LINEAR DNA WITH ENHANCED RESISTANCE AGAINST EXONUCLEASES AND METHODS FOR ITS PRODUCTION

Abstract

Linear DNA with improved resistance against exonucleases and methods for its production. The present invention relates to methods for producing a linear deoxyribonucleic acid (DNA) product (e.g., a closed linear DNA product) with improved resistance to nuclease digestion. The present invention relates to methods comprising the steps of: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adaptor molecules and intermediate adaptor molecules to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to generate a linear DNA product (e.g., a closed linear DNA product). The present invention also relates to linear deoxyribonucleic acid (DNA) products (e.g., a closed linear DNA product) with improved resistance to nuclease digestion and uses thereof.

Description

Linear DNA with enhanced resistance against exonucleases and methods for its production Technical field The present invention relates to methods for producing a linear deoxyribonucleic acid (DNA) product (e.g., a closed linear DNA product) with improved resistance to nuclease digestion. The present invention relates to methods comprising the steps of: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and intermediate adapter molecules to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to generate a linear DNA product (e.g., a closed linear DNA product). The present invention also relates to linear deoxyribonucleic acid (DNA) products (e.g., a closed linear DNA product) with improved resistance to nuclease digestion and uses thereof. Background DNA is susceptible to degradation by nucleases, which are naturally occurring enzymes within organisms that play a vital role in regulating many cellular processes, while also protecting against exogenous DNA species. Enzymatic degradation of DNA can render gene therapies ineffective and is a substantial consideration when developing gene therapies or DNA vaccines. Considerable efforts have been made to extend the effective molecular life of nucleic acids by increasing the resistance of nucleic acid molecules to both extracellular and intracellular nucleases. For linear molecules, one of the proposed solutions includes the use of nucleotides with phosphorothioate (i.e., 2-deoxynucleotides-5-(a-thio)-triphosphate). Phosphorothioate nucleotides contain a sulfur atom in place of a non-bonding oxygen atom. These modified nucleotides exhibit physical and chemical characteristics comparable to the corresponding unmodified nucleotides, but are resistant to exonuclease digestion. As such, the incorporation of the phosphorothioate functional group can extend the half-life of the nucleic acid molecule. Phosphorothioate modifications are used in nucleic acid drug development programs. In therapeutic nucleic acids, phosphorothioate-containing nucleotides are incorporated into short, single-stranded polynucleotide chains. For example, the antisense oligonucleotide fomivirsen is a 21-mer phosphorothioate oligodeoxynucleotide used to treat cytomegalovirus retinitis (Stein and Castanotto, "FDA-approved oligonucleotide therapies in 2017." Molecular Therapy 25.5 (2017): 1069–1075). Similarly, pegaptanib (trade name Macugen) is a short (27-nucleotide) phosphorothioate aptamer with a 3-3 deoxythymidine cap used to treat age-related macular degeneration of the retina. Phosphorothioate modifications have also been used in the context of a linear double-stranded polynucleotide chain (e.g., double-stranded DNA) to cap the polynucleotide chain ends to increase resistance to exonuclease digestion (Putney et al. "A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo." Proceedings of the National Academy of Sciences 78.12 (1981): 7350-7354). To cap the polynucleotide chain ends, the ends are digested with a restriction enzyme and treated with a DNA polymerase and a mixture of deoxyribonucleotide triphosphates (dNTPs), at least one type of which is a nucleotide with phosphorothioate complementary to a nucleotide in the overhanging strand.Since DNA polymerases add nucleotides in the 5 to 3 direction, the result of this treatment is a blunt-ended polynucleotide fragment with a phosphorothioate nucleotide located at the 3 end of each chain (i.e., in "the cap"). Resistance to nuclease digestion can also be achieved using closed DNA molecules, such as plasmids or minicircles. However, plasmids and minicircles have limited utility in vivo due to their frequent contamination with toxic agents derived from cellular components, fidelity problems that alter the sequence of interest, and the presence of different species (supercoiled, linear, and open circular). Alternatively, resistance to nuclease digestion can be achieved by producing closed linear DNA molecules. For example, WO2010 / 086626 A1 describes a method for producing closed linear DNA using a protelomerase. However, this method is limited because the protelomerase action produces the same sequence at both ends of the closed linear DNA molecules, allowing little flexibility. Document WO2008 / 095927 describes a system known as the "Golden Gate" assembly, whereby separately digested fragments of a desired construct are ligated into a vector. This method is inefficient due to the number of ligation events that must occur and the need to use multiple different restriction sites. Furthermore, the resulting yield is low, and the desired DNA product must be amplified using bacteria to increase the amount of DNA, requiring the presence of, for example, antibiotic resistance genes. This means that commercial scalability of the "Golden Gate" process is not possible without the use of fermentation. Therefore, there is a need for a more flexible method to produce a linear DNA product with improved resistance to nuclease digestion (e.g., exonuclease), while allowing the incorporation of additional features for different applications. Description The invention provides a method for producing a linear deoxyribonucleic acid (DNA) product (e.g., a closed linear DNA product) with improved resistance to nuclease digestion. The invention is based on the addition of adapter molecules to a double-stranded DNA molecule. The method of the invention is based on adding the adapter molecules, an endonuclease, and a ligase to the double-stranded DNA molecule in a single reaction volume (or a single contiguous aqueous volume).Therefore, the method for producing a linear DNA product comprises the steps of: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1, and n+m is at least 1; and (b) incubating the single contiguous aqueous volume to generate the linear DNA product. Preferably, the linear DNA product has improved resistance to exonuclease digestion (e.g., exonuclease I, exonuclease III, and / or exonuclease VIII). The linear DNA product may be a closed linear DNA product.The linear DNA product may comprise nuclease-resistant nucleotides (i.e., protected nucleotides), such as phosphorothioate-containing nucleotides. The linear DNA product may be a partially missed linear DNA product. The partially closed linear DNA product may comprise nuclease-resistant nucleotides (i.e., protected nucleotides), such as phosphorothioate-containing nucleotides. The linear DNA product may comprise a cassette. The cassette may comprise a coding sequence. The invention provides a method for producing a linear DNA product, wherein the method comprises: (a) bringing a double-stranded DNA molecule into contact with an endonuclease and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1;and (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region. n can be 0, 1, 2, 3, 4, 5, 6, 7 or 8. m can be 0, 1, 2, 3, 4, 5, 6, 7 or 8. Preferably, n is 0, 1, 2, 3 or 4, and m is 0, 1, 2, 3 or 4. The first and second terminal adapter molecules may be identical or different. For example, the first terminal adapter molecule and / or the second terminal adapter molecule may comprise a hairpin. The first terminal adapter molecule and / or the second terminal adapter molecule may be linear double-stranded nucleic acid molecules comprising one or more nuclease-resistant nucleotides. The first terminal adapter molecule may comprise a hairpin, and the second terminal adapter molecule may be a linear double-stranded nucleic acid molecule comprising one or more nuclease-resistant nucleotides. Therefore, the linear DNA product produced by the methods described herein is resistant to nuclease digestion (e.g., exonuclease). The intermediate adapter molecules may be the same or different. One or more of the n+m intermediate adapter molecules may comprise a spacer or a linker sequence. One or more of the n+m intermediate adapter molecules may comprise a fluorophore, a barcode, a poly-A signal, a biotinylated nucleotide, a protected nucleotide, a modified nucleotide, a spacer, a poly-A sequence, a promoter, an open reading frame, a UTR (untranslated region), a transcription factor binding site, or a termination sequence. The n intermediate adapter molecules added sequentially to one end of the double-stranded DNA molecule may be the same or different from the m intermediate adapter molecules added sequentially to the other end of the double-stranded DNA molecule. A modified nucleotide may be 2-methoxyethoxy A, 2-methoxyethoxy MeC, 2-methoxyethoxy G, 2-methoxyethoxy T, 2-O-methyl RNA bases, fluorinated bases, 2-aminopurine, 5-bromo dU, deoxyuridine, 2, 6-diaminopurine (2-amino-dA), dideoxy-C, deoxyinosine, hydroxymethyl dC, iso-dG, iso-dC, 5-methyl dC or 5-nitroindole. The step of contacting the double-stranded DNA molecule with the endonuclease and first and second terminal adapter molecules is preferably carried out in the presence of a ligase. Therefore, the invention provides a method for producing a linear deoxyribonucleic acid (DNA) product, wherein the method comprises: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region. The addition (or linking or closure) of the first terminal adapter molecule and / or the second terminal adapter molecule and / or the intermediate adapter molecules can be achieved by hybridization or ligation of the adapter molecules to the ends of the linear double-stranded region and / or to sequential adapter molecules. Therefore, n intermediate adapter molecules can hybridize to the first end of the linear double-stranded region. m intermediate adapter molecules can hybridize to the second end of the linear double-stranded region. n intermediate adapter molecules can be ligated to the first end of the linear double-stranded region. m intermediate adapter molecules can be ligated to the second end of the linear double-stranded region.The addition of the n+m intermediate adapter molecules, the first terminal adapter molecule, and the second terminal adapter molecule can occur through either hybridization or ligation of the adapter molecules to the ends of the linear double-stranded region and / or to sequentially adjacent adapter molecules. Therefore, n intermediate adapter molecules can hybridize and ligate to the first end of the linear double-stranded region. The m intermediate adapter molecules can hybridize and ligate to the second end of the linear double-stranded region. The addition can be mediated by a ligand or spacer molecule that facilitates the attachment of the adapter molecule to the first and / or second end of the linear double-stranded region or to another adapter molecule. The first terminal adapter molecules can hybridize, ligate, or hybridize and ligate to the nth intermediate adapter molecule or to the first end of the double-stranded DNA molecule.The second terminal adapter molecule can hybridize, ligate, or hybridize and ligate to the mth intermediate adapter molecule or to the second end of the double-stranded DNA molecule. n can be 0, 1, 2, 3, 4, 5, 6, 7, or 8. m can be 0, 1, 2, 3, 4, 5, 6, 7, or 8. Preferably, n is 0, 1, 2, 3, or 4, and m is 0, 1, 2, 3, or 4. The method may further comprise, prior to step (a) (i.e., the step of contacting the double-stranded DNA molecule with the endonuclease, ligase, first and second terminal adapter molecules, and the n+m intermediate adapter molecules), a step of amplifying a DNA template molecule to produce the double-stranded DNA molecule. Therefore, the invention provides a method for producing a linear DNA product, the method comprising: (a) amplification of a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, and wherein n intermediate adapter molecules are sequentially ligated to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially ligated to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is ligated to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is ligated to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region. Amplification can be in vitro or in vivo. Preferably, the amplification is in vitro. For example, amplification can be performed by rolling circle amplification (RCA), the MALBAC method, traditional polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), and recombinase polymerase amplification (RPA). Preferably, the amplification is rolling circle amplification. The invention thus provides a method for producing a linear deoxyribonucleic acid (DNA) product. The method comprises: (a) rolling circle amplification of a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and n+m is at least 1; and (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially linked to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially linked to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is linked to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is linked to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region. The method may further comprise (after the amplification step and before the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules) a heat deactivation step. Therefore, the invention provides a method for producing a linear DNA product, the method comprising: (a) amplification of a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) heat deactivation of the reaction of step (a); (c) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1 and n+m is at least 1; and (d) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially ligated to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially ligated to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is ligated to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is ligated to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region. Preferably, the amplification is rolling circle amplification. The heat deactivation step may be carried out under conditions sufficient to inactivate the reagents used during the amplification reaction. The heat deactivation step may be carried out at a temperature of at least 50 °C, at least 55 °C, at least 60 °C, at least 65 °C, at least 70 °C, at least 75 °C, at least 80 °C, at least 85 °C, at least 90 °C, at least 95 °C, or at least 100 °C. The heat deactivation step may be carried out for at least 1 min, at least 3 min, at least 5 min, at least 10 min, at least 15 min, or at least 20 min. The inventors of this application have surprisingly discovered that large concatemeric products of the rolling circle amplification reaction can be used to produce the DNA products described herein. This is surprising because the rolling circle amplification product has high viscosity and normally has to undergo purification steps before it can be used for further applications. In the method described herein, after the amplification step, the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules to form a single contiguous aqueous volume can be performed without purifying the amplification reaction product. That is, the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules can be performed directly after the amplification step. The step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules can be performed directly after the heat deactivation step. The inventors of this application have discovered a method that requires very few steps to produce the DNA product described herein. The methods described herein are very time-efficient. This is partly due to the fact that no purification step is required after the amplification reaction. Optionally, the product of the amplification reaction can be deactivated by heat.Surprisingly, the method described herein, where the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and intermediate adapter molecules is performed directly after the amplification step (i.e., without the purification step), produces higher yields of the DNA product described herein compared to a method in which the two steps are separated by purification of the amplification product. The method may also include, after the incubation stage of the single contiguous aqueous volume, a purification stage of the linear DNA product. The method may further comprise, after the incubation step in the single contiguous aqueous volume, a nuclease digestion step. The nuclease digestion may be exonuclease digestion, such as exonuclease I and / or exonuclease III. The nuclease digestion step may take place before