for Standard Assembly of Biobricks. DOI: 1721.1/21168).

DNA modules (e.g. coding for protein domains) are typically assembled by cut-and-paste mechanisms using defined flanking prefix and suffix DNA sequences. Classically, prefix and suffix sequences are coding for palindromic type II restriction sites. Type II enzymes recognize and cleave DNA at the same site and create single-stranded overhangs which can be fused to other DNA modules which are cut by the same restriction enzyme. However, DNA modules of interest have to be 5’ and 3’ terminally equipped with suitable type II restriction sites by means of DNA manipulation techniques resulting in altered/mutated

primary nucleotide sequences. Further, linear, directional DNA module assembly requires several, unique type II sites. Assembly usually requires several cloning steps as different type II restriction enzymes are often not compatible with regard to reaction conditions.

Golden Gate cloning is a frequently used molecular cloning method that allows simultane-ous and directional assembly of multiple DNA fragments into a single piece using type IIs restriction enzymes and T4 DNA ligase. Engler 2008, A one pot, one step, precision clon-ing method with high throughput capability. PloS ONE 3.11: e3647, and Engler et al.

2009, Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS ONE 4: e5553. Unlike standard type II restriction enzymes such as EcoRI and BamHI, type IIs restriction enzymes such as Bsal, BsmBI and Bbsl, cut DNA outside of their recognition sequence and therefore can create non-palindromic overhangs. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site.

Type Ils-based restriction-ligation allows for assembly of many DNA fragments in a single cloning step. For assembly, DNA fragments require stretches of 1 to 6 bp long, e.g. 3-4 bp long, complementary sequences at their 5' (prefix) and 3' (suffix) ends flanked by type IIs enzyme recognition sites in a defined distance and orientation. Upon binding to recognition sites, type IIs enzymes cleave DNA fragments at prefix and suffix nucleotide sequences, thereby removing the actual recognition site, and at the same time generating free 5’ and 3’ ends consisting of the ligation sequences. These ligation sequences are then used to fuse together DNA fragments with matching/compatible ligation sequences in a ligation reac-tion. Consequently, if5’ prefix and 3’ suffix sequences are identical between two distinct DNA fragments, these fragments can be seamlessly ligated in type Ils-based restriction-ligation reactions.

In general, DNA fragments/modules that have been used to generate a particular variant library cannot be re-used (i.e. re-assembled) in the context of a new project as they often do not display compatible prefix and suffix sequences. Instead, modules need to be re-designed to match the different cloning strategies (i.e. that prefix and suffix sequences need to be adapted to allow for ligation). This is a very time-consuming and costly aspect, especially since frequently the 4bp prefix and suffix sequences of the DNA frag-ments/modules need to be modified while the prefix- and suffix-flanked core sequences stay unaltered.

Thus, a major limitation of the cloning strategies described in the art is that they still re-quire unique and compatible prefix and suffix sequences within the DNA modules to allow

for directional assembly. These prefix and suffix sequences may not be compatible be-tween different protein domains and/or formats which limits the universal applicability of these methods.

To date, no efficient method exists to generate generic DNA fragment modules that could be re-used and assembled in type Ils-based restriction-ligation reactions independent of their 5’ and 3’ prefix and suffix sequences.

Pre-mRNA splicing is an essential process in eukaryotic gene expression. In higher verte-brates, the length of target introns that need to be recognized range from <50 nt to >500.000 nt. In humans, introns with a length of about 90 nt to about 2000 nt are most commonly found within a pre-mRNA. However, also short or even ultra-short intron se-quences have been described. For example, short introns have been found in C. elegans (<40 nt), Arabidopsis thaliana (~20-59 nt) and in human tissue (<65 nt) (Hong et al. 2006, Intron size, abundance, and distribution within untranslated regions of genes. Molecular biology and evolution, 23(12), 2392-2404; Shimada et al., 2015, Identification and valida-tion of evolutionarily conserved unusually short pre-mRNA introns in the human genome. Int. J. Mol. Sci. 2015, 16, 10376-10388).

Introns located at the boundaries between introns and exons in a pre-mRNA are also re-ferred to as spliceosomal introns. RNA splicing removes the non-coding RNA introns leav-ing behind the exons, which are then spliced and joined together to form the final mRNA (“mature mRNA”).

