Compositions of Polynucleotide Vectors and Their Use

Abstract

Disclosed herein are compositions of destination and entry vectors. Also disclosed herein are uses of the destination and entry vectors in methylation-disrupted assembly reactions, where the assembled sequence can be used as an entry vector in a subsequent assembly reaction.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62 / 790,343, filed January 9, 2019, the entire contents of which are incorporated herein by reference.

[0002] Field Disclosed herein are compositions of destination and entry vectors. Also disclosed herein are uses of the destination and entry vectors in methylation-disrupted assembly reactions, where the assembled sequence can be used as an entry vector in a subsequent assembly reaction. [Background technology]

[0003] background Various cloning strategies have been described. Traditionally, restriction ligation cloning was used to insert a nucleic acid fragment of interest into a vector. Due to limitations of this traditional approach—such as the introduction of restriction enzyme cleavage sites, "scars," restriction enzyme selection, and cloning inefficiency—other approaches have been developed to clone larger polynucleic acids in a hierarchical fashion. Examples include MoClo (Addgene) and Golden Gate (NEB) type IIS hierarchical cloning strategies and Gateway Cloning (Thermo) recombination-based hierarchical cloning strategies. Summary of the Invention

[0004] overview In some aspects, the present disclosure relates to a destination vector. In some embodiments, the polynucleic acid destination vector includes a backbone component and an insertion site component, wherein: (a) the backbone component includes a nucleic acid sequence of a selectable marker, an origin of replication, and at least one type IIS restriction site including a consensus recognition site and a corresponding cleavage site, wherein the consensus recognition site overlaps with a methylation site; and (b) the insertion site component includes a 5' type IIS dual restriction site and a 3' type IIS dual restriction site, optionally, wherein the 5' and 3' type IIS dual restriction sites are separated by at least one nucleotide; wherein each type IIS dual restriction site includes: (i) a first consensus recognition site and a corresponding cleavage site, wherein the first consensus recognition site overlaps with a methylation site that forms a boundary between the insertion site component and the backbone component, and (ii) a second consensus recognition site and a corresponding cleavage site. a cleavage site corresponding to the first consensus recognition site, wherein the second recognition site lacks an overlapping methylation site, and wherein the cleavage site corresponding to the first consensus recognition site and the cleavage site corresponding to the second consensus recognition site are both located between the first consensus recognition site and the second consensus recognition site, wherein: methylation of the destination vector at the methylation site-overlapping consensus recognition site blocks cleavage of the cleavage site corresponding to that consensus recognition site; when methylated, exposure of the destination vector to a Type IIS restriction enzyme that recognizes the consensus recognition site of the destination vector generates two polynucleic acid fragments, wherein the terminal 5' or 3' nucleic acid overhangs of the fragments, including the backbone components, differ in nucleotide sequence; and the Type IIS cleavage site in (a) differs in nucleotide sequence from the Type IIS cleavage site in (b).

[0005] In some embodiments, the cleavage sites corresponding to the first consensus recognition site and the second consensus recognition site of the 5'-type IIS dual restriction site or the 3'-type IIS dual restriction site are separated from each other by at least one nucleotide. In some embodiments, the cleavage sites corresponding to the first consensus recognition site and the second consensus recognition site of both the 5'-type IIS dual restriction site and the 3'-type IIS dual restriction site are separated from each other by at least one nucleotide.

[0006] In some embodiments, the cleavage sites corresponding to the first and second consensus recognition sites of the 5' type IIS double restriction site and the cleavage sites corresponding to the first and second consensus recognition sites of the 3' type IIS double restriction site are separated from each other by different nucleotide sequences. In some embodiments, the different nucleotide sequences comprise different nucleic acid lengths.

[0007] In some embodiments, the cleavage sites corresponding to the first and second consensus recognition sites of the 5' type IIS dual restriction site, and the cleavage sites corresponding to the first and second consensus recognition sites of the 3' type IIS dual restriction site, are separated from each other by identical nucleotide sequences. In some embodiments, the cleavage site corresponding to the first consensus recognition site and the cleavage site corresponding to the second consensus recognition site of the 5' type IIS dual restriction site and / or the 3' type IIS dual restriction site are shared cleavage sites. In some embodiments, the 5' type IIS double restriction site and the 3' type IIS double restriction site are separated by a nucleic acid sequence encoding a visual readout or a suicide cassette. In some embodiments, the visual readout is selected from the group consisting of a fluorescent protein, a chromogenic protein, LacZ, or LacZα. In some embodiments, the suicide cassette comprises the nucleic acid sequence of ccdB.

[0008] In some embodiments, the IIS-type restriction enzyme that binds to the common recognition site is selected from the group consisting of BsaI, BsmBI, BtgZI, Esp3I, FokI, HphI, BcgI, AlwI, MboII, MmeI, BsmFI, BceAI, BcoDI, BfuAI, BsmAI, EarI, EciI, FauI, HgaI, HpyAV, PleI, BbsI, SapI, and SfaNI. In some embodiments, the IIS-type restriction enzyme is a high-fidelity restriction enzyme. In some embodiments, at least one IIS-type restriction site in the backbone component of (a) is located within or adjacent to a selectable marker or an origin of replication. In some embodiments, the cleavage site of at least one IIS-type restriction site in the backbone component of (a) contains a low ligation efficiency sequence content. In some embodiments, the selectable marker contains an antibiotic resistance gene. In some embodiments, the methylation sites of (a) and (b) are methylated by the same methyltransferase.

[0009] In some embodiments, the methyltransferase is selected from the group consisting of CpG methyltransferase (optionally M.SssI), dam methyltransferase, dcm methyltransferase, GpC methyltransferase (optionally M.CviPI), AluI methyltransferase, BamHI methyltransferase, EcoRI methyltransferase, HaeIII methyltransferase, HhaI methyltransferase, HpaII methyltransferase, MspI methyltransferase, and TaqI methyltransferase. In some embodiments, when unmethylated, exposure of the destination vector to a type IIS restriction enzyme that recognizes the common recognition site of the destination vector generates at least three polynucleotide fragments, where each polynucleotide comprises a terminal 5' or 3' nucleic acid overhang, and where the terminal 5' or 3' nucleic acid overhangs of the fragments that comprise the second common recognition sequence of both the 5' type IIS double restriction site and the 3' type IIS double restriction site have different nucleotide sequences.

[0010] In other aspects, the disclosure relates to an entry vector. In some embodiments, the polynucleotide entry vector comprises a backbone component and an insert component, where (a) the backbone component comprises the backbone component of the polynucleotide destination vector disclosed herein; and (b) the insert component comprises, from 5' to 3', a first type IIS restriction site, an insert, and a second type IIS restriction site; where the first and second type IIS restriction sites each comprise: (i) a common recognition site with overlapping methylation sites, where the methylation sites form a boundary between the insert component and the backbone component, and (ii) corresponding cleavage sites, where cleavage of the cleavage sites of the first and second type IIS restriction sites generates a 5' or 3' overhang, where the 5' or 3' overhang of the first type IIS restriction site and the 5' or 3' overhang of the second type IIS restriction site have different nucleotide sequences. In some embodiments, the insert is a nucleic acid sequence that is combined in an assembly reaction.

[0011] In yet another aspect, the present disclosure relates to a method of assembling polynucleic acids into a predetermined sequence. In some embodiments, the method comprises: (a) forming a reaction mixture by combining: (i) a destination vector disclosed herein, wherein the methylation sites of both the backbone component and the insertion site component of the destination vector are methylated; (ii) at least one entry vector disclosed herein, wherein the methylation sites of the respective backbone component and insertion component of the at least one entry vector are not methylated; (iii) a type IIS restriction enzyme, wherein the type IIS restriction enzyme recognizes a common recognition site of the destination vector and the entry vector; and (iv) a ligase; (b) incubating the reaction mixture for a time sufficient for cleavage of the destination vector and the at least one entry vector via the type IIS restriction enzyme; and (c) incubating the reaction mixture for a time sufficient for the ligase to ligate each insert of the at least one entry vector to the backbone component of the destination vector, thereby generating a circular polynucleic acid; and wherein the 5' or 3' overhangs of the backbone component of the destination vector and the respective insertion components of the at least one entry vector uniquely complement each other to form a predetermined sequence comprising the backbone component of the destination vector of step (a)(i) and the respective insertion components of the at least one entry vector of step (a)(ii).