or after the purification step. The nuclease digestion step may allow for the removal of any double-stranded DNA molecules and / or adapter molecules that were not used to produce linear DNA products. Therefore, the method for producing a linear DNA product may comprise the following steps: (a) bringing a double-stranded DNA molecule into contact with an endonuclease, a ligase and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region; and (c) incubate the single contiguous aqueous volume with a nuclease (e.g., an exonuclease). n can be 0, 1, 2, 3, 4, 5, 6, 7 or 8. m can be 0, 1, 2, 3, 4, 5, 6, 7 or 8. Preferably, n is 0, 1, 2, 3 or 4, and m is 0, 1, 2, 3 or 4. The method for producing a linear DNA product may comprise the following steps: (a) amplification of a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) bringing the double-stranded DNA molecule into contact with an endonuclease, a ligase and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially ligated to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially ligated to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is ligated to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is ligated to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region; and (d) incubate the single contiguous aqueous volume with a nuclease (e.g., an exonuclease). The method for producing a linear DNA product may comprise the following steps: (a) amplification of a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) bringing the double-stranded DNA molecule into contact with an endonuclease, a ligase and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region; (d) purification of the linear DNA product; and (e) incubate the product of step (d) with a nuclease (for example, an exonuclease). The method for producing a linear DNA product may comprise the following steps: (a) amplification of a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) bringing the double-stranded DNA molecule into contact with an endonuclease, a ligase and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region; (d) incubate the single contiguous aqueous volume with a nuclease (for example, an exonuclease); and (e) purification of the linear DNA product. An endonuclease can be a restriction endonuclease enzyme. An endonuclease can be a type IIS restriction enzyme. An endonuclease can be any enzyme that recognizes a DNA sequence and cleaves it outside the recognition sequence.For example, the endonuclease can be a restriction enzyme BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuI, BaccP, Bacc, Bib, Bib BceAI, BcgI, BciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, BsgI, BsgFI, BsmFIA, BsmFII BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BtsCI, BtsI-v2, BtsMut, BspCI, CIS, CIS Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, GsuI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, MnIII, PaqI, NCIA PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI y / o TspGWI. Type IIS restriction endonucleases cleave the double-stranded DNA molecule outside of the recognition sequence (i.e., an endonuclease target sequence), meaning that the recognition sequence (i.e., endonuclease target sequence) is not included in the linear DNA product. The present inventors have surprisingly discovered methods for producing a linear DNA product with improved resistance to nuclease digestion. Specifically, the linear DNA product produced by the methods described herein has improved resistance to exonuclease digestion (e.g., exonuclease III digestion). This improved resistance to exonuclease digestion extends the shelf life of the linear DNA product both in a cell (i.e., the linear DNA product has improved resistance to intracellular exonucleases) and in a cell-free system (i.e., the linear DNA product has improved resistance to extracellular exonucleases).The present inventors have developed a method based on the addition of adapter molecules (i.e., a first terminal adapter molecule, a second terminal adapter molecule, and intermediate adapter molecules) to one or both ends of a double-stranded DNA molecule. The terminal adapter molecules may comprise a hairpin or a loop to form a closed linear DNA product with enhanced resistance to nuclease digestion (e.g., exonuclease). Furthermore, the present inventors have discovered a method for the efficient introduction of protected nucleotides in the form of a first terminal adapter molecule and a second terminal adapter molecule at both ends of a linear double-stranded region of the linear DNA product.The methods described herein may use a hairpin or loop adapter at one end of the DNA product and a linear adapter comprising protected nucleotides at the other end. That is, any type of adapter described herein may be used provided the final DNA product is protected from nuclease digestion (e.g., exonuclease). The methods of the invention provide protection against digestion by exonucleases that cleave nucleotides from the 3' end (e.g., exonuclease III) and exonucleases that cleave nucleotides from the 5' end (e.g., exonuclease VIII). Therefore, the linear DNA product produced by the methods of the invention has prolonged in vivo expression compared to a linear DNA product that does not comprise an adapter molecule described herein. The use of intermediate adapter molecules, which are added sequentially—that is, each intermediate adapter molecule is added to an adjacent intermediate adapter molecule or to the first or second terminal adapter molecule—provides greater flexibility in the structure, function, and / or length of the linear DNA product. For example, intermediate adapter molecules can comprise a tag, a targeting sequence, a coding sequence, or can be used to provide greater length to the DNA product. As used herein, the term "protected nucleotide" or "nuclease-resistant nucleotide" is intended to encompass any type of molecule that provides or enhances resistance to nuclease digestion (especially exonuclease digestion). Although adapter molecules are described herein as comprising nucleotides with phosphorothioate, a person skilled in the art would appreciate that adapter molecules can comprise any molecule that provides resistance to nuclease digestion (e.g., exonuclease III digestion). For example, adapter molecules may comprise nuclease-resistant nucleotides, that is, modified nucleotides that provide or enhance resistance to nucleases (e.g., exonuclease). Adapter molecules may comprise a peptide, polypeptide, or protein that provides or enhances resistance to nuclease digestion (e.g., exonuclease).Adapter molecules may comprise 2-O-methyl nucleotides or 2-O-methoxyethyl (MOE) nucleotides. As used herein, the term "phosphorothioate nucleotide" refers to a nucleotide having an altered phosphate backbone in which the sugar residues are linked by a phosphorothioate bond. In the phosphate backbone of an oligonucleotide sequence, the phosphorothioate bond contains a sulfur atom as a substitute for a non-bonding oxygen atom. This modification makes the bond between nucleotides resistant to degradation by nucleases. As used herein, "terminal adapter molecule" refers to an adapter molecule that is added to one end of the linear DNA region, directly or indirectly through one or more intermediate adapter molecules, to form the end of the linear DNA product. A terminal adapter molecule is nuclease-resistant once added to the linear double-stranded region, directly or indirectly. A terminal adapter molecule confers nuclease resistance to the linear DNA product. As used herein, an "intermediate adapter molecule" refers to an adapter molecule that is added to one end of another intermediate adapter molecule or to one end of the linear double-stranded region at its first end, and has another intermediate adapter molecule or a terminal adapter molecule added to its second end; i.e., an intermediate adapter molecule is hybridized or bonded to another molecule at both ends. As used herein, "sequentially" means one after the other. That is, the n intermediate adapter molecules are added end-to-end from the first end of the double-stranded DNA molecule such that the n-(n-1)th adapter molecule is added to the first end of the double-stranded DNA molecule, the nth adapter is added to the terminal adapter molecule; and the m intermediate adapter molecules are added end-to-end from the second end of the double-stranded DNA molecule such that the m-(m-1)th adapter molecule is added to the first end of the double-stranded DNA molecule, the mth adapter is added to the terminal adapter molecule.For example, in the case where n is two and m is one, the linear DNA molecule comprises, from one end to the other, a first terminal adapter molecule--n2 intermediate adapter molecule--n1 intermediate adapter molecule--double-stranded linear region--m1 intermediate adapter molecule--second terminal adapter molecule. Each adapter molecule may comprise a sequence at its first end that is compatible with a sequence at the second end of the next adapter molecule to be added or ligated sequentially. For example, when n=2, the first intermediate adapter molecule will have a sequence at its second end that is complementary to a sequence at the first end of the linear double-stranded region. The second (or nth) intermediate adapter molecule may comprise a sequence at its second end that is complementary to the sequence at the first end of the first intermediate adapter molecule and a sequence at its first end that is complementary to a sequence at one end of the first terminal adapter molecule. When each adapter molecule comprises a different complementary sequence, the adapter molecules will be added, or ligated, sequentially.Different complementary sequences can be generated using type IIS restriction endonucleases that cut at a target sequence at a location separate from the cleavage protrusion, for example, BsaI. Up to 256 different 4-nucleotide protrusions can be created using type IIS endonucleases, with intermediate and terminal adapter molecules added sequentially and directionally. As used herein, "adjacent intermediate adapter molecule" or "adjacent adapter molecule" refers to an adapter molecule that is added or bonded directly to one end of another adapter molecule. The closed DNA product comprises n intermediate adapter molecules between the first terminal adapter molecule and the first end of the linear double-stranded region, and comprises m intermediate adapter molecules between the second terminal adapter molecule and a second end of the linear double-stranded region, where nym are each 0 or an integer of at least 1, and n+m is at least 1. The first end of the linear double-stranded region and the first terminal adapter molecule can be at either the 3' or 5' end of the closed linear DNA product. The first end of the linear double-stranded DNA product and the first terminal adapter molecule can be in the 5' or 3' direction of the DNA sequence of the linear double-stranded region. The linear DNA product (e.g., the closed linear DNA product) produced by the methods of the present invention has additional advantageous properties, such as a substantial lack of a bacterial backbone and / or antibiotic resistance genes. The absence of these features is particularly beneficial in the production of a cell delivery system, such as a viral vector or a nanoparticle, for example, for cell therapy. The absence of these features makes the linear DNA product produced by the methods of the present invention particularly suitable for use in a pharmaceutical composition. Unexpectedly, the present inventors have discovered a method for producing a linear DNA product with enhanced resistance to nuclease digestion. This method allows for the efficient production of large quantities of the linear DNA product with enhanced resistance to exonuclease digestion, along with the ability to add certain characteristics to the linear DNA product. Large-scale manufacturing of the product can be performed in a cell-free system, resulting in the production of a pure sample comprising the linear DNA product substantially free of bacterial contaminants (e.g., those remaining after cell lysis). 1. Methods for producing a closed linear DNA product The methods described herein can be used to produce a closed linear DNA product, for example, a covalently closed linear DNA product. The invention provides a method for producing a closed linear DNA product, the method comprising: (a) contacting a double-stranded DNA molecule with an endonuclease and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (b) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and closed at a second end by the second terminal adapter molecule added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region. n can be 0, 1, 2, 3, 4, 5, 6, 7 or 8. m can be 0, 1, 2, 3, 4, 5, 6, 7 or 8. Preferably, n is 0, 1, 2, 3 or 4, and m is 0, 1, 2, 3 or 4. The step of contacting the double-stranded DNA molecule with the endonuclease and first and second terminal adapter molecules is preferably performed in the presence of a ligase. Therefore, the invention provides a method for producing a closed linear DNA product, wherein the method comprises: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (b) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and closed at a second end by the second terminal adapter molecule added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region. The linear double-stranded region may be the linear portion of the double-stranded DNA molecule. The invention provides a method for producing a closed linear DNA product, wherein the method comprises: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (b) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and closed at a second end by the second terminal adapter molecule added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region. A closed linear DNA product has particular utility as a therapeutic agent (i.e., therapeutic DNA) that can be used to express a gene product in vivo. This is because its closed structure (e.g., covalently closed structure) prevents attack by enzymes such as exonucleases, leading to greater stability and longevity of gene expression compared to "open" DNA molecules with exposed DNA ends. Linear double-stranded open-end cassettes have been shown to be inefficient with respect to gene expression when introduced into host tissue. This has been attributed to cassette instability due to the action of exonucleases in the extracellular space. Sequestering DNA ends within closed structures also offers other advantages. It prevents DNA ends from integrating with genomic DNA, thus providing enhanced safety for closed linear DNA products. Furthermore, the closed linear structure reduces the concatenation of DNA products within host cells, allowing for more sensitive regulation of gene product expression levels. The method of the invention can be used for the production of DNA for in vitro expression in a host cell, for example, in DNA vaccines. DNA vaccines typically encode a modified form of the DNA of an infectious organism. These vaccines are administered to a subject, where they then express the selected protein from the infectious organism, initiating an immune response against that protein, which is normally protective. DNA vaccines can also encode a tumor antigen in an immunotherapy approach against cancer. The method can produce other types of therapeutic DNA molecules, such as those used in gene therapy. For example, such DNA molecules can be used to express a functional gene in individuals with a genetic disorder caused by a dysfunctional version of that gene. Examples of such diseases include sickle cell anemia, cystic fibrosis, Huntington's disease, Duchenne muscular dystrophy, hemophilia A, alpha-1 antitrypsin deficiency, primary ciliary dyskinesia, and respiratory distress syndrome of prematurity. Other diseases where gene therapy may be useful include metabolic diseases, respiratory diseases, inflammatory diseases, autoimmune diseases, chronic and infectious diseases, including disorders such as AIDS, cancer, neurological diseases, cardiovascular disease, hypercholesterolemia, various blood disorders, including various anemias, thalassemia, and hemophilia, and emphysema.For the treatment of solid tumors, genes encoding toxic peptides (i.e., chemotherapeutic agents such as ricin, diphtheria toxin, and copper poison factor), tumor suppressor genes such as p53, genes encoding mRNA sequences that are antisense to transforming oncogenes, antineoplastic peptides such as tumor necrosis factor (TNF) and other cytokines, or transdominant negative mutants of transforming oncogenes may be expressed. The addition of intermediate adapter molecules allows for additional flexibility in the production of the closed linear DNA products of the invention. Intermediate adapter molecules can enable the production of longer closed linear DNA products. These molecules allow for the incorporation of barcodes, promoter sequences, tags, poly-A signals, and / or open reading frames into the closed linear DNA product