There remains a strong need for the fast and easy generation of recombinant DNA mole-cules that allows for expression of multiple genes of interest. Especially means and meth-ods to generate generic DNA fragment modules that could be re-used and assembled in type Ils-based restriction-ligation reactions independent of their 5’ ends and 3’ ends, i.e. prefix and suffix sequences, would be highly desirable.

SUMMARY OF THE INVENTION

The technical problem underlying the present invention can be seen as the provision of means and methods for complying with the aforementioned needs. The technical problem is solved by the embodiments characterized in the claims and herein below.

Accordingly, in certain aspects, the present invention relates to a method for producing a fusion polynucleotide encoding a polypeptide of interest. In certain embodiments, said

method comprises contacting a first and a second polynucleotide with the type IIs re-striction endonuclease and a ligase under conditions that allow for cleavage of the first polynucleotide and the second polynucleotide by said type IIs restriction endonuclease and ligation of the resulting cleavage products, thereby generating the fusion polynucleotide encoding the polypeptide of interest. In certain embodiments, each of said first and said second polynucleotide comprises an intron sequence comprising a recognition and cleav-age site for a type IIs restriction endonuclease which upon cleavage generates complemen-tary ends which can be ligated to each other such that the fusion polynucleotide encoding the polypeptide of interest is produced. In certain embodiments, the first polynucleotide comprises the 5’ portion of an intron and the second polynucleotide comprises the 3’ por-tion of an intron.

In another aspect, the present invention further relates to a composition comprising a first, second and third polynucleotide. Further envisaged by the present invention is a polynu-cleotide encoding a polypeptide of interest, which, when transcribed in a eukaryotic host cell, is transcribed into a transcript comprising at least five introns which are heterologous to said polynucleotide.

In an embodiment of the aforementioned method, the first polynucleotide comprises, in 5’ to 3’ direction, the following elements:

(i) a nucleic acid sequence encoding a first portion of the polypeptide of interest,

(ii) a nucleic acid sequence encoding a 5’ portion of a first intron,

(iii) a first cleavage sequence for a type IIs restriction endonuclease, and

(iv) a recognition sequence for said type IIs restriction endonuclease, wherein the cleavage sequence in (iii) is operably linked to the recog- nition sequence in (iv).

In an embodiment of the aforementioned method, the second polynucleotide comprises, in 5’ to 3’ direction, the following elements:

(i) a recognition sequence for the type IIs restriction endonuclease,

(ii) a second cleavage sequence for the type IIs restriction endonuclease, wherein said second cleavage sequence is complementary to the first cleavage sequence, wherein the second cleavage sequence in (ii) is operably linked to the recognition sequence in (i), i.e. to the recogni- tion sequence of the second polynucleotide,

(iii) a nucleic acid sequence encoding a 3’ portion of the first intron, and (iv) a nucleic acid sequence encoding a second portion of the polypeptide of interest.

Thus, the present invention relates to a method for producing a fusion polynucleotide en-coding a polypeptide of interest, said method comprising the steps of:

(a1) providing a first polynucleotide, said first polynucleotide comprising, in 5’ to 3’ direction,

(i) a nucleic acid sequence encoding a first portion of the polypeptide of interest,

(ii) a nucleic acid sequence encoding a 5’ portion of a first intron,

(iii)a first cleavage sequence for a type IIs restriction endonuclease, and

(iv)a recognition sequence for said type IIs restriction endonuclease, wherein the cleavage sequence in (iii) is operably linked to the recog- nition sequence in (iv), i.e. to the recognition sequence of the first polynucleotide

(a2) providing a second polynucleotide, said second polynucleotide comprising, in 5’ to 3’ direction,

(i) a recognition sequence for the type IIs restriction endonuclease,

(ii) a second cleavage sequence for the type IIs restriction endonuclease, wherein said second cleavage sequence is complementary to the first cleavage sequence, wherein the second cleavage sequence in (ii) is operably linked to the recognition sequence in (i)i.e. to the recognition sequence of the second polynucleotide,

(iii) a nucleic acid sequence encoding a 3’ portion of the first intron, and

(iv) a nucleic acid sequence encoding a second portion of the polypeptide of interest, and

(b) contacting said first polynucleotide and second polynucleotide with the type IIs restriction endonuclease and a ligase under conditions that allow for cleavage of the first polynucleotide and the second polynucleotide by said type IIs restriction endonuclease and ligation of the resulting cleavage pro- ducts, thereby producing the fusion polynucleotide encoding the polypeptide of interest.