[0012] In some embodiments, the destination vector is methylated in vitro. In some embodiments, the destination vector is methylated in vivo. In some embodiments, the destination vector is methylated in a bacterial strain that expresses a methyltransferase selected from the group consisting of CpG methyltransferase (optionally M.SssI), dam methyltransferase, dcm methyltransferase, GpC methyltransferase (optionally M.CviPI), AluI methyltransferase, BamHI methyltransferase, EcoRI methyltransferase, HaeIII methyltransferase, HhaI methyltransferase, HpaII methyltransferase, MspI methyltransferase, and TaqI methyltransferase. In some embodiments, the method further comprises isolating the ligated destination vector containing the insert from the other components of the reaction mixture. In some embodiments, the ligated destination vector is isolated by transforming bacteria with the reaction mixture and screening the bacteria for the presence of correctly ligated assemblies.

[0013] In some embodiments, the method further comprises demethylating the isolated ligated destination vector to produce a second entry vector. In some embodiments, the isolated ligated destination vector is passively demethylated via replication in a bacterial strain lacking a methyltransferase selected from the group consisting of CpG methyltransferase (optionally M.SssI), dam methyltransferase, dcm methyltransferase, GpC methyltransferase (optionally M.CviPI), AluI methyltransferase, BamHI methyltransferase, EcoRI methyltransferase, HaeIII methyltransferase, HhaI methyltransferase, HpaII methyltransferase, MspI methyltransferase, and TaqI methyltransferase.

[0014] In another aspect, the present disclosure relates to a method of cloning a nucleic acid sequence of interest. In some embodiments, the method comprises: (a) forming a reaction mixture by combining: (i) a destination vector disclosed herein, wherein the methylation sites of the backbone component are methylated; (ii) at least one polynucleic acid fragment, wherein each polynucleic acid fragment comprises an internal sequence flanked at both ends by a common recognition site and a corresponding cleavage site; (iii) a type IIS restriction enzyme, wherein the type IIS restriction enzyme recognizes the common recognition site of the destination vector and the at least one polynucleic acid fragment; and (iv) a ligase; (b) incubating the reaction mixture for a time sufficient for cleavage of the destination vector and the at least one polynucleic acid fragment via the type IIS restriction enzyme; and (c) incubating the reaction mixture for a time sufficient for the ligase to ligate each internal nucleic acid sequence of the at least one polynucleic acid fragment to the backbone component of the destination vector, thereby generating a circular polynucleic acid; and wherein the internal sequence of the at least one polynucleic acid fragment comprises the nucleic acid sequence of interest; and wherein the 5' or 3' overhangs of the backbone component of the destination vector and each internal sequence of the at least one polynucleic acid fragment uniquely complement each other to form a predetermined sequence comprising the backbone component of the destination vector of step (a)(i) and the nucleic acid sequence of interest.

[0015] In some embodiments, the destination vector is methylated in vitro. In some embodiments, the destination vector is methylated in vivo. In some embodiments, the destination vector is methylated in a bacterial strain that expresses a methyltransferase selected from the group consisting of CpG methyltransferase (optionally M.SssI), dam methyltransferase, dcm methyltransferase, GpC methyltransferase (optionally M.CviPI), AluI methyltransferase, BamHI methyltransferase, EcoRI methyltransferase, HaeIII methyltransferase, HhaI methyltransferase, HpaII methyltransferase, MspI methyltransferase, and TaqI methyltransferase. In some embodiments, the method further comprises isolating the ligated destination vector containing the insert from the other components of the reaction mixture. In some embodiments, the ligated destination vector is isolated by transforming bacteria with the reaction mixture and screening the bacteria for the presence of correctly ligated assemblies.

[0016] In some embodiments, the method further comprises demethylating the isolated ligated destination vector to generate a second entry vector. In some embodiments, the isolated ligated destination vector is passively demethylated in vivo via replication in a bacterial strain lacking a methyltransferase selected from the group consisting of CpG methyltransferase (optionally M.SssI), dam methyltransferase, dcm methyltransferase, GpC methyltransferase (optionally M.CviPI), AluI methyltransferase, BamHI methyltransferase, EcoRI methyltransferase, HaeIII methyltransferase, HhaI methyltransferase, HpaII methyltransferase, MspI methyltransferase, and TaqI methyltransferase.

[0017] In other embodiments, the method comprises: (a) forming a reaction mixture by combining: (i) at least one entry vector disclosed herein, wherein the methylation sites of the backbone components are not methylated; (ii) a polynucleotide fragment, wherein the polynucleotide fragment comprises an internal sequence flanked by common recognition sites and corresponding cleavage sites at both ends; (iii) a type IIS restriction enzyme, wherein the type IIS restriction enzyme recognizes the common recognition sites of at least one entry vector and the polynucleotide fragment; and (iv) a ligase; (b) incubating the reaction mixture for a time sufficient for cleavage of at least one entry vector and the polynucleotide fragment via the type IIS restriction enzyme; and (c) incubating the reaction mixture for a time sufficient for the ligase to ligate the internal nucleic acid sequence of the polynucleotide fragment to each insertion component of at least one entry vector, thereby generating a circular polynucleotide; and wherein the internal sequence of the polynucleotide fragment comprises a selectable marker and an origin of replication; and wherein the 5' or 3' overhangs of each insertion component of at least one entry vector and the internal sequence of at least one polynucleotide fragment are uniquely complementary to each other to form a predetermined sequence comprising the nucleic acid sequence of interest.

[0018] In some embodiments, the polynucleotide fragment is a PCR product. In some embodiments, the polynucleotide fragment is methylated in vitro. In some embodiments, the predetermined sequence further comprises the sequence of the entry vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The following drawings form a part of this specification and are included to further demonstrate certain embodiments of the present disclosure, which can be better understood by reference in combination with the detailed description of the specific embodiments presented herein with one or more of these drawings. It should be understood that the data shown in the drawings are in no way intended to limit the scope of the disclosure.

[0020]

Figure 1

Figure 2

Figure 3

[0021] Detailed Description Disclosed herein are compositions of a destination vector and an entry vector, as well as a composition of a kit for assembling a polynucleotide having a predetermined sequence. Further disclosed herein is a method for assembling a polynucleotide having a predetermined sequence, which includes a methylation-inhibited assembly reaction, wherein the assembled predetermined sequence can be used as an entry vector for a subsequent assembly reaction. These compositions and methods are configured based on the hierarchical cloning strategy described above. In particular, the disclosed compositions and methods can eliminate the need to switch restriction enzymes and / or selection mechanisms during the cloning steps. Further, the disclosed method may show a reduction in background because the backbone of the donor vector can be degraded during the cloning process.

[0022] Destination Vector and Compositions In some aspects, the present disclosure relates to destination vector polynucleic acids and compositions comprising destination vector polynucleic acids. In some embodiments, the destination vector is a linear vector. In other embodiments, the destination vector is a circular vector. The destination vector comprises a backbone component and an insertion site component. As used herein, the term "backbone component" refers to the portion of the destination vector (or the portion of the entry vector adjacent to the insertion component, see "Entry Vectors and Compositions" below) adjacent to the insertion component and includes at least an origin of replication. In some embodiments, the backbone component further comprises a selectable marker gene.

[0023] In some embodiments, the selectable marker comprises a visible reporter gene such as a fluorescent protein or a chromogenic protein. Examples of fluorescent proteins are known to those of skill in the art and include, without limitation: TagBFP, mTagBFP2, Azurite, EBFP2, mKalama1, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFP1, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, mNeonGreen, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira-Orange, mKOκ, mKO2, mOrange, mOrange2, mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mRuby2, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, and iRFP. Examples of chromogenic proteins are known to those of skill in the art. See, for example, U.S. Patent No. 9,771,402, which describes various chromogenic proteins. In some embodiments, the selectable marker comprises an auxotrophic complementation gene. In some embodiments, the selectable marker comprises an antibiotic resistance gene. Examples of selectable markers are known to those of skill in the art and include, without limitation: AmpR, NeoR, mFabI, ZeoR, NAT, HygR, SpcR (AadA), Pac, Ura3, His3, Leu2, and Trp1. In some embodiments, the backbone component further comprises at least one type IIS restriction site.