of the invention. An intermediate adapter molecule may comprise a cassette. For example, if it is advantageous to include several genes of interest, or open reading frames, in the closed linear DNA product, the intermediate adapter molecules may comprise one or more cassettes, and the linear DNA region may comprise one or more cassettes. An intermediate adapter molecule may comprise a tag, a signaling sequence, a targeting sequence, modified nucleotides, or a binding residue.An intermediate adapter molecule can comprise a promoter, a UTR, a transcription factor binding site, or a termination sequence. For example, intermediate adapter molecules that are added or ligated in the 5' direction of the linear double-stranded region can be used to add or change a promoter sequence, or to add or change UTRs. Intermediate adapter molecules added or ligated in the 3' direction of the linear double-stranded region can be used to add poly-A signals or sequences, terminators, or to change or add UTRs. Intermediate adapter molecules can also be used to add barcodes, fluorophores, or modified nucleotides in either the 5' or 3' direction of the linear double-stranded region. A modified nucleotide can be 2-methoxyethoxy A, 2-methoxyethoxy MeC, 2-methoxyethoxy G, 2-methoxyethoxy T, 2-O-methyl RNA bases, fluorinated bases, 2-aminopurine, 5-bromo dU, deoxyuridine, 2, 6-diaminopurine (2-amino-dA), dideoxy-C, deoxyinosine, hydroxymethyl dC, iso-dG, iso-dC, 5-methyl dC or 5-nitroindole. In addition to providing nuclease resistance to the closed linear DNA product, terminal adapter molecules can also provide additional functional features. A terminal adapter molecule can enable the production of longer closed linear DNA products. Terminal adapter molecules can allow barcodes, promoter sequences, tags, poly-A signals, and / or open reading frames to be incorporated into the closed linear DNA product of the invention. A terminal adapter molecule can comprise a tag, a signaling sequence, a targeting sequence, or a binding residue. A terminal adapter molecule can comprise a UTR or a termination sequence. The closure of the linear double-stranded region by the first terminal adapter molecule and / or the second terminal adapter molecule (directly or indirectly through one or more intermediate adapter molecules) can be achieved by hybridization or ligation of the adapter molecules to the ends of the linear double-stranded region and / or to sequential adapter molecules. Therefore, n intermediate adapter molecules can hybridize sequentially to the first end of the linear double-stranded region. m intermediate adapter molecules can hybridize sequentially to the second end of the linear double-stranded region. n intermediate adapter molecules can ligate sequentially to the first end of the linear double-stranded region. m intermediate adapter molecules can ligate sequentially to the second end of the linear double-stranded region.The addition of the n+m intermediate adapter molecules, the first terminal adapter molecule, and the second terminal adapter molecule can occur through either hybridization or ligation of the adapter molecules to the ends of the linear double-stranded region and / or adjacent adapter molecules. Therefore, intermediate adapter molecules can hybridize and ligate sequentially to the first end of the linear double-stranded region. The m intermediate adapter molecules can hybridize and ligate sequentially to the second end of the linear double-stranded region. The addition can be facilitated by a ligand or spacer molecule that enables the adapter molecule to bind to the first and / or second end of the linear double-stranded region or to an adjacent adapter molecule.The first terminal adapter molecules can hybridize, ligate, or hybridize and ligate to the nth intermediate adapter molecule or to the first end of the double-stranded DNA molecule. The second terminal adapter molecule can hybridize, ligate, or hybridize and ligate to the mth intermediate adapter molecule or to the second end of the double-stranded DNA molecule. n can be 0, 1, 2, 3, 4, 5, 6, 7, or 8. m can be 0, 1, 2, 3, 4, 5, 6, 7, or 8. Preferably, n is 0, 1, 2, 3, or 4, and m is 0, 1, 2, 3, or 4. The step of incubating the single contiguous aqueous volume to generate the closed linear DNA product may comprise generating the linear portion of the double-stranded DNA molecule by subjecting the double-stranded DNA molecule to digestion with the endonuclease. The step of incubating the single contiguous aqueous volume can be carried out under conditions that promote the addition (or binding) of the first and second terminal adapter molecules and / or intermediate adapter molecules to the linear double-stranded region to produce the closed linear DNA product. The addition can be achieved by creating a covalent bond between the first and / or second terminal adapter molecule and the first and / or second end of the linear double-stranded region. The addition can be achieved by creating a covalent bond between the first and / or second terminal adapter molecule and an nth and / or nth intermediate adapter molecule. The addition can be achieved by creating a covalent bond between an intermediate adapter molecule and the first or second end of the linear double-stranded region. The addition can be achieved by creating a covalent bond between an intermediate adapter molecule and an adjacent adapter molecule. The incubation step of the single contiguous aqueous volume can be carried out under conditions that promote the digestion of the double-stranded DNA molecule to produce the linear portion. The digestion of the double-stranded DNA molecule to produce the linear portion can be performed at an initial temperature of 1°C–100°C, 1°C–80°C, 5°C–70°C, 10°C–60°C, 15°C–55°C, 20°C–50°C, 25°C–45°C, 30°C–40°C, 35°C–39°C, 36°C–38°C, or at approximately 37°C. The digestion can be by endonuclease digestion, preferably by type IIS endonuclease digestion. The step of incubating the single contiguous aqueous volume can be carried out under conditions that promote the binding of the linear double-stranded region to the first and second terminal adapter molecules and / or to intermediate adapter molecules. The step of incubating the single contiguous aqueous volume can be carried out under conditions that promote the binding of the intermediate adapter molecules to the first and second terminal adapter molecules and / or adjacent intermediate adapter molecules and / or to the first and / or second end of the linear double-stranded region. The ligation can have an efficiency of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95%.For example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% of the linear double-stranded regions (or portions of double-stranded DNA molecules) can be incorporated into closed linear DNA products. Preferably, the ligation has an efficiency of at least 15%. The ligation efficiency can be established based on the DNA quantification values ​​before and after the digestion / ligation reaction. Therefore, the ligation efficiency can be established based on the equation: (initial amount of amplified DNA) / (final amount of linear DNA) x 100%. Ligation efficiency can also be established based on DNA quantification values ​​before and after the digestion / ligation reaction and subsequent exonuclease treatment to remove remaining open DNA constructs and excess adapter molecules. For example, the double-stranded DNA molecule generated by rolling circle amplification is first quantified so that the amount of double-stranded DNA used as starting material during the digestion / ligation reaction is known. After all enzymatic reactions, the linear DNA product is quantified to calculate the ligation efficiency according to the equation above. DNA quantification methods are known to an expert in the technique. For example, DNA quantifications can be carried out using the ThermoFisher Qubit dsDNA BR assay (https: / / www.thermofisher.com / order / catalog / product / Q32850# / Q32850). The step of linking the linear double-stranded region to the first and second terminal adapter molecules can be performed at a second temperature of 1 °C-90 °C, 2 °C-70 °C, 5 °C-60 °C, 8 °C-55 °C, 9 °C-50 °C, 10 °C-45 °C, 11 °C-40 °C, 12 °C-37 °C, 13 °C-30 °C, 14 °C-25 °C, 15 °C-20 °C or approximately 16 °C. The step of incubating the single contiguous aqueous volume may comprise incubating at a first temperature and then incubating at a second temperature. The first temperature may be 1°C–100°C, 1°C–80°C, 5°C–70°C, 10°C–60°C, 15°C–55°C, 20°C–50°C, 25°C–45°C, 30°C–40°C, 35°C–39°C, 36°C–38°C, or approximately 37°C. The second temperature can be 1°C–90°C, 2°C–70°C, 5°C–60°C, 8°C–55°C, 9°C–50°C, 10°C–45°C, 11°C–40°C, 12°C–37°C, 13°C–30°C, 14°C–25°C, 15°C–20°C, or approximately 16°C. Preferably, the first temperature is 35°C–39°C and the second temperature is 14°C–18°C. Under these conditions, the endonuclease can be a type IIS restriction endonuclease (e.g., Bsal), and the ligase can be T4 DNA ligase, T7 DNA ligase, mammalian DNA ligase I, III, and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase. The step of incubating the single contiguous aqueous volume can be performed isothermally. The step of incubating the single contiguous aqueous volume can comprise incubation at a constant temperature. The constant temperature promotes the simultaneous digestion of the double-stranded DNA molecule to produce the linear portion of the double-stranded DNA molecule and the ligation of the linear double-stranded region to the n+m intermediate adapter molecules and / or the first and second terminal adapter molecules. For example, the constant temperature can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. Preferably, the constant temperature is 30 °C. The constant temperature is intended to mean that the temperature does not change significantly during the reaction.Constant temperature is intended to mean that the temperature variation during the incubation stage of the single contiguous aqueous volume is less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, or less than 1°C. In a preferred embodiment, the temperature during the incubation stage of the single contiguous aqueous volume does not deviate by more than 5°C, preferably by no more than 3°C, and even more preferably by no more than 1°C. Therefore, the constant temperature can be a temperature in the range of 20°C–30°C, 22°C–32°C, 24°C–34°C, 26°C–36°C, 28°C–38°C, 30°C–40°C, 22°C–28°C, 32°C–38°C, 25°C–35°C, 26°C–34°C, 27°C–33°C, 27.5°C–32.5°C, 28°C–32°C, 28.5°C–31.5°C, 29°C–31°C, or 29.5°C–30.5°C. Preferably, the constant temperature is a temperature in the range of 27.5°C–32.5°C.Alternatively, the constant temperature can be a temperature in the range of 32°C–42°C, 33°C–41°C, 34°C–40°C, 35°C–39°C, or 36°C–38°C. Preferably, the constant temperature is a temperature in the range of 34.5°C–39.5°C. The step of incubating the single contiguous aqueous volume may comprise subjecting it to cycling between the first temperature and the second temperature. The step of incubating the single contiguous aqueous volume may comprise subjecting it to cycling between the first temperature and the second temperature at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 80, at least 90, or at least 100 times, preferably at least 20 times. The incubation stage of the single contiguous aqueous volume may comprise subjection to cycles between the first temperature and the second temperature less than 40, less than 35, less than 30 times, less than 29, less than 25 times.The incubation stage of the single contiguous aqueous volume may comprise subjection to cycling between the first and second temperatures 2-100, 5-80, 10-70, 20-60, or 30-60 times. The incubation stage of the single contiguous aqueous volume may comprise subjection to cycling between the first and second temperatures 2-20, 5-29, 61-100, or 65-80 times. The method may further comprise, prior to step (a) (i.e., the step of contacting the double-stranded DNA molecule with the endonuclease, ligase, and the first and second terminal adapter molecules), a step of amplifying a DNA template molecule to produce the double-stranded DNA molecule. Therefore, the invention provides a method for producing a closed linear DNA product, the method comprising: (a) amplifying a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (c) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and closed at a second end by the second terminal adapter molecule added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region. The amplification step can be performed by in vitro or in vivo amplification. Preferably, the amplification step is performed by in vitro amplification. For example, the amplification step can be performed by rolling circle amplification (RCA), the MALBAC method, traditional polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), and polymerase recombinase amplification (RPA). Preferably, the amplification step is performed by rolling circle amplification. Therefore, the invention provides a method for producing a closed linear DNA product, the method comprising: (a) amplifying a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule, wherein the DNA template molecule is amplified by rolling circle amplification; (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (c) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and closed at a second end by the second terminal adapter molecule added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region. Rolling circle amplification can be performed without a primer, or with one or more primers. For example, the primer can be a synthetic primer. The primers can be random primers. Rolling circle amplification can be performed in the presence of a primase. The primase can be TthPrimPol. Preferably, if rolling circle amplification is performed without a primer, it is performed in the presence of a primase, such as TthPrimPol. Similarly, if a primer is used during the amplification reaction, a primase is not used. The double-stranded DNA product can be generated by rolling circle amplification in vitro under isothermal conditions using a suitable nucleic acid polymerase, such as Phi29 DNA polymerase. In the methods described herein, the DNA template molecule may comprise at least one cleavable target sequence. The cleavable target sequence may be an endonuclease target sequence. Preferably, the DNA template molecule comprises at least two endonuclease target sequences. The endonuclease target sequences may be the same or different. Preferably, at least one endonuclease target sequence is a restriction endonuclease target sequence. Different restriction endonuclease target sequences shall be known to the person skilled in the art. The cleavable target sequence may be a type IIS restriction endonuclease target sequence.For example, the target sequence of restriction endonuclease can be a target sequence of BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuHPI, BaeI, BarI, BbvI, BccI, 1999; BceAI, BcgI, BciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, BsgI, BslFI, BsmAI, BsmFI, . BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BtsCI, BtsI-v2, BtsMutI, Bvel, Csel, CspCI, Eam1104I, EarI, Ecil, Eco31I, Eco57I, Esp3I, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1109l, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, NmeAIII, PaqCI, PciSI, Pctl, Piel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI and / or TspGWI. The at least one cleavable sequence (e.g., endonuclease target sequence) can be a native cleavable sequence (that is, a cleavable sequence present in the mold molecule) .Alternatively, at least one cleavable sequence (e.g., endonuclease target sequence) can be introduced into the DNA template molecule prior to the production of the closed linear DNA product. An endonuclease can be a restriction endonuclease enzyme. An endonuclease can be a type IIS restriction enzyme. An endonuclease can be any enzyme that recognizes a DNA sequence and cleaves it outside the recognition sequence.For example, the endonuclease can be a restriction enzyme BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuI, BaccP, Bacc, Bib, Bib BceAI, BcgI, BciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, BsgI, BsgFI, BsmFIA, BsmFII BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BtsCI, BtsI-v2, BtsMut, BspCI, CIS, CIS Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, GsuI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, MnIII, PaqI, NCIA PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI y / o TspGWI. The ligase can be a DNA ligase, such as a DNA T4 ligase, DNA T7 ligase, mammalian DNA ligase I, III and IV; DNA Taq ligase, DNA Tth ligase, or E. coli DNA ligase. The DNA template molecule used in the methods described herein may be single-stranded or double-stranded. Preferably, the DNA template molecule is double-stranded. The DNA template molecule may be a naturally occurring circular DNA molecule. For example, the DNA template molecule may be (i) a plasmid, (ii) a minicircle, (iii) a cosmid, (iv) a bacterial artificial chromosome (BAC), or (v) a molecular inversion probe (MIP). The DNA template molecule may be an enzymatically produced circular DNA molecule. For example, the DNA template molecule may be (i) a circular DNA molecule obtained from a recombinase reaction, preferably the Cre recombinase reaction, or (ii) a circular DNA molecule obtained from a ligase reaction, preferably using the Golden Gate assembly. The DNA template molecule may be an enzymatically produced covalently closed linear DNA molecule.For example, the DNA template molecule can be (i) a DNA molecule processed with TelN proteomerase; or (ii) a DNA molecule generated by ligating the DNA ends with an adapter. The DNA template molecule can comprise a double-stranded element and a single-stranded element. For example, the DNA template molecule can comprise a double-stranded DNA molecule and a single-stranded hairpin loop. The DNA template molecule can be linear. If the DNA template molecule is linear, prior to amplification (e.g., rolling circle amplification), a DNA template molecule can be circularized to produce a DNA template molecule suitable for use in the methods described herein. The DNA template molecule may comprise a cassette. The cassette may be a mammalian expression cassette. The cassette may further comprise a promoter. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence. The cassette may further comprise a LoxP sequence, preferably two LoxP sequences. If the two LoxP sequences are oriented in the same direction, the DNA sequence between the two LoxP sequences is cut as a circular loop of DNA. If the two LoxP sequences are oriented in opposite directions, the DNA sequence between the two LoxP sequences is inverted. Therefore, preferably, the two LoxP sequences are in the same orientation (i.e., the same direction) in the DNA template molecule. The DNA template molecule may comprise a homopolymeric sequence at a 5' end, a 3' end, or both. The homopolymeric sequence may be added to the DNA template molecule before circularization. The homopolymeric sequence may be a polyA, polyC, polyG, or polyT sequence. The homopolymeric sequence may be 3–200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the linear DNA product, in which case the homopolymeric sequence may be 4–12 nucleotides in length, or 5–10 nucleotides in length. The homopolymeric sequence may be used to enhance mRNA expression, in which case the homopolymeric sequence may be 10–200 nucleotides in length, preferably 80–150 nucleotides in length.The homopolymeric sequence may be at least 10, at least 20, 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 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides long. Preferably, the homopolymeric sequence is at least 100 nucleotides long. More preferably, the homopolymeric sequence is at least 120 nucleotides long. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides. The method may further comprise (after the amplification step and before the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules) a heat deactivation step. Therefore, the invention provides a method for producing a closed linear DNA product, the method comprising: (a) amplification of a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) heat deactivation of the reaction of step (a); (c) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (d) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and closed at a second end by the second terminal adapter molecule added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region. Preferably, the amplification is rolling circle amplification. The heat deactivation step may be carried out under conditions sufficient to inactivate the reagents used during the amplification reaction. The heat deactivation step may be carried out at a temperature of at least 50 °C, at least 55 °C, at least 60 °C, at least 65 °C, at least 70 °C, at least 75 °C, at least 80 °C, at least 85 °C, at least 90 °C, at least 95 °C, or at least 100 °C. The heat deactivation step may be carried out for at least 1 min, at least 3 min, at least 5 min, at least 10 min, at least 15 min, or at least 20 min. In the method described herein, after the amplification step, the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules to form a single contiguous aqueous volume can be performed without purifying the amplification reaction product. That is, the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules can be performed directly after the amplification step. The step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules can be performed directly after the heat deactivation step. The method may further comprise, after the incubation stage of the single contiguous aqueous volume, a purification stage of the closed linear DNA product. The method may further comprise, after the incubation step in the single contiguous aqueous volume, a nuclease digestion step. The nuclease digestion may be exonuclease digestion, such as exonuclease I and / or exonuclease III. The nuclease digestion step may take place before or after the purification step. This step allows for the removal of any double-stranded DNA and / or adapter molecules that were not used during the execution of the method. Therefore, the method may comprise the following steps: (a) amplifying a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) bringing the double-stranded DNA molecule into contact with an endonuclease, a ligase and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; (c) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and closed at a second end by the second terminal adapter molecule added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region; and (d) incubate the single contiguous aqueous volume with a nuclease (e.g., exonuclease). In the method described herein, after the amplification step, the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules to form a single contiguous aqueous volume can be performed without purifying the amplification reaction product. That is, the step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules can be performed directly after the amplification step. The step of contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules can be performed directly after the heat deactivation step. The method may include the following stages: (a) amplifying a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) bringing the double-stranded DNA molecule into contact with an endonuclease, a ligase and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; (c) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and closed at a second end by the second terminal adapter molecule added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region; (d) purify the closed linear DNA product; and (e) incubate the purified product from step (d) with a nuclease (e.g., exonuclease). The method may include the following stages: (a) amplifying a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) heat deactivation of the reaction of step (a); (c) bringing the double-stranded DNA molecule into contact with an endonuclease, a ligase and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; (d) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and closed at a second end by the second terminal adapter molecule added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region; (e) purify the closed linear DNA product; and (f) incubate the purified product from step (d) with a nuclease (e.g., exonuclease). The method may include the following stages: (a) amplifying a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) bringing the double-stranded DNA molecule into contact with an endonuclease, a ligase and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; (c) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and closed at a second end by the second terminal adapter molecule added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region; (d) incubate the single contiguous aqueous volume with a nuclease (for example, exonuclease); and (e) purify the closed linear DNA product. The method may include the following stages: (a) amplifying a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) heat deactivation of the reaction of step (a); (c) bringing the double-stranded DNA molecule into contact with an endonuclease, a ligase and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; (d) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein the closed linear DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and closed at a second end by the second terminal adapter molecule added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region; (e) incubate the single contiguous aqueous volume with a nuclease (for example, exonuclease); and (f) purify the closed linear DNA product. The step of incubating the single contiguous aqueous volume (or the purified product from step (d)) with a nuclease may be carried out at a temperature of 5–90 °C, 10–80 °C, 15–70 °C, 20–60 °C, 25–50 °C, 30–45 °C, or 35–40 °C. The step of incubating the single contiguous aqueous volume (or the purified product from step (d)) with a nuclease may be carried out for at least 10, at least 20, at least 30, at least 40, at least 50, or at least 60 min. The step of incubating the single contiguous aqueous volume (or the purified product from step (d)) may be carried out at two different temperatures. For example, the step of incubating the single contiguous aqueous volume (or the purified product from step (d)) can be carried out at 15-40 °C for 10-60 minutes followed by a temperature of 60-90 °C for 10-30 minutes. The higher temperature normally inactivates the nuclease (e.g., exonuclease).Therefore, the method also provides a nuclease (e.g., exonuclease) inactivation step. The step of incubating the single contiguous aqueous volume (or the purified product from step (d)) can be carried out at 37 °C for 30 min and 80 °C for 20 min. Preferably, the nuclease (e.g., exonuclease) inactivation step is carried out at a temperature of 70–80 °C. The nuclease (e.g., exonuclease) inactivation step can be carried out for at least 1, at least 5, at least 10, at least 20, or at least 30 min. Preferably, the nuclease (e.g., exonuclease) inactivation step is carried out for at least 5 min. The method can be a cell-free method. The closed linear DNA product may be partially double-stranded and / or partially single-stranded. The closed linear DNA product may comprise a portion that is double-stranded and a portion that is single-stranded. The closed linear DNA product may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example, a gene that encodes a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosome binding site, and a translation termination sequence. The cassette may further comprise sequences that aid protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas-mediated homology-directed repair (HDR). The cassette may encode the CRISPR guide RNA.The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, polyC, polyT, or polyG sequence. The homopolymeric sequence may be 3–200 nucleotides in length. The homopolymeric sequence may be used to facilitate cassette purification, in which case the homopolymeric sequence may be 4–12 nucleotides in length, or 5–10 nucleotides in length. The homopolymeric sequence may be used to enhance mRNA expression, in which case the homopolymeric sequence may be 10–200 nucleotides in length, preferably 80–150 nucleotides in length.The homopolymeric sequence can be at least 10, at least 20, 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 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides long. For example, the homopolymeric sequence can comprise a polyA sequence of at least 120 nucleotides. The closed linear DNA product may comprise a spacer. The spacer may be at least 10, at least 20, 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 125, at least 150, at least 175, or at least 200 base pairs in length. The closed linear DNA product may comprise an inverted terminal repeat sequence. The closed linear DNA product may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the closed linear DNA product is at least 50 base pairs long. The double-stranded DNA molecule can be circular, or branched. The double-stranded DNA molecule may not comprise an adapter. The double-stranded DNA molecule may not comprise a hairpin, loop, or stem-loop structure. The double-stranded DNA molecule may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example, a gene that encodes a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosome binding site, and a translation termination sequence. The cassette may further comprise sequences that aid protein expression, such as a cap-independent translation element. The cassette may comprise (or modify) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas-mediated homology-directed repair (HDR). The cassette may encode the CRISPR guide RNA.The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, polyC, polyT, or polyG sequence. The homopolymeric sequence may be 3–200 nucleotides in length. The homopolymeric sequence may be used to facilitate cassette purification, in which case the homopolymeric sequence may be 4–12 nucleotides in length, or 5–10 nucleotides in length. The homopolymeric sequence may be used to enhance mRNA expression, in which case the homopolymeric sequence may be 10–200 nucleotides in length, preferably 80–150 nucleotides in length.The homopolymeric sequence may be at least 10, at least 20, 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 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides long. Preferably, the homopolymeric sequence is at least 100 nucleotides long. More preferably, the homopolymeric sequence is at least 120 nucleotides long. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides. The double-stranded DNA molecule may include a spacer. The spacer may be at least 10, at least 20, 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 125, at least 150, at least 175, or at least 200 base pairs long. The spacer may improve the amplification performance of the double-stranded DNA molecule. The double-stranded DNA molecule can be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long. The double-stranded DNA molecule may comprise one or more cleavable target sequences (e.g., endonuclease). The double-stranded DNA molecule may comprise two cleavable target sequences (e.g., endonuclease). The one or more cleavable target sequences (e.g., endonuclease) may be type IIS endonuclease target sequences.The one or more cleavable diana sequences (e.g., endonuclease) may be diana sequences of BBSI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AARI, Acc36I, AclWI, ACUI, AjuI, ArHPI, AlwI, AlwWI BAEI, ​​BARI, BVI, BCCI, BCEAI, BCGI, BCVI, BCODI, BFUI, BFUI, BMRI, BMSI, BMUI, BP1I, BPMI, BSEGI, BSAXI, BSEI, BSEI, BSEGI, BSEMI, BSEMI, BSEI, BslFI, BsmAI, BsmFI, BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, Bstv1I, BstV2I, BsuI, BtCigZI, Btsv, Bts CseI, CspCI, EAM1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, GsuI, HGAI, HphI, HpyAV, LGUI, LmnI, Lsp1109I, LweI, MboII,MboII, M2A6, MlyIII,I PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI and / or TspGWI. The bicatenary DNA molecule may be an amplification product. Preferably, the amplification is amplified by rolling circle. The linear double-stranded region (i.e., the linear portion of the double-stranded molecule) can be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long. The linear double-stranded region (e.g., the linear portion of the double-stranded molecule) may comprise a sequence that is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the sequence of the double-stranded DNA molecule. The first and second ends of the linear double-stranded region (e.g., the linear portion of the double-stranded molecule) may be resistant to nuclease digestion. Preferably, the first and second ends of the linear double-stranded region are resistant to exonuclease digestion, such as exonuclease III and / or exonuclease I. The linear double-stranded region may comprise a 3-OH group at the first and / or second end. The 3-OH group may facilitate ligation to the first and / or second terminal adaptor molecule(s) and / or n+m intermediate adapter molecules (which may comprise a 5-phosphate). The linear double-stranded region may comprise a 5-phosphate at the first and / or second end. The 5-phosphate may facilitate ligation to the first and / or second terminal adaptor molecule(s) (which may comprise a 3-OH group). The linear double-stranded region (e.g., the linear portion of the double-stranded molecule) may include a protrusion. For example, the linear double-stranded region may include a 5-protrusion or a 3-protrusion. The linear double-stranded region may include one or more blunt ends. The linear double-stranded region may include: one 5-protrusion and one blunt end, two 5-protrusions, one 3-protrusion and one blunt end, two 3-protrusions, or one 5-protrusion and one 3-protrusion. The protrusion may be at least 3 nucleotides long (preferably 4 to 8 nucleotides). The