The produced polynucleotide comprises a first intron, i.e. shall encode the first intron. Said first intron shall be functional and shall comprise the nucleic acid sequence encoding the 5’ portion of the first intron and the nucleic acid sequence encoding the 3’ portion of the first intron.

Accordingly, the produced fusion polynucleotide shall comprise, in5’ to 3’ direction,

(aa) the nucleic acid sequence encoding the first portion of the polypeptide of in- terest,

(bb) a nucleic acid sequence encoding a first intron, wherein said first intron is functional, and wherein said first intron comprises the nucleic acid sequence encoding the 5’ portion of the first intron and the nucleic acid sequence en- coding the 3’ portion of the first intron, and

(cc) the nucleic acid sequence encoding the second portion of the polypeptide of interest.

The method of the present invention allows for the production of a fusion polynucleotide encoding a polypeptide of interest. Thus, the method is a cloning method. Such methods are typically carried out in vitro. The method of the present invention is not limited to the steps explicitly mentioned above and, thus, may comprise steps in addition to these steps. For example, further steps may relate to the ligation of additional polynucleotide sequences to the fusion polynucleotide of the present invention. E.g., as described herein below, the method of the present invention does not only allow for the production of a fusion polynu-cleotide comprising the elements (aa), (bb) and (cc) as set forth above, but also the produc-tion of a fusion polynucleotide comprising additional elements such as nucleic acid se-quences encoding a the third, fourth, fifth, sixth, seventh, eighth, ninth, tenth etc. portion of the polypeptide of interest. Further, additional polynucleotides can be used for the ligation. Such polynucleotides may encode the 5’ untranslated region (5’ UTR) and 3’ untranslated region (3’ UTR) of the polynucleotide encoding the polypeptide of interest. Since these sequences are transcribed, but not translated, it is possible to apply the intron-based clon-ing approach as described herein.

Moreover, further steps may relate to the cloning of the fusion polynucleotide into a vector such as an expression vector, the introduction of the fusion polynucleotide, or the vector comprising said fusion polynucleotide into a suitable host cell such as a mammalian cell, and/or the isolation, i.e. the purification, of the polypeptide from the host cell or the culture medium/supernatant. The purification process might be supported by the presence of suita-ble tags in the protein of interest, e.g. a His tag.

The term “polynucleotide” as used herein shall refer to a ribonucleic acid (RNA), or in particular to a desoxyribonucleic acid (DNA). Unless stated otherwise, the term “polynu-cleotide” herein refers to a single strand of a DNA polynucleotide or, in particular to a double-stranded DNA polynucleotide. Said double-stranded DNA shall during step (b) of the method of the present invention temporarily comprise one or two single-stranded over-hangs at the end(s). This is depending on the number of cleavage sequences, i.e. cleavage

sites, present in the polynucleotide: if one cleavage site is present, the polynucleotide com-prises one single-stranded overhang at one end, if two cleavage sequences are present, the polynucleotide comprises two single-stranded overhangs (one at each end). The overhangs result from cleavage with the type IIs endonuclease and allow for the ligation of fragments in a predetermined order (as described elsewhere herein).

The length of a polynucleotide is designated by the number of base pairs or nucleotides. Unless otherwise stated, both terms are used interchangeably, regardless whether or not the respective nucleic acid is a single- or double-stranded nucleic acid. Also, as polynucleo-tides are defined by their respective nucleotide sequence, the terms nucleo-tide/polynucleotide and nucleotide sequence/polynucleotide sequence are used inter-changeably.