[0024] As used herein, the term "restriction site" refers to a polynucleotide sequence that includes a recognition site and a corresponding cleavage site. As used herein, the term "recognition site" refers to a polynucleotide sequence that is recognized and specifically bound by a type IIS restriction enzyme. The "corresponding cleavage site" refers to the site that is cleaved when the type IIS restriction enzyme binds to the recognition site. In some embodiments, the cleavage site is only 2 nucleotides in length and cleavage occurs between the two nucleotides (corresponding to a blunt-end cleavage site). In some embodiments, the cleavage site is at least 3 nucleotides in length (corresponding to a 5' or 3' overhang site that includes at least 1 nucleotide of single-stranded overhang), at least 4 nucleotides in length (corresponding to a 5' or 3' overhang site that includes at least 2 nucleotides of single-stranded overhang), at least 5 nucleotides in length (corresponding to a 5' or 3' overhang site that includes at least 3 nucleotides of single-stranded overhang), at least 6 nucleotides in length (corresponding to a 5' or 3' overhang site that includes at least 4 nucleotides of single-stranded overhang), at least 7 nucleotides in length (corresponding to a 5' or 3' overhang site that includes at least 5 nucleotides of single-stranded overhang), at least 8 nucleotides in length (corresponding to a 5' or 3' overhang site that includes at least 6 nucleotides of single-stranded overhang), or at least 9 nucleotides in length (corresponding to a 5' or 3' overhang site that includes at least 7 nucleotides of single-stranded overhang). In some embodiments, cleavage of the cleavage site generates a single-stranded overhang. The length of the overhang can vary (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 nucleotides in length). In some embodiments, the overhang is a 5' overhang. In other embodiments, the overhang is a 3' overhang.

[0025] Type IIS restriction enzymes recognize asymmetric DNA sequences and cleave outside of their recognition sequences. Examples of known Type IIS restriction enzymes include, but are not limited to: AcuI, AlwI, BaeI, BbsI, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, BmrI, BpmI, BpuEI, BsaI, BsaXI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, BsmI, BspCNI, BspMI, BspQI, BsrDI, BsrI, BtgZI, BtsCI, BtsI, BtsIMutI, CspCI, EarI, EciI, Esp3I, FauI, FokI, HgaI, HphI, HpyAV, MboII, MlyI, MmeI, MnII, NmeAIII, PleI, SapI, and SfaNI. In some embodiments, the Type IIS restriction enzyme is a high-fidelity restriction enzyme.

[0026] Some Type IIS restriction enzymes are methylation-sensitive and include, but are not limited to: AlwI (dam methylation-sensitive), BceAI (CpG methylation-sensitive), BcgI (dam and CpG methylation-sensitive), BcoDI (CpG methylation-sensitive), BfuAI (CpG methylation-sensitive), BsaI (dcm and CpG methylation-sensitive), BsmAI (CpG methylation-sensitive), BsmBI (CpG methylation-sensitive), BsmFI (dcm and CpG methylation-sensitive), BtgZI (CpG methylation-sensitive), EarI (CpG methylation-sensitive), EciI (CpG methylation-sensitive), Esp3I (CpG methylation-sensitive), FauI (CpG methylation-sensitive), FokI (dcm and CpG methylation-sensitive), HgaI (CpG methylation-sensitive), HphI (dam and dcm methylation-sensitive), HpyAV (CpG methylation-sensitive), MboII (dam methylation-sensitive), MmeI (CpG methylation-sensitive), SapI (CpG methylation-sensitive), PleI (CpG methylation-sensitive), and SfaNI (CpG methylation-sensitive).

[0027] In some embodiments, the recognition sites are common recognition sites. Each “common recognition site” of a destination vector (or an entry vector as described in “Entry Vectors and Compositions” below) comprises (i) consists of the same nucleic acid sequence and / or (ii) comprises a nucleic acid sequence recognized and / or bound by the same type IIS restriction enzyme (some type IIS restriction enzymes recognize diverse sequences; for example, MmeI recognizes the sequence 5′-TCCRAC-3′, where R represents A or G). In some embodiments, at least one common recognition site of the backbone component is an overlapping methylation site (i.e., a sequence recognized and methylated by a methyltransferase enzyme, such as, for example, CpG methyltransferase (optionally M.SssI), dam methyltransferase, dcm methyltransferase, GpC methyltransferase (optionally M.CviPI), AluI methyltransferase, BamHI methyltransferase, EcoRI methyltransferase, HaeIII methyltransferase, HhaI methyltransferase, HpaII methyltransferase, MspI methyltransferase, and TaqI methyltransferase). In some embodiments, the methylation site comprises a dcm, dam, and / or CpG methylation site.

[0028] In some embodiments, the Type IIS restriction enzyme that binds to a consensus recognition site is selected from the group consisting of AcuI, AlwI, BaeI, BbsI, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, BmrI, BpmI, BpuEI, BsaI, BsaXI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, BsmI, BspCNI, BspMI, BspQI, BsrDI, BsrI, BtgZI, BtsCI, BtsI, BtsIMutI, CspCI, EarI, EciI, Esp3I, FauI, FokI, HgaI, HphI, HpyAV, MboII, MlyI, MmeI, MnII, NmeAIII, PleI, SapI, and SfaNI. In some embodiments, the type IIS restriction enzyme that binds to the consensus recognition site is a methylation-sensitive type IIS restriction enzyme selected from the group consisting of AlwI, BceAI, BcgI, BcoDI, BfuAI, BsaI, BsmAI, BsmBI, BsmFI, BtgZI, EarI, EciI, Esp3I, FauI, FokI, HgaI, HphI, HpyAV, MboII, MmeI, PleI, SapI, and SfaNI. Methylation-sensitive restriction enzymes do not cleave cleavage sites corresponding to methylated recognition sites. In some embodiments, at least one Type IIS restriction site is located within or adjacent to the selectable marker and / or origin of replication of the backbone component.

[0029] In some embodiments, the scaffold component comprises 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, or more than 10 Type IIS restriction sites. In some embodiments, at least two of the Type IIS restriction sites of the scaffold component comprise a consensus recognition site. In some embodiments, each of the at least two consensus recognition sites of the scaffold component is adjacent to a methylation site. In some embodiments, the cleavage site of at least one IIS-type restriction site in the backbone component (e.g., the cleavage site corresponding to the consensus recognition site) contains a sequence content with low ligation efficiency. For example, in some embodiments, the sequence with low ligation efficiency includes blunt-end cleavage sites. In other embodiments, the sequence with low ligation efficiency includes overhangs of two or fewer nucleotides. Additional examples of low-efficiency ligation sequences are known in the art and include, but are not limited to, TNNA, TTTT, and AAAA. See, for example: Potapov V. et al., A single-molecule sequencing assay for the comprehensive profiling of T4 DNA ligase fidelity and bias during DNA end-joining. Nucleic Acids Res. 2018 Jul 27; 46(13):e79; Vladimir P. et al., Optimization of Golden Gate assembly through application of ligation sequence-dependent fidelity and bias profiling. BioRxiv. 2018 May 15; doi: 10.1101 / 322297; the entireties of which are incorporated herein by reference.

[0030] As used herein, the term "insertion site component" refers to a polynucleotide comprising a 5'IIS-type double restriction site and a 3'IIS-type double restriction site (see, e.g., Figure 1). As used herein, the term "double restriction site" refers to a nucleic acid sequence comprising a pair of inward-facing IIS-type restriction sites (i.e., the cleavage sites corresponding to each - first and second - recognition site are both located between the two recognition sites). In some embodiments, the nucleic acid sequence of the 3'IIS-type double restriction site is the reverse complement of the 5'IIS-type double restriction site, excluding the nucleic acid sequences of one or more cleavage sites. For example, in some embodiments, the nucleic acid sequence of the 5'IIS-type double restriction site is CCGGTCTCNNNNNNGAGACC (SEQ ID NO: 1), the nucleic acid sequence of the 3'IIS-type double restriction site is GGTCTCNNNNNNGAGACCGG (SEQ ID NO: 2), and N represents A, T, G, or C.

[0031] In some embodiments, the first and / or second recognition sites of the double restriction site are common recognition sites. In some embodiments, the common recognition site of the double restriction site overlaps with a methylation site (i.e., a sequence recognized and methylated by a methyltransferase enzyme). In some embodiments, the methylation site includes dcm, dam, and / or CpG methylation sites. In some embodiments, the double restriction site includes: (i) a first recognition site and a corresponding cleavage site, where the first recognition site overlaps with a methylation site that forms a boundary between the insertion site component and the backbone component, and (ii) a second recognition site and a corresponding cleavage site, where the second recognition site lacks the overlapping methylation site.