protrusion may be on the sense strand or the antisense strand of the linear double-stranded region. The linear portion of the double-stranded DNA molecule (e.g., the linear portion of the double-stranded molecule) can be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more or n+m of the n+m intermediate adapter molecules may be a synthetic adapter molecule. The first terminal adapter molecule may be a nucleic acid adapter molecule. The second terminal adapter molecule may be a nucleic acid adapter molecule. One or more, or n+m, of the n+m intermediate adapter molecules may be nucleic acid adapter molecules. The first terminal adapter molecule and / or the second terminal adapter molecule may comprise a self-complementary element that creates a loop, such as a hairpin loop or a stem-loop. Therefore, the first terminal adapter molecule may comprise a hairpin or a stem-loop. The second terminal adapter molecule may comprise a hairpin or a stem-loop. Both the first and second terminal adapter molecules may comprise a hairpin or a stem-loop.The terminal or intermediate adapter molecules may each comprise a double-stranded portion comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are joined together at a hairpin such that the sense strand hybridizes with the antisense strand. The double-stranded portion of an adapter may comprise a 3- or 5-prong of at least 1, at least 2, at least 3, at least 4, or at least 5 nucleotides. Preferably, the 3- or 5-prong is 4–8 nucleotides long. Each end of the linear double-stranded region (or linear portion of the double-stranded DNA molecule) may comprise a 3- or 5-prong. A portion of the first terminal adapter molecule (e.g., the protruding portion) may be complementary to the first end of the linear double-stranded region or to a first end of an intermediate adapter molecule, preferably the nth intermediate adapter molecule. A portion of the second terminal adapter molecule (e.g., the protruding portion) may be complementary to the second end of the linear double-stranded region or to a first end of an intermediate adapter molecule, preferably the mth adapter molecule. A portion of the first end of the linear double-stranded region may be complementary to a first end of an intermediate adapter molecule, preferably an n-(n-1) intermediate adapter molecule. A portion of the second end of the linear double-stranded region may be complementary to a first end of an intermediate adapter molecule, preferably an m-(m-1) intermediate adapter molecule.A portion of a second end of an intermediate adapter molecule may be complementary to a first end of an adjacent intermediate adapter molecule; for example, a portion of the second end of the nth intermediate adapter molecule may be complementary to a portion of the first end of the n-1st intermediate adapter molecule, and a portion of the second end of the n-1st adapter molecule may be complementary to a portion of the first end of the n-2nd intermediate adapter molecule, and so on.A portion of the second end of an intermediate adapter molecule may be complementary to a first end of an adjacent intermediate adapter molecule; for example, a portion of the second end of the m-th intermediate adapter molecule may be complementary to a portion of the first end of the m-1st intermediate adapter molecule, and a portion of the second end of the m-1st adapter molecule may be complementary to a portion of the first end of the m-2nd intermediate adapter molecule, and so on. n may be 0, 1, 2, 3, 4, 5, 6, 7, or 8. m may be 0, 1, 2, 3, 4, 5, 6, 7, or 8. Preferably, n is 0, 1, 2, 3, or 4, and m is 0, 1, 2, 3, or 4. The closed linear DNA product can be a covalently closed linear DNA product. Therefore, in embodiments where the terminal adapter molecules comprise a loop (e.g., a hairpin), the terminal adapter molecules close the ends of the linear double-stranded region (or close the ends of the nth and mth intermediate adapter molecules) forming a covalently closed linear DNA product. The first and / or second terminal adapter molecule may comprise or consist of the sequence of SEQ ID NO: 9 or a portion thereof. The first and / or second terminal adapter molecule may comprise at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides of SEQ ID NO: 9. The double-stranded portion of the first and / or second terminal adapter molecule may comprise the sequence of SEQ ID NO: 10 or a portion thereof. The double-stranded portion of the first and / or second terminal adapter molecule may comprise at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 contiguous nucleotides of SEQ ID NO: 10. The single-stranded portion of the first and / or second terminal adapter molecule may comprise an ACTCA sequence.The single-stranded portion of the first terminal adapter molecule and / or the second terminal adapter molecule may comprise at least 1, at least 2, at least 3, at least 4, or at least 5 contiguous nucleotides of the ACTCA sequence. The first and / or second terminal adapter molecule may comprise the sequence SEQ ID NO: 12. The first and / or second adapter molecule may comprise at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides of SEQ ID NO: 12. The invention provides a method for producing a covalently closed linear DNA product, wherein the method comprises: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1, and wherein n+m is at least 1, wherein the first and second terminal adapter molecules are nucleic acid adapter molecules each comprising a hairpin; and (b) incubating the single contiguous aqueous volume to generate the covalently closed linear DNA product, wherein the covalently closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, wherein the closed linear DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and closed at a second end by the second terminal adapter molecule added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,wherein (i) the first terminal adapter molecule comprises a protrusion that is complementary and hybridizes to a protrusion at the first end of the linear double-stranded region or to the nth intermediate adapter molecule, thereby closing the first end of the linear double-stranded region, and (ii) the second terminal adapter molecule comprises a protrusion that is complementary and hybridizes to a protrusion at the second end of the linear double-stranded region or to the nth intermediate adapter molecule, thereby closing the second end of the linear double-stranded region. The invention provides a method for producing a covalently closed linear DNA product, wherein the method comprises: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1, and wherein n+m is at least 1, wherein the first and second terminal adapter molecules are nucleic acid adapter molecules each comprising a hairpin; and (b) incubating the single contiguous aqueous volume to generate the covalently closed linear DNA product, wherein the covalently closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and closed at a second end by the second terminal adapter molecule added to the mth intermediate adapter molecule, or when m is 0,to the second end of the linear double-stranded region, wherein (i) the first terminal adapter molecule comprises a protrusion that is complementary to and hybridizes with a protrusion at the first end of the linear double-stranded region or with the nth intermediate adapter molecule, thereby closing the first end of the linear double-stranded region, and (ii) the second terminal adapter molecule comprises a protrusion that is complementary to and hybridizes with a protrusion at the second end of the linear double-stranded region or with the nth intermediate adapter molecule, thereby closing the second end of the linear double-stranded region, and wherein the first terminal adapter molecule is linked to the first end of the linear double-stranded region and the second terminal adapter molecule is linked to the second end of the linear double-stranded region. The invention provides a method for producing a covalently closed linear DNA product, wherein the method comprises: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1, and wherein n+m is at least 1, wherein the first and second terminal adapter molecules are nucleic acid adapter molecules each comprising a hairpin; and (b) incubating the single contiguous aqueous volume to generate the covalently closed linear DNA product, wherein the covalently closed linear DNA product comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and closed at a second end by the second terminal adapter molecule added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,wherein (i) the first terminal adapter molecule comprises a protrusion that is complementary and hybridizes to a protrusion at the first end of the linear portion of the double-stranded DNA molecule or to the nth intermediate adapter molecule, thereby closing the first end of the linear portion of the double-stranded DNA molecule and (ii) the second terminal adapter molecule comprises a protrusion that is complementary and hybridizes to a protrusion at the second end of the linear portion of the double-stranded DNA molecule or to the nth intermediate adapter molecule, thereby closing the second end of the linear portion of the double-stranded DNA molecule, and wherein the first terminal adapter molecule is linked to the first end of the linear portion of the double-stranded DNA molecule or to the nth intermediate adapter molecule,and the second terminal adapter molecule is linked to the second end of the linear portion of the double-stranded DNA molecule or to the m intermediate adapter molecule. n can be 0, 1, 2, 3, 4, 5, 6, 7 or 8. m can be 0, 1, 2, 3, 4, 5, 6, 7 or 8. Preferably, n is 0, 1, 2, 3 or 4, and m is 0, 1, 2, 3 or 4. The first terminal adapter molecule and / or the second terminal adapter molecule and / or the n+m intermediate adapter molecules may not be a plasmid or a vector DNA. The first terminal adapter molecule and / or the second terminal adapter molecule may comprise a single-stranded portion. The single-stranded portion may form a hairpin or a stem-loop. Therefore, the first terminal adapter molecule and / or the second terminal adapter molecule may comprise a loop portion. The single-stranded portion may comprise fewer than 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides. Preferably, the single-stranded portion comprises 5 nucleotides. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise a double-stranded portion. The double-stranded portion may comprise less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, or less than 10 base pairs. The double-stranded portion may comprise 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, or at least 15 base pairs. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise a 5-phosphate. The 5-phosphate may facilitate ligation to the linear double-stranded region or to adjacent terminal or intermediate adapter molecules (which may comprise a 3-OH group at the first and / or second end). The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise a 3-OH group. The 3-OH group may facilitate ligation to the linear double-stranded region and / or to adjacent intermediate adapter molecules (which may comprise a 5-phosphate at the first and / or second end). The first terminal adapter molecule and / or the second terminal adapter molecule may comprise the sequence of SEQ ID NO: 1 or a portion thereof. The first terminal adapter molecule and / or the second terminal adapter molecule may comprise at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides of SEQ ID NO: 1. The double-stranded portion of the first terminal adapter molecule and / or the second terminal adapter molecule may comprise the sequence of SEQ ID NO: 2 or a portion thereof. The double-stranded portion of the first terminal adapter molecule and / or the second terminal adapter molecule may comprise at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 contiguous nucleotides of SEQ ID NO: 2.The single-stranded portion of the first terminal adapter molecule and / or the second terminal adapter molecule may comprise an ACTCA sequence. The single-stranded portion of the first terminal adapter molecule and / or the second terminal adapter molecule may comprise at least 1, at least 2, at least 3, at least 4, or at least 5 contiguous nucleotides of the ACTCA sequence. The first and second adapter molecules may comprise an identical nucleic acid sequence. The first and second adapter molecules may comprise a different nucleic acid sequence. The first terminal adapter molecule may comprise a portion that is complementary to the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second terminal adapter molecule may comprise a portion that is complementary to the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The first terminal adapter molecule may comprise a portion that hybridizes to the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second terminal adapter molecule may comprise a portion that hybridizes to the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule).The first terminal adapter molecule may comprise a portion that is complementary to and hybridizes with the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second terminal adapter molecule may comprise a portion that is complementary to and hybridizes with the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The portion that is complementary to or hybridizes with the first or second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule) can be a 5' or 3' protrusion of the first and / or second terminal adapter molecule. The protrusion of the first terminal adapter molecule can be complementary to the first end of the linear double-stranded region, and / or the protrusion of the second terminal adapter molecule can be complementary to the second end of the double-stranded region. The protrusion of the first terminal adapter molecule can hybridize with the first end of the linear double-stranded region, and / or the protrusion of the second terminal adapter molecule can hybridize with the second end of the linear double-stranded region.The protrusion of the first terminal adapter molecule can be complementary and hybridize with the first end of the linear double-stranded region and / or the protrusion of the second terminal adapter molecule can be complementary and hybridize with the second end of the linear double-stranded region. The first terminal adapter molecule may comprise a portion that is complementary to the first end of the nth intermediate adapter molecule. The second terminal adapter molecule may comprise a portion that is complementary to the first end of the mth intermediate adapter molecule. The first terminal adapter molecule may comprise a portion that hybridizes to the first end of the nth intermediate adapter molecule. The second terminal adapter molecule may comprise a portion that hybridizes to the first end of the mth intermediate adapter molecule. The first terminal adapter molecule may comprise a portion that is complementary to and hybridizes to the first end of the nth intermediate adapter molecule. The second terminal adapter molecule may comprise a portion that is complementary to and hybridizes to the first end of the mth intermediate adapter molecule. The portion that is complementary to or hybridizes with the first end of the nth or mth intermediate adapter molecule can be a 5-protrusion or a 3-protrusion of the first and / or second terminal adapter molecule. The protrusion of the first terminal adapter molecule can be complementary to the first end of the nth intermediate adapter molecule, and / or the protrusion of the second terminal adapter molecule can be complementary to the first end of the mth intermediate adapter molecule. The protrusion of the first terminal adapter molecule can hybridize with the first end of the nth intermediate adapter molecule, and / or the protrusion of the second terminal adapter molecule can hybridize with the first end of the mth intermediate adapter molecule.The protrusion of the first terminal adapter molecule can be complementary and hybridize with the first end of the nth intermediate adapter molecule and / or the protrusion of the second terminal adapter molecule can be complementary and hybridize with the first end of the mth intermediate adapter molecule. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may not comprise a type IIS endonuclease target sequence.The first terminal adapter molecule and / or the second terminal adapter molecule may not comprise target sequences of BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw2I, ArwI, ASUI, ASUI BaeI, BarI, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMIX, BseMIX, BsegI, BsegI, BseI, BseMI BslFI, BsmAI, BsmFI, BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BcCI, BcI-Mut-I, Bsp-MutI BveI, CseI, CspCI, Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, GsuI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, Mly, MnI, MnI, MnI, 1269 NmeAIII, PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI y / o TspGWI. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise one or more locked nucleic acids (LNAs). The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise one or more protected nucleotides (i.e., nuclease-resistant nucleotides), such as phosphorothioate-containing nucleotides. The protected nucleotides may be located in the single-stranded portion (e.g., hairpin portion) of the terminal adapter molecules or in the double-stranded portion of the terminal or intermediate adapter molecules. The protected nucleotides may also be located in the protruding portion of the terminal or intermediate adapter molecules. The closed linear DNA product may comprise a plurality of nucleotides with phosphorothioate in internal positions on each strand. For example, the closed linear DNA product may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, 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 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g., nucleotides with phosphorothioate) in internal positions on each strand. Preferably, the closed linear DNA product comprises at least 2 protected nucleotides (e.g., phosphorothioate nucleotides) in internal positions on each strand. Terminal and / or intermediate adapter molecules may comprise at least two types of phosphorothioate nucleotides. For example, the at least two types of phosphorothioate nucleotides are: aS-dATP and aS-dCTP, aS-dATP and aS-dGTP, aS-dATP and aS-dTTP, aS-dCTP and aS-dGTP, aS-dCTP and aS-dTTP, or aS-dGTP and aS-dTTP. Terminal and / or intermediate adapter molecules may comprise at least three types of phosphorothioate nucleotides. For example, the at least three types of phosphorothioate nucleotides are: (a) aS-dATP, aS-dCTP and aS-dGTP; (b) aS-dATP, aS-dCTP and aS-dTTP; (c) aS-dATP, aS-dGTP and aS-dTTP; either (d) aS-dCTP, aS-dGTP and aS-dTTP. Terminal and / or intermediate adapter molecules may comprise at least four types of phosphorothioate nucleotides. For example, the at least four types of protected nucleotides are αS-dATP, αS-dCTP, αS-dGTP, and αS-dTTP. Internal positions may not be located between the second and penultimate nucleotide of the closed linear DNA product. The linear double-stranded region (or linear portion of the double-stranded molecule) may comprise a plurality of nucleotides with phosphorothioate in internal positions in each chain. For example, the linear double-stranded region (or linear portion of the double-stranded molecule) may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, 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 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g., phosphorothioate-containing nucleotides) in internal positions in each strand. Preferably, the linear double-stranded region (or linear portion of the double-stranded molecule) comprises at least 2 protected nucleotides (e.g., phosphorothioate-containing nucleotides) in internal positions on each strand.Internal positions may not be located between the second and penultimate nucleotide of the linear double-stranded region (or linear portion of the double-stranded molecule). One or more of the n+m intermediate adapter molecules may comprise a plurality of nucleotides with phosphorothioate in internal positions in each chain. For example, one or more of the n+m intermediate adapter molecules may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, 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 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least or 500 protected nucleotides (e.g., phosphorothioate nucleotides) in internal positions in each chain. Nucleotides resistant to exonuclease digestion (i.e., protected nucleotides) suitable for use in the methods described herein may be phosphorothioate-containing nucleotides. For example, phosphorothioate-containing nucleotides may include aS-dATP (i.e., 2-deoxyadenosine-5-(a-thio)-triphosphate), aS-dCTP (i.e., 2-deoxycytidine-5-(a-thio)-triphosphate), aS-dGTP (i.e., 2-deoxyguanosine-5-(a-thio)-triphosphate), aS-dTTP (i.e., 2-deoxythymidine-5-(a-thio)-triphosphate), aS-dUTP (i.e., 2-deoxyuridine-5-(a-thio)-triphosphate), and / or uridine 2,3-cyclophosphorothioate. Nucleotides containing phosphorothioate can be Sp isomers, Rp isomers, or a mixture of both Sp and Rp isomers. Nucleotides resistant to exonuclease digestion (i.e., protected nucleotides) can be 2-O-methyl nucleotides or 2-O-methoxyethyl nucleotides (MOE). For example, MOE nucleotides can be 2-O-methoxyethyl guanosine, 2-O-methoxyethyl cytidine, 2-O-methoxyethyl adenosine, and / or 2-O-methoxyethyl thymidine. The first end of the linear double-stranded region can be complementary to a portion of the first terminal adapter molecule or to a portion of an intermediate adapter molecule, preferably the n-(n-1)th adapter molecule. The second end of the linear double-stranded region can be complementary to a portion of the second terminal adapter molecule or to a portion of an intermediate adapter molecule, preferably the m-(m-1)th adapter molecule. The first and / or second ends of the linear double-stranded region can be generated by endonuclease digestion. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise a functional portion. The functional portion may be a binding molecule, a targeting sequence, a signal sequence, or a probe. The functional portion may be a cassette, an open reading frame, or a coding sequence. The functional portion may be a promoter and enhancer, an NLS sequence, modified nucleotides, a terminator, a transcription factor binding site, a barcode, or a fluorophore. The functional portion may be a probe. As used herein, the term "probe" refers to a fragment of DNA, RNA, or DNA / RNA chimera of variable length (e.g., 3–1000 bases), used to detect the presence of target nucleotide sequences that are complementary to the sequence in the probe. Typically, the probe hybridizes to single-stranded nucleic acid whose base sequence allows probe-target base pairing due to the complementarity between the probe and the target. Therefore, the functional portion may be a DNA sequence, an RNA sequence, or a DNA / RNA chimera sequence. As used herein, the term "complementary" refers to the pairing of nucleotide sequences according to the Watson / Crick pairing rules. For example, a sequence 5-GCGGTCCCA-3 has the complementary sequence 5-TGGGACCGC-3.A complementary sequence can also be an RNA sequence complementary to the DNA sequence. The functional portion may be a binding molecule. The term "binding molecule" refers to any molecule capable of binding to the linear DNA product described herein and / or capable of binding to an additional molecule or target. The binding molecule may be a protein, a polypeptide, or a peptide. The binding molecule may be an antibody, such as a monoclonal or polyclonal antibody. The binding molecule may be an antibody fragment. The functional portion can facilitate detection of the DNA product by binding to capture molecules (e.g., capture antibodies bound by protein-protein interactions). The functional portion can bind to a cellular target, for example, a cell receptor. The functional portion can be a tag. The tag can be any chemical entity that allows the detection of the double-stranded nucleic acid molecule through physical, chemical, and / or biological means. The tag can be a chromophore, a fluorophore, and / or a radioactive molecule. The functional portion may be a targeting sequence. The targeting sequence can be a fragment of DNA or RNA of variable length, used to direct the DNA product to a specific location within a cell. The targeting sequence can be used to increase the transfection efficiency of non-viral gene delivery by enhancing nuclear import of the closed linear DNA product. For example, the targeting sequence may be a nuclear DNA targeting sequence (i.e., a recognition sequence for endogenous DNA-binding proteins), such as the SV40 enhancer sequence (preferably in the 3' direction of the cassette). The targeting sequence may also be a proteomerase targeting sequence. To facilitate the detection and / or quantification of the DNA product, the functional portion may comprise a fluorophore, a radioactive compound, or a barcode. A signal corresponding to the presence, absence, and / or level of the closed linear DNA product can be measured using a barcode. The barcode may comprise at least one binding residue attached to a barcode portion, wherein the barcode portion comprises at least one nucleotide (i.e., the barcode portion comprises a nucleotide sequence at least one nucleotide in length), and wherein the binding residue is capable of binding to the 3', 5', or blunt end of the closed linear DNA product. The signal can be measured by determining the presence, absence, and / or level of the barcode portion of the barcode (e.g., by sequencing or PCR).The barcode portion may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides. The barcode may comprise at least two linker residues (e.g., a first linker residue and a second linker residue). For example, the first linker residue attached to the first barcode portion may be attached to the 3' end of the closed linear DNA product, and the second linker residue attached to the second barcode portion may be attached to the 5' end of the closed linear DNA product. The 3' and 5' ends may comprise a 3' protrusion, a 5' protrusion, or a blunt end. A signal corresponding to the presence, absence, and / or level of the closed linear DNA product can be measured using a fluorophore (i.e., a fluorescently labeled molecule) attached to or connected to protrusion 3, protrusion 5, or the blunt end of the closed linear DNA product. The signal can be measured by flow cytometry and / or fluorescence-activated cell sorting. The functional portion can also facilitate DNA sequencing. For example, the functional portion can be a sequencing adapter.The term "sequencing adapter" is intended to encompass one or more nucleic acid domains that include at least a portion of a nucleic acid sequence (or complement thereof) used by a sequencing platform of interest, such as a sequencing platform provided by Illumina® (e.g., the HiSeq™, MiSeq™ and / or Genome Analyzer™ sequencing systems), Oxford Nanopore™ Technologies (e.g., the MinlON sequencing system), Ion Torrent™ (e.g., the Ion PGMTM and / or Ion Proton™ sequencing systems), Pacific Biosciences (e.g., the PACBIO RS II sequencing system); Life Technologies™ (e.g., a SOLiD sequencing system), Roche (e.g., the 454 GS FLX+ and / or GS Junior sequencing systems), or any other sequencing platform of interest. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise an inverted terminal repeat sequence. The inverted terminal repeat sequences of the first terminal adapter molecule and the second terminal adapter molecule may be symmetrical (i.e., have the same symmetrical three-dimensional organization) or asymmetrical (i.e., have different three-dimensional organizations). The inverted terminal repeat sequences of the first terminal adapter molecule and the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may be of the same or different serotypes. An inverted terminal repeat sequence may comprise a terminal resolution site and a Rep-binding site. One or more of the n+m intermediate adapter molecules (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 intermediate adapter molecules) may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, e.g., a gene that encodes a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosome binding site, and a translation termination sequence. The cassette may further comprise sequences that aid protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas-mediated homology-directed repair (HDR).The cassette may encode the CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, polyC, polyT, or polyG sequence. The homopolymeric sequence may be 3–200 nucleotides in length. The homopolymeric sequence may be used to facilitate cassette purification, in which case the homopolymeric sequence may be 4–12 nucleotides in length, or 5–10 nucleotides in length. The homopolymeric sequence may be used to enhance mRNA expression, in which case the homopolymeric sequence may be 10–200 nucleotides in length, preferably 80–150 nucleotides in length.The homopolymeric sequence may be at least 10, at least 20, 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 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides long. Preferably, the homopolymeric sequence is at least 100 nucleotides long. More preferably, the homopolymeric sequence is at least 120 nucleotides long. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides. One or more of the n+m intermediate adapter molecules (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 intermediate adapter molecules) may comprise a spacer. The spacer may be at least 10, at least 20, 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 125, at least 150, at least 175, at least 200, or at least 300 base pairs in length. One or more of the n+m intermediate adapter molecules (for example, 1, 2, 3, 4, 5, 6, 7, or 8 intermediate adapter molecules) can be 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 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs in length. Preferably, one or more of the n+m intermediate adapter molecules (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 intermediate adapter molecules) are at least 10 base pairs in length.A termination adapter molecule can be 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 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100 base pairs in length. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise a polyA signal sequence. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise an aptamer. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules can confer resistance to nuclease digestion, such as exonuclease digestion (e.g., exonuclease I and / or exonuclease III digestion). The closure of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule) at the first end can generate a closed first end of the closed linear DNA product. The closure of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule) at the second end can generate a closed second end of the closed linear DNA product. The closed first end and the closed second end of the closed linear DNA product may be resistant to nuclease digestion. Nuclease digestion may be exonuclease digestion. Preferably, nuclease digestion is exonuclease III and / or exonuclease I digestion. 2. Methods for producing a linear DNA product comprising nuclease-resistant nucleotides The methods described herein can be used to produce a linear DNA product comprising nuclease resistance (i.e., protected nucleotides). The invention provides a method for producing a linear deoxyribonucleic acid (DNA) product, wherein the method comprises: (a) contacting a double-stranded DNA molecule with an endonuclease and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is added to a first end of the linear double-stranded region and the second terminal adapter molecule is added to a second end of the linear double-stranded region, and wherein the first and second terminal adapter molecules are nucleic acid molecules comprising one or more nuclease-resistant nucleotides (i.e., protected nucleotides). The step of bringing the double-stranded DNA molecule into contact with the endonuclease and the first and second terminal adapter molecules is preferably performed in the presence of a ligase. Therefore, the method for producing a linear DNA product may comprise the following steps: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is added to a first end of the linear double-stranded region and the second terminal adapter molecule is added to a second end of the linear double-stranded region, and wherein the first and second terminal adapter molecules are nucleic acid molecules comprising one or more nuclease-resistant nucleotides (i.e., protected nucleotides). The linear DNA product produced by the methods described herein has improved resistance to nuclease digestion (e.g., exonuclease). For example, the linear DNA product has prolonged in vivo expression compared to a linear DNA product that does not contain protected nucleotides. The addition of intermediate adapter molecules allows for additional flexibility in the production of the linear DNA products of the invention. Intermediate adapter molecules can enable the production of longer linear DNA products. These molecules allow for the incorporation of barcodes, promoter sequences, tags, poly-A signals, and / or open reading frames into the linear DNA product of the invention. An intermediate adapter molecule may comprise a cassette. For example, if it is advantageous to include several genes of interest, or open reading frames, in the linear DNA product, the intermediate adapter molecules may comprise one or more cassettes, and the linear double-stranded region may comprise one or more cassettes. An intermediate adapter molecule may comprise a tag, a signaling sequence, a targeting sequence, a modified nucleotide, or a linker.An intermediate adapter molecule may comprise a