The polynucleotide that shall be produced by the present invention shall be a fusion poly-nucleotide and thus shall be produced by the fusion of various polynucleotides. In particu-lar, said fusion polynucleotide shall be produced by contacting the first polynucleotide herein and second polynucleotide, and optionally, at least one further polynucleotide (such as a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth etc. polynucleotide) with a type IIs restriction endonuclease and a ligase under conditions that allow for the cleavage of the first polynucleotide and second polynucleotide (and, if present, of the at least one further polynucleotide, such as the third, fourth, fifth, sixth, seventh, eighth, ninth, tenth etc. poly-nucleotide) by said type IIs restriction endonuclease and the ligation of the resulting cleav-age products, thereby producing the fusion polynucleotide encoding the polypeptide of interest.

According to steps (a1) and (a2) of the method of the present invention, a first and second polynucleotide shall be provided. How to provide a polynucleotide is well known in the art. In an embodiment, the provided polynucleotides are derived from (i.e. produced by) polymerase chain reaction (PCR). In another embodiment, said polynucleotides are derived from (i.e. produced by) artificial gene synthesis. The same applies to the third, fourth, fifth, sixth, seventh, eighth, ninth, and tenth etc. polynucleotide as referred to herein elsewhere. In an embodiment, the provided polynucleotides are present in a vector, i.e. are comprised by a vector. In an alternative embodiment, the polynucleotides are provided as linear DNA fragments.

The first polynucleotide as referred to in step (a1) of the method of the present invention shall comprise, in 5’ to 3’ direction, the following elements

(i) a nucleic acid sequence encoding a first portion of the polypeptide of interest,

(ii) a nucleic acid sequence encoding a 5’ portion of a first intron,

(iii) a first cleavage sequence for a type IIs restriction endonuclease, and

(iv) a recognition sequence for said type IIs restriction endonuclease, wherein the first cleavage sequence in (iii) is operably linked to the recognition sequence in (iv).

The second polynucleotide as referred to in step (a1) of the method of the present invention shall comprise, in 5’ to 3’ direction, the following elements

(i) a recognition sequence for the type IIs restriction endonuclease

(ii) a second cleavage sequence for the type IIs restriction endonuclease, wherein said second cleavage sequence is complementary to first cleavage sequence, wherein the second cleavage sequence in (ii) is operably linked to the recognition sequence in (i), i.e. to the recogni- tion sequence of the second polynucleotide,

(iii) a nucleic acid sequence encoding a 3’ portion of the first intron, and

(iv) a nucleic acid sequence encoding a second portion of the polypeptide of interest.

The nucleic acid sequence as set forth under item (i) of step (a1) shall encode a first por-tion of the polypeptide of interest, and the nucleic acid sequence under item (iv) of step (a2) for a second portion of said polypeptide. Typically, a nucleic acid sequence encoding shall encode a portion of the polypeptide of interest as referred to herein in accordance with the present invention has a length of at least 10, at least 50, or at least 200 bp. Said nucleic acid sequence shall comprise a portion of the coding sequence. Thus, the portion of the polypeptide may have a length of at least 1 amino acid, at least about 3 amino acids, at least about 15 amino acids, or at least about 50 amino acids. Thus, the portion may have a length from a minimum 1 amino acid to any feasible length, e.g. 100 amino acids, 500 amino acids, 1000 amino acids, or more. In an embodiment of the method of the present invention, the portion comprises a protein domain. Thus, the portion may have a length of e.g. 100 to 150 amino acids.

Claims

1. A method for producing a fusion polynucleotide encoding a polypeptide of interest, said method comprising the steps of:

(a1) providing a first polynucleotide, said first polynucleotide comprising, in 5’ to 3’ direction,

(i) a nucleic acid sequence encoding a first portion of the polypeptide of interest,

(ii) a nucleic acid sequence encoding a 5’ portion of a first intron,

(iii) a first cleavage sequence for a type IIs restriction endonuclease, and

(iv) a recognition sequence for the type IIs restriction endonuclease, wherein the first cleavage sequence in (iii) is operably linked to said recognition sequence,

(a2) providing a second polynucleotide, said second polynucleotide comprising, in 5’ to 3’ direction,