[0032] In some embodiments, the cleavage site corresponding to the first recognition site of the double restriction site and the cleavage site corresponding to the second recognition site are separated from each other by at least 1, at least 2, 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 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides. In some embodiments, the cleavage site corresponding to the first common recognition site and the cleavage site corresponding to the second common recognition site of the 5'IIS type double restriction site or the 3'IIS type double restriction site are separated from each other by at least one nucleotide. In some embodiments, the cleavage site corresponding to the first common recognition site and the cleavage site corresponding to the second common recognition site of both the 5'IIS type double restriction site and the 3'IIS type double restriction site are separated from each other by at least one nucleotide.

[0033] In some embodiments, the cleavage site corresponding to the first common recognition site and the cleavage site corresponding to the second common recognition site of the 5'IIS type double restriction site, and the cleavage site corresponding to the first common recognition site and the cleavage site corresponding to the second common recognition site of the 3'IIS type double restriction site are separated from each other by the same nucleotide sequence (i.e., the same nucleotide sequence). In some embodiments, the cleavage site corresponding to the first common recognition site and the cleavage site corresponding to the second common recognition site of the 5'IIS type double restriction site, and the cleavage site corresponding to the first common recognition site and the cleavage site corresponding to the second common recognition site of the 3'IIS type double restriction site are separated from each other by different nucleotide sequences. The different nucleotide sequences can differ in the identity of one or more nucleotides. In some embodiments, the different nucleotide sequences are of the same length. In other embodiments, the different nucleotide sequences are of different lengths.

[0034] In some embodiments, the cleavage site corresponding to the first recognition site and the cleavage site corresponding to the second recognition site of a dual restriction site comprise a shared cleavage site (i.e., a Type IIS restriction enzyme that binds to the first recognition site cleaves the same cleavage site as a Type IIS restriction enzyme that binds to the second recognition site). In some embodiments, the cleavage site corresponding to the first consensus recognition site and the cleavage site corresponding to the second consensus recognition site of a 5'-type IIS dual restriction site or a 3'-type IIS dual restriction site comprise a shared cleavage site. In some embodiments, the cleavage site corresponding to the first consensus recognition site and the cleavage site corresponding to the second consensus recognition site of both a 5'-type IIS dual restriction site and a 3'-type IIS dual restriction site comprise a shared cleavage site.

[0035] In some embodiments, the cleavage site corresponding to the first recognition site and the cleavage site corresponding to the second recognition site are identical (even if the cleavage sites are not shared). In some embodiments, the cleavage site corresponding to the first recognition site and the cleavage site corresponding to the second recognition site differ in nucleotide sequence. In some embodiments, the cleavage site corresponding to the first recognition site and the cleavage site corresponding to the second recognition site differ in nucleotide length. In some embodiments, the cleavage site corresponding to the first recognition site and / or the second recognition site of the 5' type IIS double restriction site and the first recognition site and / or the second recognition site of the 3' type IIS restriction site differ in length.

[0036] In some embodiments, the 5'IIS-type double restriction site and the 3'IIS-type double restriction site of the insertion site component are separated by at least 1, at least 2, 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 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, or at least 5000 nucleotides. In some embodiments, the nucleic acid sequence separating the 5'IIS-type double restriction site and the 3'IIS-type double restriction site comprises a counter-selection marker (e.g., sacB, rpsL (strA), tatAR, pheS, thyA, lacY, lacZ, gata-1, ccdB, galK, or ePheSA294G). In some embodiments, the counter-selection marker is a visual readout or a suicide cassette (i.e., a lethal counterselector). In some embodiments, the visual readout is selected from the group consisting of a fluorescent protein or LacZ. In some embodiments, the suicide cassette comprises the nucleic acid sequence of ccdB.

[0037] In some embodiments, at least one IIS-type restriction site in the backbone component has a nucleotide sequence different from at least one IIS-type restriction site in the insertion component. In some embodiments, at least one cleavage site corresponding to the common recognition site of the backbone component has a nucleotide sequence different from at least one cleavage site corresponding to the common recognition site of the insertion component. In some embodiments, each cleavage site corresponding to the common recognition site in the backbone component has a nucleotide sequence different from each cleavage site corresponding to the common recognition site in the insertion component. In some embodiments, each cleavage site corresponding to the common recognition site in the destination vector is unique.

[0038] In some embodiments, the methylation sites of each common recognition site with overlapping methylation sites in the destination vector are the same (i.e., each methylation site with overlapping common recognition sites in the destination vector can be methylated by the same methyltransferase enzyme). In some embodiments, the methylation sites of at least two common recognition sites with overlapping methylation sites in the destination vector are unique (i.e., at least two methylation sites with overlapping common recognition sites in the destination vector are methylated by different methyltransferase enzymes). Examples of methyltransferase enzymes include, but are not limited to: CpG methyltransferase (optionally M.SssI), dam methyltransferase, dcm methyltransferase, GpC methyltransferase (optionally M.CviPI), AluI methyltransferase, BamHI methyltransferase, EcoRI methyltransferase, HaeIII methyltransferase, HhaI methyltransferase, HpaII methyltransferase, MspI methyltransferase, and TaqI methyltransferase.

[0039] Figure 1 provides a schematic diagram showing one aspect of a destination vector. In this aspect, the 5'IIS type double restriction site and the 3'IIS type double restriction site each include a first common recognition site and a second common recognition site where the methylation sites overlap. In this case, the common recognition site is that of BsaI. The BsaI restriction site includes a recognition site of 5'-GGTCTC-3' and a cleavage site of (N1) / (N5) (i.e., 5'-NNNNNN-3', where N represents A, T, G, or C, and generates a 4-base pair 5' overhang upon cleavage). In this case, the cleavage sites of the first common recognition site and the second common recognition site are shared cleavage sites. Those skilled in the art will recognize that the nucleic acid sequences of the shared cleavage sites of the 5'IIS type double restriction site and the 3'IIS type double restriction site can be different from each other. Those skilled in the art will also understand that the BsaI common recognition site (and its corresponding cleavage site) can be replaced with any other IIS type restriction enzyme recognition site. Furthermore, those skilled in the art will understand that the cleavage sites of the first common recognition site and the second common recognition site of the 5'IIS type double restriction site and / or the 3'IIS type double restriction site do not necessarily have to be shared cleavage sites. Furthermore, this aspect depicts an MspI methylation site where the BsaI recognition site overlaps. Those skilled in the art will understand that the MspI methylation site can be replaced with any other methylation site known in the art.

[0040] In some aspects, when methylated, exposure of the destination vector to an IIS type restriction enzyme that recognizes the common recognition site of the destination vector generates at least two polynucleotide fragments, where the 5' or 3' nucleic acid overhangs of the fragments containing the backbone component have different nucleotide sequences. In some embodiments, when not methylated, exposure of the destination vector to an IIS-type restriction enzyme that recognizes a common recognition site of the destination vector generates at least three polynucleotide fragments, where each polynucleotide contains a terminal 5' or 3' nucleic acid overhang, and the terminal 5' or 3' nucleic acid overhangs of the fragments containing both the 5' IIS-type double restriction site and the second common recognition sequence of the 3' IIS-type double restriction site have different nucleotide sequences.

[0041] In some embodiments, a polynucleotide destination vector comprising a backbone component and an insertion site component, wherein: (a) the backbone component comprises a nucleic acid sequence of a selectable marker, an origin of replication, and at least one IIS-type restriction site comprising a common recognition site and a corresponding cleavage site, where the common recognition site overlaps with a methylation site; (b) the insertion site component comprises a 5' IIS-type double restriction site and a 3' IIS-type double restriction site, optionally where the 5' and 3' IIS-type double restriction sites are separated by at least one nucleotide; where each IIS-type double restriction site comprises: (i) a first common recognition site and a corresponding cleavage site, where the first common recognition site overlaps with a methylation site that forms a boundary between the insertion site component and the backbone component, and (ii) a second common recognition site and a corresponding cleavage site, where the second recognition site lacks an overlapping methylation site, where the cleavage site corresponding to the first common recognition site and the cleavage site corresponding to the second common recognition site are both located between the first common recognition site and the second common recognition site; where methylation of the destination vector at the common recognition site with overlapping methylation sites blocks cleavage at the cleavage site corresponding to that common recognition site; when methylated, exposure of the destination vector to an IIS-type restriction enzyme that recognizes a common recognition site of the destination vector generates two polynucleotide fragments, where the terminal 5' or 3' nucleic acid overhang of the fragment containing the backbone component has a different nucleotide sequence; and the IIS-type cleavage site of (a) and the IIS-type cleavage site of (b) have different nucleotide sequences.