promoter, a UTR, a transcription factor binding site, or a termination sequence. A modified nucleotide may be 2-methoxyethoxy A, 2-methoxyethoxy MeC, 2-methoxyethoxy G, 2-methoxyethoxy T, 2-O-methyl RNA bases, fluorinated bases, 2-aminopurine, 5-bromo dU, deoxyuridine, 2, 6-diaminopurine (2-amino-dA), dideoxy-C, deoxyinosine, hydroxymethyl dC, iso-dG, iso-dC, 5-methyl dC or 5-nitroindole. In addition to providing nuclease resistance to the linear DNA product, terminal adapter molecules can also provide additional functional features. A termination adapter molecule can enable the production of longer linear DNA products. Termination adapter molecules can allow barcodes, promoter sequences, tags, poly-A signals, and / or open reading frames to be incorporated into the linear DNA product of the invention. A terminal adapter molecule can comprise a tag, a signaling sequence (e.g., an NLS), a targeting sequence, or a binding residue. A terminal adapter molecule can comprise a promoter, a UTR, or a termination sequence. The addition of the first terminal adapter molecule and / or the second terminal adapter molecule to the first and / or second end of the linear double-stranded region (directly or indirectly through one or more intermediate adapter molecules) can be accomplished by hybridization or ligation of the adapter molecules to the ends of the linear double-stranded region and / or to sequential adapter molecules. Therefore, n intermediate adapter molecules can hybridize sequentially to the first end of the linear double-stranded region. m intermediate adapter molecules can hybridize sequentially to the second end of the linear double-stranded region. n intermediate adapter molecules can ligate sequentially to the first end of the linear double-stranded region. m intermediate adapter molecules can ligate sequentially to the second end of the linear double-stranded region.The addition of the n+m intermediate adapter molecules, the first terminal adapter molecule, and the second terminal adapter molecule can occur through either hybridization or ligation of the adapter molecules to the ends of the linear double-stranded region and / or adjacent adapter molecules. Therefore, n intermediate adapter molecules can hybridize and ligate sequentially to the first end of the linear double-stranded region. The m intermediate adapter molecules can hybridize and ligate sequentially to the second end of the linear double-stranded region. The addition can be facilitated by a ligand or spacer molecule that enables the adapter molecule to bind to the first and / or second end of the linear double-stranded region or to an adjacent adapter molecule.The first terminal adapter molecules can hybridize, ligate, or hybridize and ligate to the nth intermediate adapter molecule or to the first end of the double-stranded DNA molecule. The second terminal adapter molecule can hybridize, ligate, or hybridize and ligate to the mth intermediate adapter molecule or to the second end of the double-stranded DNA molecule. Hybridization is based on the complementarity of a portion of the first and / or second terminal adapter molecules and / or a portion of the first and / or second end of the n+m intermediate adapter molecules with the first and / or second end of the linear double-stranded region. The method for producing a linear DNA product may comprise the following steps: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,and wherein the first terminal adapter molecule and the second terminal adapter molecules are nucleic acid molecules comprising one or more nuclease-resistant nucleotides (i.e., protected nucleotides). The method for producing a linear DNA product may comprise the following steps: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially ligated to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially ligated to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is ligated to the nth intermediate adapter molecule or, when m is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is ligated to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region, and wherein the first terminal adapter molecule and the second terminal adapter molecules are nucleic acid molecules comprising one or more nuclease-resistant nucleotides (i.e., protected nucleotides).n can be 0, 1, 2, 3, 4, 5, 6, 7 or 8. m can be 0, 1, 2, 3, 4, 5, 6, 7 or 8. Preferably, n is 0, 1, 2, 3 or 4, and m is 0, 1, 2, 3 or 4. As used herein, the term "complementary" refers to the pairing of nucleotide sequences according to the Watson / Crick pairing rules. For example, a sequence 5-GCGGTCCCA-3 has the complementary sequence 5-TGGGACCGC-3. A complementary sequence can also be an RNA sequence complementary to the DNA sequence. Preferably, the step of bringing the double-stranded DNA molecule into contact with the endonuclease, ligase, and first and second terminal adapter molecules is carried out in a single reaction (i.e., a single step). The step of incubating the single contiguous aqueous volume to generate the linear DNA product may comprise generating the linear portion of the double-stranded DNA molecule by subjecting the double-stranded DNA molecule to digestion with the endonuclease. The step of incubating the single contiguous aqueous volume can be carried out under conditions that promote the addition (or binding) of the first and second terminal adapter molecules and / or intermediate adapter molecules to the linear double-stranded region to produce the linear DNA product. The addition can be achieved by creating a covalent bond between the first and / or second terminal adapter molecule and the first and / or second end of the linear double-stranded region. The addition can be achieved by creating a covalent bond between the first and / or second terminal adapter molecule and an nth and / or nth intermediate adapter molecule. The addition can be achieved by creating a covalent bond between an intermediate adapter molecule and the first or second end of the linear double-stranded region. The addition can be achieved by creating a covalent bond between an intermediate adapter molecule and an adjacent adapter molecule. The incubation step of the single contiguous aqueous volume can be carried out under conditions that promote the digestion of the double-stranded DNA molecule to produce the linear portion. The digestion of the double-stranded DNA molecule to produce the linear portion can be performed at an initial temperature of 1°C–100°C, 1°C–80°C, 5°C–70°C, 10°C–60°C, 15°C–55°C, 20°C–50°C, 25°C–45°C, 30°C–40°C, 35°C–39°C, 36°C–38°C, or at approximately 37°C. The digestion can be by endonuclease digestion, preferably by type IIS endonuclease digestion. The step of incubating the single contiguous aqueous volume can be carried out under conditions that promote the binding of the linear double-stranded region to the first and second terminal adapter molecules and / or to intermediate adapter molecules. The step of incubating the single contiguous aqueous volume can be carried out under conditions that promote the binding of the intermediate adapter molecules to the first and second terminal adapter molecules and / or adjacent intermediate adapter molecules and / or to the first and / or second end of the linear double-stranded region. The ligation can have an efficiency of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95%.For example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% of the linear double-stranded regions (or portions of double-stranded DNA molecules) can be incorporated into closed linear DNA products. Preferably, the ligation has an efficiency of at least 15%. The ligation step of the linear double-stranded region to the first and second terminal adapter molecules and / or n+m intermediate adapter molecules can be performed at a second temperature of 1 °C-90 °C, 2 °C-70 °C, 5 °C-60 °C, 8 °C-55 °C, 9 °C-50 °C, 10 °C-45 °C, 11 °C-40 °C, 12 °C-37 °C, 13 °C-30 °C, 14 °C-25 °C, 15 °C-20 °C or approximately 16 °C. The step of incubating the single contiguous aqueous volume may comprise incubating at a first temperature and then incubating at a second temperature. The first temperature may be 1°C–100°C, 1°C–80°C, 5°C–70°C, 10°C–60°C, 15°C–55°C, 20°C–50°C, 25°C–45°C, 30°C–40°C, 35°C–39°C, 36°C–38°C, or approximately 37°C. The second temperature can be 1°C–90°C, 2°C–70°C, 5°C–60°C, 8°C–55°C, 9°C–50°C, 10°C–45°C, 11°C–40°C, 12°C–37°C, 13°C–30°C, 14°C–25°C, 15°C–20°C, or approximately 16°C. Preferably, the first temperature is 35°C–39°C. Preferably, the second temperature is 14°C–18°C. The step of incubating the single contiguous aqueous volume may comprise subjecting it to cycling between the first temperature and the second temperature. The step of incubating the single contiguous aqueous volume may comprise subjecting it to cycling between the first temperature and the second temperature at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 80, at least 90, or at least 100 times, preferably at least 20 times. The incubation stage of the single contiguous aqueous volume may comprise subjection to cycles between the first temperature and the second temperature less than 40, less than 35, less than 30 times, less than 29, less than 25 times.The incubation stage of the single contiguous aqueous volume may comprise subjection to cycling between the first and second temperatures 2-100, 5-80, 10-70, 20-60, or 30-60 times. The incubation stage of the single contiguous aqueous volume may comprise subjection to cycling between the first and second temperatures 2-20, 5-29, 61-100, or 65-80 times. The step of incubating the single contiguous aqueous volume can be performed isothermally. The step of incubating the single contiguous aqueous volume can comprise incubation at a constant temperature. The constant temperature promotes the simultaneous digestion of the double-stranded DNA molecule to produce the linear portion of the double-stranded DNA molecule and the ligation of the linear double-stranded region to the n+m intermediate adapter molecules and / or the first and second terminal adapter molecules. For example, the constant temperature can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. Preferably, the constant temperature is 30 °C. The constant temperature is intended to mean that the temperature does not change significantly during the reaction.Constant temperature is intended to mean that the temperature variation during the incubation stage of the single contiguous aqueous volume is less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, or less than 1°C. In a preferred embodiment, the temperature during the incubation stage of the single contiguous aqueous volume does not deviate by more than 5°C, preferably by no more than 3°C, and even more preferably by no more than 1°C. Therefore, the constant temperature can be a temperature in the range of 20°C–30°C, 22°C–32°C, 24°C–34°C, 26°C–36°C, 28°C–38°C, 30°C–40°C, 22°C–28°C, 32°C–38°C, 25°C–35°C, 26°C–34°C, 27°C–33°C, 27.5°C–32.5°C, 28°C–32°C, 28.5°C–31.5°C, 29°C–31°C, or 29.5°C–30.5°C. Preferably, the constant temperature is a temperature in the range of 27.5°C–32.5°C.Alternatively, the constant temperature can be a temperature in the range of 32°C–42°C, 33°C–41°C, 34°C–40°C, 35°C–39°C, or 36°C–38°C. Preferably, the constant temperature is a temperature in the range of 34.5°C–39.5°C. The first and second terminal adapter molecules and / or one or more of the n+m intermediate adapter molecules may comprise one or more phosphorothioate nucleotides, such that, upon addition of the terminal adapter molecules (e.g., ligated) to the linear double-stranded region, the linear DNA product is resistant to nuclease digestion or has enhanced or potentiated resistance to nuclease digestion. The linear DNA product may be resistant to 3-end exonuclease digestion (e.g., by exonuclease III) and / or 5-end exonuclease digestion (e.g., by exonuclease VIII). The first and second adapter molecules and / or one or more of the n+m intermediate adapter molecules may comprise a plurality of phosphorothioate nucleotides. For example, the terminal or intermediate adapter molecules may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 phosphorothioate nucleotides in each chain. A terminal and / or intermediate adapter molecule may be a nucleic acid adapter molecule. The terminal or intermediate adapter molecule may be double-stranded. The terminal or intermediate adapter molecule may comprise a portion that is double-stranded. The first and / or second terminal adapter molecules may comprise 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, or at least 16 base pairs. The terminal and / or intermediate adapter molecules may comprise a plurality of phosphorothioate nucleotides in each chain. For example, the adapter molecules may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 phosphorothioate nucleotides in each chain. One or more of the n+m intermediate adapter molecules may comprise a plurality of phosphorothioate nucleotides in internal positions in each chain.For example, one or more of the n+m intermediate adapter molecules may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, 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 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least or 500 protected nucleotides (e.g., phosphorothioate nucleotides) in internal positions in each chain. The terminal adapter molecules may comprise a plurality of nucleotides with phosphorothioate in internal positions on each chain. For example, the adapter molecules may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 nucleotides with phosphorothioate in internal positions on each chain. Preferably, the adapter molecule comprises at least 2 nucleotides with phosphorothioate in internal positions on each chain. Internal positions may not be located between the second and penultimate nucleotides of the adapter molecule. Internal positions can be any position in the adapter molecules other than the last nucleotide at the end of each chain. Terminal or intermediate adapter molecules may comprise at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% protected nucleotides. Once terminal adapter molecules are added to the linear double-stranded region (directly or indirectly through intermediate adapter molecules), the linear DNA product may comprise a protected nucleotide (e.g., a phosphorothioate nucleotide) at the 5' end (or in the 5' end region) of one or both strands. Preferably, the linear DNA product comprises a phosphorothioate nucleotide at the 5' end (or in the 5' end region) of one or both strands. The linear DNA product may comprise a phosphorothioate nucleotide at the 5' end (or in the 5' end region) of one or both strands. Since most exonucleases, e.g., exonuclease III, remove nucleotides from the 3' end of the polynucleotide chain, the linear DNA product may comprise a protected nucleotide at the 3' end (or in the 3' end region) of one or both strands.Preferably, the linear DNA product comprises a nucleotide with phosphorothioate at the 3' end (or the 3' end region) of one or both strands. The linear DNA product may comprise at least one nucleotide with phosphorothioate at the 3' end (or the 3' end region) and at least one nucleotide with phosphorothioate at the 5' end (or the 5' end region) of one or both strands. The linear DNA product may comprise a nucleotide with phosphorothioate at the 3' end (or the 3' end region) and the 5' end (or the 5' end region) of one or both strands. The linear DNA product may further comprise a plurality of protected nucleotides (e.g., phosphorothioate nucleotides) in internal positions in each strand. For example, the linear DNA product may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, 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 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g., phosphorothioate-containing nucleotides) in internal positions on each strand. Preferably, the linear DNA product comprises at least 2 protected nucleotides (e.g., phosphorothioate nucleotides) in internal positions on each strand. Internal positions may not be located between the second and penultimate nucleotides of the linear DNA product. Internal positions can be any position in the adapter molecules other than the last nucleotide at the end of each strand. The linear double-stranded region (or linear portion of the double-stranded molecule) may comprise a plurality of nucleotides with phosphorothioate in internal positions in each chain. For example, the linear double-stranded region (or linear portion of the double-stranded molecule) may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, 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 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g., nucleotides with osphorothioate) in internal positions in each strand. Preferably, the linear double-stranded region (or linear portion of the double-stranded molecule) comprises at least 2 protected nucleotides (e.g., phosphorothioate-containing nucleotides) in internal positions on each strand.Internal positions may not be located between