(i) a recognition sequence for the type IIs restriction endonuclease,

(ii) a second cleavage sequence for the type IIs restriction endonuclease, wherein said second cleavage sequence is complementary to the first cleavage sequence, and wherein said second cleavage sequence is op- erably linked to the recognition sequence in (a2) (i),

(iii) a nucleic acid sequence encoding a 3’ portion of the first intron,

(iv) a nucleic acid sequence encoding a second portion of the polypeptide of interest,

(v) a nucleic acid sequence encoding a 5’ portion of a second intron,

(vi) a third cleavage sequence for the type IIs restriction endonuclease, which differs from the first cleavage sequence of the first polynucleo- tide, and

(vii) a recognition sequence for the type IIs restriction endonuclease, wherein said third cleavage sequence is operably linked to the recog- nition sequence in (a2)(vii), and

(a3) providing a third polynucleotide, said third polynucleotide comprising, in 5’ to 3’ direction,

(i) a recognition sequence for the type IIs restriction endonuclease,

(ii) a fourth cleavage sequence for the type IIs restriction endonuclease, wherein said fourth cleavage sequence is complementary to the third

cleavage sequence, wherein the fourth cleavage sequence is operably linked to the recognition sequence in (a3)(i),

(iii) a nucleic acid sequence encoding a 3’ portion of the second intron, and

(iv) a nucleic acid sequence encoding a third portion of the polypeptide of interest, and

(b) contacting said first, second and third polynucleotides with the type IIs re- striction endonuclease and a ligase under conditions that allow for cleavage of the first, second and third polynucleotide by said type IIs restriction endo- nuclease and ligation of the resulting cleavage products, thereby producing the fusion polynucleotide encoding the polypeptide of interest.

2. The method of claim 1, wherein said fusion polynucleotide comprises, in 5’ to 3’ direction:

(aa) the nucleic acid sequence encoding the first portion of the polypeptide of in- terest,

(bb) a nucleic acid sequence encoding the first intron, wherein said first intron is functional, and wherein said first intron comprises the nucleic acid sequence encoding the 5’ portion of the first intron and the nucleic acid sequence en- coding the 3’ portion of the first intron,

(cc) the nucleic acid sequence encoding the second portion of the polypeptide of interest,

(dd) a nucleic acid sequence encoding the second intron, wherein said second in- tron is functional, and wherein said second intron comprises the nucleic acid sequence encoding the 5’ portion of the second intron and the nucleic acid sequence encoding the 3’ portion of the second intron, and

(ee) the nucleic acid sequence encoding the third portion of the polypeptide of interest.

3. The method of claims 1 and 2, wherein said fusion polynucleotide, when transcribed in a eukaryotic host cell, is transcribed into a transcript that is processed in said cell so that each intron is spliced out of said transcript, thereby producing a mRNA en- coding the polypeptide of interest.

4. The method of any one of claims 1 to 3, wherein the first intron and/or the second intron is/are heterologous to the fusion polynucleotide.

5. The method of any one of claims 1 to 4, wherein the polynucleotide encoding the first intron and/or the polynucleotide encoding the second intron has a length of 40 to 2000 bp.

6. The method of any one of claims 1 to 5, wherein the polynucleotide encoding the first and/or second intron has a length of 50 to 200 bp, in particular of 50 to 150 bp.

7. The method of any one of claims to 1 to 6, wherein said first and/or second intron comprise(s) an internal stop codon in frame with the open reading frame of the fu- sion polynucleotide encoding the polypeptide of interest.

8. A method for producing a polypeptide of interest, comprising the steps of

(i) producing a fusion polynucleotide encoding the polypeptide of interest by the method according to any one of claims 1 to 7, and

(ii) expressing said fusion polynucleotide in a eukaryotic host cell, thereby pro- ducing said polypeptide of interest, and optionally

(iii)isolating the produced polypeptide of interest from said eukaryotic host cell.