[0042] Entry Vectors and Compositions In some aspects, the present disclosure relates to entry vector polynucleic acids and compositions comprising entry vector polynucleic acids. In some embodiments, the entry vector is a linear vector. In other embodiments, the entry vector is a circular vector. The entry vector comprises a backbone component (described in "Destination Vectors and Compositions" above) and an insert component. As used herein, the term "insert component" refers to a polynucleic acid comprising a first IIS-type restriction site, an insert, and a second IIS-type restriction site (see, e.g., Figure 2). As used herein, the term "insert" refers to a nucleic acid sequence adjacent to the first and second IIS-type restriction sites. The nucleotide length of the insert can vary. For example, the insert can be at least 20, at least 50, at least 100, at least 200, at least 500, at least 1000, at least 2000, at least 5000, at least 10,000, or at least 20,000 nucleotides in length.

[0043] In some embodiments, the first IIS-type restriction site and the second IIS-type restriction site of the insert component are inward-facing (i.e., the cleavage sites corresponding to the first and second recognition sites are located between the two recognition sites). In some embodiments, the recognition site of the first IIS-type restriction site and the recognition site of the second IIS-type restriction site are both common recognition sites. In some embodiments, the cleavage site of the first IIS-type restriction site of the insert component and the cleavage site of the second IIS-type restriction site of the insert component have different nucleotide sequences. In some embodiments, the nucleic acid sequence of the first restriction site, excluding the sequence of the cleavage site, is the reverse complement of the second restriction site. For example, in some embodiments, the nucleic acid sequence of the first IIS-type restriction site is CCGGTCTCNNNNNN (SEQ ID NO: 3), and the nucleic acid sequence of the second IIS-type restriction site is NNNNNNGAGACCGG (SEQ ID NO: 4), where N represents A, T, G, or C.

[0044] In some embodiments, at least one IIS-type restriction site in the backbone component of the entry vector has a different nucleotide sequence from at least one IIS-type restriction site in the insertion component of the entry vector. In some embodiments, at least one cleavage site corresponding to the common recognition site of the backbone component has a different nucleotide sequence from at least one cleavage site corresponding to the common recognition site of the insertion component. In some embodiments, each cleavage site corresponding to the common recognition site in the backbone component has a different nucleotide sequence from each cleavage site corresponding to the common recognition site in the insertion component. In some embodiments, each cleavage site corresponding to the common recognition site is unique.

[0045] In some embodiments, the methylation sites of each common recognition site with overlapping methylation sites in the entry vector are the same (i.e., each methylation site with overlapping common recognition sites in the destination vector can be methylated by the same methyltransferase enzyme). In some embodiments, the methylation sites of at least two common recognition sites with overlapping methylation sites in the entry vector are unique (i.e., at least two methylation sites with overlapping common recognition sites in the destination vector are methylated by different methyltransferase enzymes). In some embodiments, methylation of the entry vector at a common recognition site with overlapping methylation sites blocks cleavage of the cleavage site corresponding to that common recognition site.

[0046] In some embodiments, when not methylated, exposure of the entry vector to an IIS-type restriction enzyme that recognizes the common recognition site of the entry vector generates at least two polynucleotide fragments, where each polynucleotide fragment includes a terminal 5' or 3' nucleic acid overhang, and where at least one of the at least two polynucleotide fragments includes the insert of the entry vector. In some embodiments, the 5' or 3' overhang of the polynucleotide fragment containing the insert has a different nucleotide sequence. In some embodiments, the polynucleotide entry vector comprises a backbone component and an insert component, where (a) the backbone component comprises the backbone component of the polynucleotide destination vectors disclosed herein; and (b) the insert component comprises, from 5' to 3', a first IIS-type restriction site, an insert, and a second IIS-type restriction site; where the first and second IIS-type restriction sites each comprise: (i) a common recognition site with overlapping methylation sites, where the methylation sites form a boundary between the insert component and the backbone component, and (ii) corresponding cleavage sites, where cleavage of the cleavage sites of the first and second IIS-type restriction sites generates a 5' or 3' overhang, where the 5' or 3' overhang of the first IIS-type restriction site and the 5' or 3' overhang of the second IIS-type restriction site have different nucleotide sequences from each other.

[0047] Figure 2 provides a schematic diagram showing one embodiment of the entry vector. In this embodiment, the first IIS-type restriction site and the second IIS-type double restriction site each comprise a common recognition site with overlapping methylation sites. In this case, the common recognition site is that of BsaI. The BsaI restriction site comprises a recognition site of 5'-GGTCTC-3' and a cleavage site of (N1) / (N5) (i.e., 5'-NNNNNN-3', where N represents A, T, G, or C, and generates a 4-base pair 5' overhang upon cleavage). One of ordinary skill in the art will understand that the BsaI common recognition site (and its corresponding cleavage site) can be replaced with that of any other IIS-type restriction enzyme recognition site. Further, one of ordinary skill in the art will understand that the cleavage site of the first IIS-type restriction site and the cleavage site of the second IIS-type restriction site need not be the same. Further, this embodiment depicts an MspI methylation site overlapping the BsaI recognition site. One of ordinary skill in the art will understand that the MspI methylation site can be replaced with any number of methylation sites known in the art.

[0048] Composition of a kit for assembling a polynucleotide having a predetermined sequence In some aspects, the present disclosure relates to a kit composition for assembling a polynucleic acid having a predetermined sequence. In some embodiments, the kit includes a set of destination vectors described above in "Destination Vectors and Compositions," wherein: (i) the cleavage site corresponding to the first common recognition site of the 5' type IIS dual restriction site and the cleavage site corresponding to the first recognition site of the 3' type IIS dual restriction site of each destination vector in the set of destination vectors differ in nucleotide sequence; (ii) the cleavage site of the 5' type IIS dual restriction site of at least one destination vector in the set of destination vectors is identical to the 3' type IIS dual restriction site of at least one other destination vector in the set of destination vectors; and (iii) the cleavage site of the 3' type IIS dual restriction site of at least one destination vector in the set of destination vectors is identical to the 5' type IIS dual restriction site of at least one other destination vector in the set of destination vectors.

[0049] In some embodiments, each destination vector in the set of destination vectors comprises a backbone component and an insertion site component, wherein: (a) the backbone component comprises a nucleic acid sequence of a selectable marker, an origin of replication, and at least one type IIS restriction site comprising a consensus recognition site and a corresponding cleavage site, where the consensus recognition site overlaps with a methylation site; and (b) the insertion site component comprises a 5' type IIS dual restriction site and a 3' type IIS dual restriction site, optionally, where the 5' and 3' type IIS dual restriction sites are separated by at least one nucleotide; where each type IIS dual restriction site comprises: (i) a first consensus recognition site and a corresponding cleavage site, where the first consensus recognition site overlaps with a methylation site that forms a boundary between the insertion site component and the backbone component, and (ii) a second a consensus recognition site and a corresponding cleavage site, wherein the second recognition site lacks an overlapping methylation site, and wherein the cleavage site corresponding to the first consensus recognition site and the cleavage site corresponding to the second consensus recognition site are both located between the first consensus recognition site and the second consensus recognition site, wherein: methylation of the destination vector at the methylation site-overlapping consensus recognition site blocks cleavage of the cleavage site corresponding to that consensus recognition site; when methylated, exposure of the destination vector to a Type IIS restriction enzyme that recognizes the consensus recognition site of the destination vector generates two polynucleic acid fragments, wherein the terminal 5' or 3' nucleic acid overhangs of the fragments, including the backbone components, differ in nucleotide sequence; and the Type IIS cleavage site in (a) differs in nucleotide sequence from the Type IIS cleavage site in (b).