9. A composition, comprising a first, second and third polynucleotide,

wherein said first polynucleotide comprises, in 5’ to 3’ direction:

(i) a nucleic acid sequence encoding a first portion of the polypeptide of interest,

(ii) a nucleic acid sequence encoding a 5’ portion of a first intron,

(iii) a first cleavage sequence for a type IIs restriction endonuclease , and

(iv) a recognition sequence for said type IIs restriction endonuclease, wherein the first cleavage sequence is operably linked to said recogni- tion sequence,

wherein said second polynucleotide comprises, in 5’ to 3’ direction:

(i) a recognition sequence for the type IIs restriction endonuclease,

(ii) a second cleavage sequence for the type IIs restriction endonuclease, wherein said second cleavage sequence is complementary to the first cleavage sequence, wherein the second cleavage sequence is operably linked to said recognition sequence (i) of the second polynucleotide,

(iii) a nucleic acid sequence encoding a 3’ portion of the first intron,

(iv) a nucleic acid sequence encoding a second portion of the polypeptide of interest,

(v) a nucleic acid sequence encoding a 5’ portion of a second intron,

(vi) a third cleavage sequence for the type IIs restriction endonuclease, which differs from the first cleavage sequence of the first polynucleo- tide, and

(vii) a recognition sequence for the type IIs restriction endonuclease, wherein the third cleavage sequence is operably linked to said recog- nition sequence,

and

wherein said third polynucleotide comprises, in 5’ to 3’ direction:

(i) a recognition sequence for the type IIs restriction endonuclease

(ii) a fourth cleavage sequence for the type IIs restriction endonuclease, wherein said cleavage sequence is complementary to the third cleav- age sequence, wherein the fourth cleavage sequence in (ii) is operably linked to the recognition sequence (i) of said third polynucleotide,

(iii)a nucleic acid sequence encoding a 3’ portion of the second intron, and

(iv)a nucleic acid sequence encoding a third portion of the polypeptide of interest.

10. The composition of claim 9, wherein said composition further comprises the type IIs restriction endonuclease and a ligase, in particular wherein said type IIs restriction endonuclease and said ligase allow for cleavage of the first polynucleotide, the sec- ond polynucleotide and the third polynucleotide and ligation of the resulting cleavage products, thereby generating the fusion polynucleotide encoding the polypeptide of interest.

11. A kit comprising a first, second and third polynucleotide as defined in claim 9, a type IIs restriction endonuclease that allows for cleavage of the first polynucleotide, the second polynucleotide and the third polynucleotide, and a ligase that allows for the ligation of cleavage products that result from the cleavage with said type IIs re- striction endonuclease.

12. The method of any one of claims 1 to 8, the composition of any one of claims 9 or 10, or the kit of claim 11, wherein said type IIs restriction endonuclease is selected from Acul, Alwl, Bael, Bbsl, Bbvl, Bccl, BceAI, Bcgl, BciVI, BcoDI, BfuAI, Bmrl, Bpml, BpuEI, Bsal, BsaXI, BseRI, Bsgl, BsmAI, BsmBI, BsmFI, Bsml, BspCNI, BspMI, BspQI, BsrDI, Bsrl, BtgZI, BtsCI, Btsl, BtsIMutI, CspCI, Earl, Ecil, Faul, Fokl, Hgal, Hphl, HpyAV, Mboll, Mlyl, Mmel, Mnll, NmeAIII, Plel, Sapl, and SfaNI.

13. A polynucleotide encoding a polypeptide of interest, which, when transcribed in a eukaryotic host cell, is transcribed into a transcript comprising at least five introns which are heterologous to said polynucleotide.

14. The polynucleotide of claim 13, wherein each of said introns preferably has a length of 50 to 200 nt, more preferably a length of 50 to 150 nt, and most preferably a length of 50 to 100 nt.

15. The polynucleotide of claim 13, wherein each of said introns preferably has a length of 80 to 200 nt, such as a length of 90 to 150 nt or 90 to 120 nt.

16. The polynucleotide according to any one of claims 13 to 17, wherein all introns comprise an internal stop codon in frame with the open reading frame of the nucleic acid sequence encoding the polypeptide of interest.

17. Use of a composition comprising a ligase and a type IIs restriction endonuclease for producing a fusion polynucleotide encoding a polypeptide of interest by cleavage of

(a1) the first polynucleotide as defined in claim 1,

(a2) the second polynucleotide as defined in claim 1, and

(a3) the third polynucleotide as defined in claim 1,

with said endonuclease, and ligation of the resulting cleavage products.