[0050] In some embodiments, the set of destination vectors includes 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 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 20, at least 25, at least 30, at least 40, at least 50, or more than 50 different destination vectors. In some embodiments, each destination vector within the set of destination vectors includes the same backbone component. In some embodiments, at least one destination vector within the set of destination vectors includes a unique backbone component.

[0051] In some embodiments, the kit further comprises: at least one reaction buffer (e.g., digestion buffer, ligase buffer, methyltransferase buffer, and / or universal buffer), at least one type IIS restriction enzyme (e.g., AlwI, BbsI, BceAI, BcgI, BcoDI, BfuAI, BsaI, BsmAI, BsmBI, BsmFI, BtgZI, EarI, EciI, Esp3I, FauI, FokI, HgaI, HphI, HpyAV, MboII, MmeI, PleI, SapI, SfaNI, and / or functional variants thereof), at least one methyltransferase (e.g., CpG methyltransferase (optionally M.SssI), dam methyltransferase, dcm methyltransferase, GpC methyltransferase (optionally M.CviPI), AluI methyltransferase, BamHI methyltransferase, EcoRI methyltransferase, HaeIII methyltransferase, HhaI methyltransferase, HpaII methyltransferase, MspI methyltransferase, and TaqI methyltransferase, and / or functional variants thereof), at least one ligase (e.g., T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, taq DNA ligase, and / or functional variants thereof), and / or at least one preparation of competent cells.

[0052] In some embodiments, the kit comprises at least two preparations of competent cells, wherein at least one preparation of competent cells comprises cells that express a methyltransferase capable of methylating a destination vector at a methylation site that overlaps a common recognition site, and wherein at least one preparation of competent cells comprises cells that are unable to methylate a destination vector at a methylation site that overlaps a common recognition site. In some embodiments, the competent cells are prokaryotic cells. In some embodiments, the competent cells are eukaryotic cells.

[0053] Method for assembling a polynucleotide having a predetermined sequence In some aspects, the present disclosure relates to a method for assembling a polynucleotide having a predetermined sequence. In some embodiments, the method for assembling a polynucleotide having a predetermined sequence includes forming a first reaction mixture, where the first reaction mixture includes at least two polynucleotides and a type IIS restriction enzyme, where forming the first reaction mixture results in the production of at least two polynucleotide cleavage products that include 5' or 3' overhangs, and where the at least two polynucleotide cleavage products together include a polynucleotide having a predetermined sequence. In some embodiments, the method further includes forming a second reaction mixture, where the second reaction mixture includes at least two polynucleotide cleavage products and a ligase, where the 5' or 3' overhangs of the at least two polynucleotide cleavage products uniquely complement each other to form a predetermined sequence by ligation.

[0054] In some embodiments, the first reaction mixture and the second reaction mixture are formed sequentially (i.e., the reaction mixture containing the type IIS restriction enzyme is formed first, followed by the reaction mixture containing the ligase). In some embodiments, the at least two polynucleotide cleavage products are purified before forming the second reaction mixture. In some embodiments, the first reaction mixture and the second reaction mixture are the same (i.e., cleavage and ligation of the destination vector and the entry vector occur in a single reaction volume).

[0055] In some embodiments, the at least two polynucleic acids of the first reaction mixture comprise a destination vector, as described above in "Destination Vectors and Compositions," and at least one entry vector, as described above in "Entry Vectors and Compositions." For example, in some embodiments, the method includes: (a) forming a reaction mixture by combining: (i) a destination vector, wherein the methylation sites in both the backbone component and the insertion site component of the destination vector are methylated; (ii) at least one entry vector, wherein the methylation sites in each of the backbone component and the insertion component of the at least one entry vector are unmethylated; (iii) a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme recognizes a common recognition site in the destination vector and the entry vector; and (iv) a ligase; (b) ligating the reaction mixture to combine the destination vector and the at least one entry vector. (a) incubating the vector for a time sufficient for cleavage via a Type IIS restriction enzyme; and (c) incubating the reaction mixture for a time sufficient for the ligase to ligate the insert of each of the at least one Entry vector to the backbone component of the destination vector, thereby generating a circular polynucleic acid; and wherein the 5' or 3' overhangs of the backbone component of the destination vector and the insert component of each of the at least one Entry vector are uniquely complementary to each other to form a predetermined sequence comprising the backbone component of the destination vector of step (a)(i) and the insert component of each of the at least one Entry vector of step (a)(ii).

[0056] In some embodiments, the first reaction mixture comprises 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, or more than 10 entry vectors. In some embodiments, the destination vector is methylated in vitro (e.g., by forming a reaction mixture comprising the destination vector and a methyltransferase enzyme, and incubating the reaction mixture for a time sufficient for the methyltransferase enzyme to methylate the destination vector). In other embodiments, the destination vector is methylated in vivo. For example, in some embodiments, the destination vector is methylated in a bacterial strain that expresses a methyltransferase selected from the group consisting of: CpG methyltransferase (optionally M.SssI), dam methyltransferase, dcm methyltransferase, GpC methyltransferase (optionally M.CviPI), AluI methyltransferase, BamHI methyltransferase, EcoRI methyltransferase, HaeIII methyltransferase, HhaI methyltransferase, HpaII methyltransferase, MspI methyltransferase, and TaqI methyltransferase.

[0057] In some embodiments, the method further comprises isolating the ligated destination vector containing the insert from the other components of the reaction mixture. In some embodiments, the ligated destination vector is isolated by transforming bacteria with a second reaction mixture and screening the bacteria for the presence of correctly ligated assemblies. In some embodiments, the method further comprises demethylating the isolated ligated destination vector to generate a second entry vector. In some embodiments, the isolated ligated destination vector is passively demethylated. For example, the destination vector can be passively demethylated via in vitro amplification (e.g., PCR). Alternatively, the destination vector can be passively demethylated in vivo via replication in a bacterial strain lacking a methyltransferase selected from the group consisting of CpG methyltransferase (optionally M.SssI), dam methyltransferase, dcm methyltransferase, GpC methyltransferase (optionally M.CviPI), AluI methyltransferase, BamHI methyltransferase, EcoRI methyltransferase, HaeIII methyltransferase, HhaI methyltransferase, HpaII methyltransferase, MspI methyltransferase, and TaqI methyltransferase.

[0058] In some embodiments, the backbone of the destination vector and the backbone of at least one entry vector are the same. In some embodiments, the backbone of the destination vector and the backbone of each entry vector are the same. Figure 3 provides a schematic diagram showing one embodiment where at least two polynucleic acids of the first reaction mixture include a destination vector as described above and at least one entry vector as described above. One of ordinary skill in the art will understand that the described method can be modified by the use of different destination vectors and / or entry vectors.

[0059] In other embodiments, at least two polynucleic acids of the first reaction mixture comprise a destination vector as described in the above "Destination Vectors and Compositions" and at least one polynucleic acid fragment (e.g., a PCR product or other synthetic fragment). For example, in some embodiments, the method comprises: (a) forming a reaction mixture by combining: (i) a destination vector, wherein the methylation sites of the backbone component are methylated; (ii) at least one polynucleic acid fragment, wherein each polynucleic acid fragment comprises an internal sequence flanked at both ends by a common recognition site and a corresponding cleavage site; (iii) a type IIS restriction enzyme, wherein the type IIS restriction enzyme recognizes the common recognition site of the destination vector and at least one polynucleic acid fragment; and (iv) a ligase; (b) incubating the reaction mixture for a time sufficient for cleavage of the destination vector and at least one polynucleic acid fragment via the type IIS restriction enzyme; and (c) incubating the reaction mixture for a time sufficient for the ligase to ligate each internal nucleic acid sequence of the at least one polynucleic acid fragment to the backbone component of the destination vector, thereby generating a circular polynucleic acid; and wherein the internal sequence of the at least one polynucleic acid fragment comprises the nucleic acid sequence of interest; and wherein the 5' or 3' overhangs of the backbone component of the destination vector and each internal sequence of the at least one polynucleic acid fragment uniquely complement each other to form a predetermined sequence comprising the backbone component of the destination vector of step (a)(i) and the nucleic acid sequence of interest.

[0060] In some embodiments, the destination vector is methylated in vitro (e.g., by forming a reaction mixture comprising the destination vector and a methyltransferase enzyme, and incubating the reaction mixture for a time sufficient for the methyltransferase enzyme to methylate the destination vector). In other embodiments, the destination vector is methylated in vivo. For example, in some embodiments, the destination vector is methylated in a bacterial strain that expresses a methyltransferase selected from the group consisting of: CpG methyltransferase (optionally M.SssI), dam methyltransferase, dcm methyltransferase, GpC methyltransferase (optionally M.CviPI), AluI methyltransferase, BamHI methyltransferase, EcoRI methyltransferase, HaeIII methyltransferase, HhaI methyltransferase, HpaII methyltransferase, MspI methyltransferase, and TaqI methyltransferase.

[0061] In some embodiments, the method further comprises isolating the ligated destination vector containing the insert from the other components of the second reaction mixture. In some embodiments, the ligated destination vector is isolated by transforming cells (e.g., bacteria) with the second reaction mixture and screening the cells or progeny of the cells for the presence of a correctly ligated assembly. In some embodiments, the method further comprises demethylating an isolated ligated destination vector to generate a second entry vector. In some embodiments, the isolated ligated destination vector is passively demethylated in vivo via replication in a bacterial strain lacking a methyltransferase selected from the group consisting of: CpG methyltransferase (optionally M.SssI), dam methyltransferase, dcm methyltransferase, GpC methyltransferase (optionally M.CviPI), AluI methyltransferase, BamHI methyltransferase, EcoRI methyltransferase, HaeIII methyltransferase, HhaI methyltransferase, HpaII methyltransferase, MspI methyltransferase, and TaqI methyltransferase.

[0062] In other embodiments, at least two polynucleic acids of the first reaction mixture comprise an entry vector as described in "Entry Vectors and Compositions" above, and a polynucleic acid fragment (e.g., a PCR product or other synthetic fragment). For example, in some embodiments, the method comprises: (a) forming a reaction mixture by combining: (i) at least one entry vector disclosed herein, wherein the methylation sites of the backbone components are not methylated; (ii) a polynucleic acid fragment, wherein the polynucleic acid fragment comprises an internal sequence flanked by common recognition sites and corresponding cleavage sites at both ends; (iii) a type IIS restriction enzyme, wherein the type IIS restriction enzyme recognizes the common recognition sites of at least one entry vector and the polynucleic acid fragment; and (iv) a ligase; (b) incubating the reaction mixture for a time sufficient for cleavage of at least one entry vector and the polynucleic acid fragment via the type IIS restriction enzyme; and (c) incubating the reaction mixture for a time sufficient for the ligase to ligate the internal nucleic acid sequence of the polynucleic acid fragment to each insertion component of at least one entry vector, thereby generating a circular polynucleic acid; and wherein the internal sequence of the polynucleic acid fragment comprises a selectable marker and an origin of replication; and wherein the 5' or 3' overhangs of each insertion component of at least one entry vector and the internal sequence of at least one polynucleic acid fragment uniquely complement each other to form a predetermined sequence comprising the nucleic acid sequence of interest.

[0063] In some embodiments, the first reaction mixture comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more than ten entry vectors. In some embodiments, the backbones of each entry vector are the same. In some embodiments, the polynucleotide fragment comprises the sequence of the backbone component of the destination vector or entry vector as described above. In some embodiments, the polynucleotide fragment is methylated in vitro. In some embodiments, the predetermined sequence further comprises the sequence of the entry vector.

[0064] Other embodiments All features disclosed herein can be combined in any combination. Each feature disclosed herein can be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless specifically stated otherwise, each feature disclosed is only an example of a general series of equivalent or similar features. From the above description, those skilled in the art can readily ascertain the essential characteristics of the present disclosure and, without departing from the spirit and scope thereof, make various changes and modifications to adapt the present disclosure to various usages and conditions. Accordingly, other embodiments are also within the scope of the claims.

[0065] Equivalents While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and each such variation and / or modification is deemed to be within the scope of the embodiments of the present invention described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or applications for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Accordingly, it is to be understood that the foregoing embodiments are presented by way of example only, and that, within the scope of the appended claims and equivalents thereof, embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the inventive scope of the present disclosure, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0066] All definitions defined and used herein should be understood to govern dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms. All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, and in some cases may be included in their entirety. The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0067] As used in this specification and the claims, the phrase "and / or" is to be understood to mean "either or both" of the elements so joined, i.e., elements that may be present conjunctively in some cases and disjunctively in other cases. Multiple elements listed with "and / or" are to be construed in the same manner, i.e., "one or more" of the elements so joined. Other elements may optionally be present whether or not specifically identified by the "and / or" clause, depending on whether or not they are related to the elements specifically identified. Thus, by way of non-limiting example, reference to "A and / or B" when used in combination with open-ended terms such as "comprising" may, in one aspect, refer to only A (optionally including elements other than B); in another aspect, to only B (optionally including elements other than A); in yet another aspect, to both A and B (optionally including other elements); and so on.

[0068] As used in this specification and the claims, "or" is to be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" is to be interpreted as inclusive, i.e., including at least one of a number of elements or a list of elements, but also including more than one, and optionally, additional unlisted items. Terms that clearly indicate the contrary, such as "only one" or "exactly one", or "consisting of" when used in the claims, refer to exactly one of a number of elements or a list of elements. In general, the term "or" as used in this specification is to be interpreted as indicating exclusive alternatives only when preceded by exclusive terms such as "either", "one of", "only one of", or "exactly one" (i.e., "either one but not both"). When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0069] As used in this specification and claims, the phrase "at least one," in reference to a list of one or more elements, should be interpreted to mean at least one element selected from any one or more elements in the list of elements, but need not include at least one of each and every element specifically listed in the list of elements, nor does it exclude any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related to the specifically identified elements or not, may optionally be present. Thus, as non-limiting examples, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") means, in one embodiment, at least one, optionally two or more, A, and no B (and optionally including elements other than B); in another embodiment, at least one, optionally two or more, B, and no A (and optionally including elements other than A); in yet another embodiment, at least one, optionally two or more, A, and at least one, optionally two or more, B (and optionally including other elements); and so forth.

[0070] It should also be understood that, unless expressly stated to the contrary, in any method claimed herein that includes multiple steps or acts, the order of the method steps or acts is not necessarily limited to the described order of the method steps or acts. In the claims and the above specification, all transitional phrases, such as "comprising", "including", "possessing", "having", "containing", "involving", "retaining", "constituting", etc., are to be understood as being open-ended, i.e., meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" are to be considered closed or semi-closed transitional phrases, respectively, as described in the Patent Examination Procedure Manual of the United States Patent and Trademark Office, Section 2111.03. It should be understood that embodiments described in this document using an open-ended transitional phrase (e.g., "comprising") are also contemplated in alternative embodiments as "consisting of" and "consisting essentially of" the features described by the open-ended transitional phrase. For example, if this disclosure describes a "composition comprising A and B", this disclosure also contemplates alternative embodiments of a "composition consisting of A and B" and a "composition consisting essentially of A and B".

Claims

**Claim 1** A polynucleotide destination vector comprising a backbone component and an insertion site component, wherein: (a) the backbone component comprises a nucleic acid sequence of a selectable marker, an origin of replication, and at least one IIS-type restriction site comprising a common recognition site and a corresponding cleavage site, wherein the common recognition site overlaps with a methylation site; and (b) the insertion site component comprises a 5'IIS-type double restriction site and a 3'IIS-type double restriction site; wherein each IIS-type double restriction site comprises: (i) a first common recognition site and a corresponding cleavage site, wherein the first common recognition site overlaps with a methylation site forming a boundary between the insertion site component and the backbone component, and (ii) a second common recognition site and a corresponding cleavage site, wherein the second recognition site lacks the overlapping methylation site, wherein the cleavage site corresponding to the first common recognition site and the cleavage site corresponding to the second common recognition site are both located between the first common recognition site and the second common recognition site, wherein: methylation of the destination vector at a common recognition site with overlapping methylation sites blocks cleavage at the cleavage site corresponding to that common recognition site; when methylated, exposure of the destination vector to an IIS-type restriction enzyme that recognizes the common recognition site of the destination vector generates two polynucleotide fragments, wherein the terminal 5' or 3' nucleic acid overhangs of the fragment containing the backbone component have different nucleotide sequences; and the IIS-type cleavage site of (a) has a different nucleotide sequence from the IIS-type cleavage site of (b), said polynucleotide destination vector. **Claim 2** the cleavage site corresponding to the first common recognition site and the cleavage site corresponding to the second common recognition site of the 5'IIS-type double restriction site and / or the 3'IIS-type double restriction site are separated from each other by at least one nucleotide, or, the cleavage site corresponding to the first common recognition site and the cleavage site corresponding to the second common recognition site of the 5'IIS-type double restriction site and / or the 3'IIS-type double restriction site are a shared cleavage site, the destination vector according to claim 1. **Claim 3** the cleavage site corresponding to the first common recognition site and the cleavage site corresponding to the second common recognition site of both the 5'IIS-type double restriction site and the 3'IIS-type double restriction site are separated from each other by at least one nucleotide, wherein, The cleavage site corresponding to the first common recognition site and the cleavage site corresponding to the second common recognition site of the 5'IIS type double restriction site, and the cleavage site corresponding to the first common recognition site and the cleavage site corresponding to the second common recognition site of the 3'IIS type double restriction site are separated from each other by different nucleotide sequences, or, The destination vector according to claim 1, wherein the cleavage site corresponding to the first common recognition site and the cleavage site corresponding to the second common recognition site of the 5'IIS type double restriction site, and the cleavage site corresponding to the first common recognition site and the cleavage site corresponding to the second common recognition site of the 3'IIS type double restriction site are separated from each other by the same nucleotide sequence.

4. The destination vector according to any one of claims 1 to 3, wherein the 5'IIS type double restriction site and the 3'IIS type double restriction site are separated by a nucleic acid sequence encoding a visual readout or a suicide cassette.

5. The IIS type restriction enzyme that binds to the common recognition site is selected from the group consisting of BsaI, BsmBI, BtgZI, Esp3I, FokI, HphI, BcgI, AlwI, MboII, MmeI, BsmFI, BceAI, BcoDI, BfuAI, BsmAI, EarI, EciI, FauI, HgaI, HpyAV, PleI, BbsI, SapI, and SfaNI. At least one IIS type restriction site in the backbone component of (a) is located within or adjacent to the selection marker or the origin of replication. The cleavage site of at least one IIS type restriction site in the backbone component of (a) contains a sequence content selected from the group consisting of overhangs of two or fewer nucleotides and overhangs of the sequences TNNN, TTTT, or AAAA. The selection marker contains an antibiotic resistance gene. The methylation sites of (a) and (b) are methylated by the same methyltransferase. When not methylated, exposure of the destination vector to a type IIS restriction enzyme that recognizes the common recognition site of the destination vector generates at least three polynucleotide fragments, where each polynucleotide contains a terminal 5' or 3' nucleic acid overhang, and where the terminal 5' or 3' nucleic acid overhang of the fragment containing the second common recognition sequence of both the 5' type IIS double restriction site and the 3' type IIS double restriction site has a different nucleotide sequence, or, The destination vector according to any one of claims 1 to 4, which is a combination thereof.

6. A polynucleotide entry vector comprising a backbone component and an insertion component, wherein, (a) the backbone component comprises the backbone component of the polynucleotide destination vector according to any one of claims 1 to 5; and (b) the insertion component comprises, from 5' to 3', a first type IIS restriction site, an insert, and a second type IIS restriction site; wherein the first and second type IIS restriction sites each comprise: (i) a common recognition site with overlapping methylation sites, where the methylation sites form a boundary between the insertion component and the backbone component, and (ii) corresponding cleavage sites, where cleavage of the cleavage sites of the first and second type IIS restriction sites generates a 5' or 3' overhang, and where the 5' or 3' overhang of the first type IIS restriction site and the 5' or 3' overhang of the second type IIS restriction site have different nucleotide sequences from each other, Said polynucleotide entry vector.

7. The entry vector according to claim 6, wherein the insert is a nucleic acid sequence that is combined in an assembly reaction.

8. A method for assembling a polynucleotide into a predetermined sequence, comprising the following: (a) Forming a reaction mixture by combining: (i) a destination vector according to any one of claims 1 to 5, wherein the methylation sites of both the backbone component and the insertion site component of the destination vector are methylated; (ii) at least one entry vector according to claim 6 or 7, wherein the methylation sites of the respective backbone component and insertion component of the at least one entry vector are not methylated; (iii) a type IIS restriction enzyme, wherein the type IIS restriction enzyme recognizes a common recognition site of the destination vector and the entry vector; and (iv) a ligase; (b) Incubating the reaction mixture for a time sufficient for cleavage of the destination vector and at least one entry vector via the type IIS restriction enzyme; (c) Incubating the reaction mixture for a time sufficient for the ligase to ligate each insert of the at least one entry vector to the backbone component of the destination vector, thereby generating a circular polynucleotide; and wherein the 5' or 3' overhangs of the backbone component of the destination vector and the respective insert components of the at least one entry vector uniquely complement each other to form a predetermined sequence comprising the backbone component of the destination vector of step (a)(i) and the respective insert components of the at least one entry vector of step (a)(ii); The method as described above, including the above steps. **Claim 9** The method according to claim 8, wherein the destination vector is methylated in vitro or in vivo. **Claim 10** Further comprising isolating the ligated destination vector containing the insert from the other components of the reaction mixture, Further comprising demethylating the isolated ligated destination vector to generate a second entry vector, or a combination thereof, The method according to claim 8 or 9, which is a combination thereof. **Claim 11** A method for cloning a nucleic acid sequence of interest, comprising: (a) Forming a reaction mixture by combining: (i) a destination vector according to any one of claims 1 to 5, wherein the methylation site of the backbone component is methylated; (ii) at least one polynucleotide fragment, wherein each polynucleotide fragment comprises an internal sequence flanked at both ends by a common recognition site and a corresponding cleavage site; (iii) a type IIS restriction enzyme, wherein the type IIS restriction enzyme recognizes the common recognition site of the destination vector and the at least one polynucleotide fragment; and (iv) a ligase; (b) Incubating the reaction mixture for a time sufficient for cleavage of the destination vector and the at least one polynucleotide fragment via the type IIS restriction enzyme; (c) Incubating the reaction mixture for a time sufficient for the ligase to ligate each internal nucleic acid sequence of the at least one polynucleotide fragment to the backbone component of the destination vector, thereby generating a circular polynucleotide; and wherein the internal sequence of the at least one polynucleotide fragment comprises the nucleic acid sequence of interest; and wherein the 5' or 3' overhangs of the backbone component of the destination vector and each internal sequence of the at least one polynucleotide fragment are uniquely complementary to each other to form a predetermined sequence comprising the backbone component of the destination vector and the nucleic acid sequence of interest in step (a)(i); The method as described above. **Claim 12** The method according to claim 11, wherein the destination vector is methylated in vitro or in vivo. **Claim 13** Further comprising isolating the ligated destination vector containing the predetermined sequence from the other components of the reaction mixture, Further comprising demethylating the isolated ligated destination vector to generate a second entry vector, or, A combination thereof, the method according to claim 11 or 12. **Claim 14** A method for cloning a nucleic acid sequence of interest, comprising: (a) forming a reaction mixture by combining: (i) at least one entry vector according to claim 1, wherein the methylation site of the backbone component is not methylated; (ii) a polynucleotide fragment, wherein the polynucleotide fragment comprises an internal sequence flanked by common recognition sites and corresponding cleavage sites at both ends; (iii) a type IIS restriction enzyme, wherein the type IIS restriction enzyme recognizes the common recognition sites of at least one entry vector and the polynucleotide fragment; and (iv) a ligase; (b) incubating the reaction mixture for a time sufficient for cleavage of at least one entry vector and the polynucleotide fragment via the type IIS restriction enzyme; and (c) incubating the reaction mixture for a time sufficient for the ligase to ligate the internal nucleic acid sequence of the polynucleotide fragment to each insertion component of at least one entry vector, thereby generating a circular polynucleotide; and wherein the internal sequence of the polynucleotide fragment comprises a selectable marker and an origin of replication; and wherein the 5' or 3' overhangs of each insertion component of at least one entry vector and the internal sequence of at least one polynucleotide fragment are uniquely complementary to each other to form a predetermined sequence comprising the nucleic acid sequence of interest, the method comprising. **Claim 15** wherein the polynucleotide fragment is a PCR product, wherein the polynucleotide fragment is methylated in vitro, wherein the predetermined sequence further comprises the sequence of the entry vector, or, a combination thereof, the method according to claim 14.

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