the second and penultimate nucleotide of the linear double-stranded region (or linear portion of the double-stranded molecule). Nucleotides resistant to exonuclease digestion (i.e., protected nucleotides) can be nucleotides containing phosphorothioate of at least one type. For example, at least one type of nucleotide containing phosphorothioate is aS-dATP (i.e., 2-deoxyadenosine-5-(a-thio)-triphosphate), aS-dCTP (i.e., 2-deoxycytidine-5-(a-thio)-triphosphate), aS-dGTP (i.e., 2-deoxyguanosine-5-(a-thio)-triphosphate), aS-dTTP (i.e., 2-deoxythymidine-5-(a-thio)-triphosphate), aS-dUTP (i.e., 2-deoxyuridine-5-(a-thio)-triphosphate), and / or uridine 2,3-cyclophosphorothioate. Terminal and / or intermediate adapter molecules may comprise at least two types of phosphorothioate nucleotides. For example, the at least two types of phosphorothioate nucleotides are: aS-dATP and aS-dCTP, aS-dATP and aS-dGTP, aS-dATP and aS-dTTP, aS-dCTP and aS-dGTP, aS-dCTP and aS-dTTP, or aS-dGTP and aS-dTTP. Terminal and / or intermediate adapter molecules may comprise at least three types of phosphorothioate nucleotides. For example, the at least three types of phosphorothioate nucleotides are: (e) aS-dATP, aS-dCTP and aS-dGTP; (f) aS-dATP, aS-dCTP and aS-dTTP; (g) aS-dATP, aS-dGTP and aS-dTTP; either (h) aS-dCTP, aS-dGTP and aS-dTTP. Terminal and / or intermediate adapter molecules may comprise at least four types of phosphorothioate nucleotides. For example, the at least four types of protected nucleotides are aS-dATP, aS-dCTP, aS-dGTP, and aS-dTTP. Nucleotides containing phosphorothioate can be Sp isomers, Rp isomers, or a mixture of both Sp and Rp isomers. Nucleotides resistant to exonuclease digestion (i.e., protected nucleotides) can be MOE nucleotides of at least one type, or at least two, three, or four types. For example, MOE nucleotides can be 2-O-methoxyethylguanosine, 2-O-methoxyethylcytidine, 2-O-methoxyethyladenosine, and / or 2-O-methoxyethylthymidine. The method may further comprise, prior to step (a) (i.e., the step of contacting the double-stranded DNA molecule with the endonuclease, ligase, and the first and second terminal adapter molecules and n+m intermediate adapter molecules), a step of amplifying a DNA template molecule to produce the double-stranded DNA molecule. Therefore, the invention provides a method for producing a linear DNA product, the method comprising: (a) amplifying a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,and wherein the first terminal adapter molecule and the second terminal adapter molecules are nucleic acid molecules comprising one or more nuclease-resistant nucleotides (i.e., protected nucleotides). The amplification step can be performed by in vitro or in vivo amplification. Preferably, the amplification step is performed by in vitro amplification. For example, the amplification step can be performed by rolling circle amplification (RCA), the MALBAC method, traditional polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), and recombinase polymerase amplification (RPA). Preferably, the amplification step is performed by rolling circle amplification. Therefore, the invention provides a method for producing a linear DNA product, the method comprising: (a) amplifying a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule, wherein the DNA template molecule is amplified by rolling circle amplification; (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,and wherein the first terminal adapter molecule and the second terminal adapter molecules are nucleic acid molecules comprising one or more nuclease-resistant nucleotides (i.e., protected nucleotides). Rolling circle amplification can be performed without a primer, or with one or more primers. For example, the primer can be a synthetic primer. The primers can be random primers. Rolling circle amplification can be performed in the presence of a primase. The primase can be TtfiPrimPol. Preferably, if rolling circle amplification is performed without a primer, it is performed in the presence of a primase, such as TtfiPrimPol. Similarly, if a primer is used during the amplification reaction, a primase is not used. The double-stranded DNA product can be generated by in vitro rolling circle amplification under isothermal conditions using a suitable nucleic acid polymerase, such as Phi29 DNA polymerase. In the methods described herein, the DNA template molecule may comprise at least one cleavable target sequence. The cleavable target sequence may be an endonuclease target sequence. Preferably, the DNA template molecule comprises at least two endonuclease target sequences. The endonuclease target sequences may be the same or different. Preferably, at least one endonuclease target sequence is a restriction endonuclease target sequence. Different restriction endonuclease target sequences shall be known to the person skilled in the art. The cleavable target sequence may be a type IIS restriction endonuclease target sequence.For example, the target sequence of restriction endonuclease can be a target sequence of BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuHPI, BaeI, BarI, BbvI, BccI, 1999; BceAI, BcgI, BciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, BsgI, BslFI, BsmAI, BsmFI, . BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BtsCI, BtsI-v2, BtsMutI, BveI, CseI, CspCI, Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, GsuI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, Mva1269I, NmeAIII, PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI and / or TspGWI. The at least one cleavable sequence (e.g., endonuclease target sequence) can be a native cleavable sequence (that is, a cleavable sequence present in the mold molecule) .Alternatively, at least one cleavable sequence (e.g., endonuclease target sequence) can be introduced into the DNA template molecule prior to the production of the linear DNA product. An endonuclease can be a restriction endonuclease enzyme. An endonuclease can be a type IIS restriction enzyme. An endonuclease can be any enzyme that recognizes a DNA sequence and cleaves it outside the recognition sequence.For example, the endonuclease can be a restriction enzyme BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, Asu BarHPI, BHPI, BBC, BBC, Bcc BceAI, BcgI, BciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, BselI, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, BsgI, BsgI, BsmFII, BsmFIA, BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstVII, BstV2I, BsuI, BtgZI, BcCI, BcI-v2, BcMut CI, BcI, Eam, C114 EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, GsuI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, Mva1269I, NmeI, Paq, PCI, PCI, PCI PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI y / o TspGWI. The ligase can be a DNA ligase, such as a DNA T4 ligase, DNA T7 ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase. The DNA template molecule used in the methods described herein may be single-stranded or double-stranded. Preferably, the DNA template molecule is double-stranded. The DNA template molecule may be a naturally occurring circular DNA molecule. For example, the DNA template molecule may be (i) a plasmid, (ii) a minicircle, (iii) a cosmid, (iv) a bacterial artificial chromosome (BAC), or (v) a molecular inversion probe (MIP). The DNA template molecule may be an enzymatically produced circular DNA molecule. For example, the DNA template molecule may be (i) a circular DNA molecule obtained from a recombinase reaction, preferably the Cre recombinase reaction, or (ii) a circular DNA molecule obtained from a ligase reaction, preferably using the Golden Gate assembly. The DNA template molecule may be an enzymatically produced covalently closed linear DNA molecule.For example, the DNA template molecule can be (i) a DNA molecule processed with TelN proteomerase; or (ii) a DNA molecule generated by ligating the DNA ends with an adapter. The DNA template molecule can comprise a double-stranded element and a single-stranded element. For example, the DNA template molecule can comprise a double-stranded DNA molecule and a single-stranded hairpin loop. The DNA template molecule can be linear. If the DNA template molecule is linear, prior to amplification (e.g., rolling circle amplification), a DNA template molecule can be circularized to produce a DNA template molecule suitable for use in the methods described herein. The DNA template molecule may comprise a cassette. The cassette may be a mammalian expression cassette. The cassette may further comprise a promoter. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence. The cassette may further comprise a LoxP sequence, preferably two LoxP sequences. If the two LoxP sequences are oriented in the same direction, the DNA sequence between the two LoxP sequences is cut as a circular loop of DNA. If the two LoxP sequences are oriented in opposite directions, the DNA sequence between the two LoxP sequences is inverted. Therefore, preferably, the two LoxP sequences are in the same orientation in the DNA template molecule. The DNA template molecule may comprise a homopolymeric sequence at a 5' end, a 3' end, or both. The homopolymeric sequence may be added to the DNA template molecule before circularization. The homopolymeric sequence may be a polyA, polyC, polyG, or polyT sequence. The homopolymeric sequence may be 3–200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the linear DNA product, in which case the homopolymeric sequence may be 4–12 nucleotides in length, or 5–10 nucleotides in length. The homopolymeric sequence may be used to enhance mRNA expression, in which case the homopolymeric sequence may be 10–200 nucleotides in length, preferably 80–150 nucleotides in length.The homopolymeric sequence may be at least 10, at least 20, 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 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides long. Preferably, the homopolymeric sequence is at least 100 nucleotides long. More preferably, the homopolymeric sequence is at least 120 nucleotides long. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides. The method may also include, after the incubation stage of the single contiguous aqueous volume, a purification stage of the linear DNA product. The method may further comprise, after the incubation stage of the single contiguous aqueous volume, a nuclease digestion stage. The nuclease digestion may be exonuclease digestion, such as exonuclease I and / or exonuclease III digestion. The nuclease digestion stage may take place before or after the purification stage. This step allows for the removal of any double-stranded DNA and / or adapter molecules that were not used during the execution of the method. Therefore, the method may comprise the following steps: (a) amplifying a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) bringing the double-stranded DNA molecule into contact with an endonuclease, a ligase and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least; (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,and wherein the first terminal adapter molecule and the second terminal adapter molecules are nucleic acid molecules comprising one or more nuclease-resistant nucleotides (i.e., protected nucleotides); and, (d) incubate the single contiguous aqueous volume with a nuclease (e.g., exonuclease). The method may include the following stages: (a) amplifying a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) bringing the double-stranded DNA molecule into contact with an endonuclease, a ligase and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each an integer number of at least 1, and wherein n+m is at least 1; (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,and wherein the first terminal adapter molecule and the second terminal adapter molecules are nucleic acid molecules comprising one or more nuclease-resistant nucleotides (i.e., protected nucleotides); (d) purify the closed linear DNA product; and (e) incubate the purified product from step (d) with a nuclease (e.g., exonuclease). The method may include the following stages: (a) amplifying a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) bringing the double-stranded DNA molecule into contact with an endonuclease, a ligase and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; (c) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,and wherein the first terminal adapter molecule and the second terminal adapter molecules are nucleic acid molecules comprising one or more nuclease-resistant nucleotides (i.e., protected nucleotides); (d) incubate the single contiguous aqueous volume with a nuclease (for example, exonuclease); and (e) purify the closed linear DNA product. The step of incubating the single contiguous aqueous volume (or the purified product from step (d)) with a nuclease may be carried out at a temperature of 5–90 °C, 10–80 °C, 15–70 °C, 20–60 °C, 25–50 °C, 30–45 °C, or 35–40 °C. The step of incubating the single contiguous aqueous volume (or the purified product from step (d)) with a nuclease may be carried out for at least 10, at least 20, at least 30, at least 40, at least 50, or at least 60 min. The step of incubating the single contiguous aqueous volume (or the purified product from step (d)) may be carried out at two different temperatures. For example, the step of incubating the single contiguous aqueous volume (or the purified product from step (d)) can be carried out at 15-40 °C for 10-60 minutes followed by a temperature of 60-90 °C for 10-30 minutes. The higher temperature normally inactivates the nuclease (e.g., exonuclease).Therefore, the method also provides a nuclease (e.g., exonuclease) inactivation step. The step of incubating the single contiguous aqueous volume (or the purified product from step (d)) can be carried out at 37 °C for 30 min and 80 °C for 20 min. Preferably, the nuclease (e.g., exonuclease) inactivation step is carried out at a temperature of 70–80 °C. The nuclease (e.g., exonuclease) inactivation step can be carried out for at least 1, at least 5, at least 10, at least 20, or at least 30 min. Preferably, the nuclease (e.g., exonuclease) inactivation step is carried out for at least 5 min. A portion of the first terminal adapter molecule (e.g., the protruding portion) may be complementary to the first end of the linear double-stranded region or to a first end of an intermediate adapter molecule, preferably the nth intermediate adapter molecule. A portion of the second terminal adapter molecule (e.g., the protruding portion) may be complementary to the second end of the linear double-stranded region or to a first end of an intermediate adapter molecule, preferably the mth adapter molecule. A portion of the first end of the linear double-stranded region may be complementary to a first end of an intermediate adapter molecule, preferably an n-(n-1) intermediate adapter molecule. A portion of the second end of the linear double-stranded region may be complementary to a first end of an intermediate adapter molecule, preferably an m-(m-1) intermediate adapter molecule.A portion of a second end of an intermediate adapter molecule may be complementary to a first end of an adjacent intermediate adapter molecule; for example, a portion of the second end of the nth intermediate adapter molecule may be complementary to a portion of the first end of the n-1st intermediate adapter molecule, and a portion of the second end of the n-1st adapter molecule may be complementary to a portion of the first end of the n-2nd intermediate adapter molecule, and so on.A portion of the second end of an intermediate adapter molecule may be complementary to a first end of an adjacent intermediate adapter molecule; for example, a portion of the second end of the m-th intermediate adapter molecule may be complementary to a portion of the first end of the m-1st intermediate adapter molecule, and a portion of the second end of the m-1st adapter molecule may be complementary to a portion of the first end of the m-2nd intermediate adapter molecule, and so on. n may be 0, 1, 2, 3, 4, 5, 6, 7, or 8. m may be 0, 1, 2, 3, 4, 5, 6, 7, or 8. Preferably, n is 0, 1, 2, 3, or 4, and m is 0, 1, 2, 3, or 4. The first and second ends of the linear double-stranded region may be resistant to nuclease digestion. Preferably, the first and second ends of the linear double-stranded region are resistant to exonuclease digestion, such as exonuclease III and / or exonuclease I. The linear DNA product can be partially double-stranded and / or partially single-stranded. The linear DNA product can comprise a portion that is double-stranded and a portion that is single-stranded. The linear DNA product may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example, a gene that encodes a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosome binding site, and a translation termination sequence. The cassette may further comprise sequences that aid protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas-mediated homology-directed repair (HDR). The cassette may encode the CRISPR guide RNA.The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, polyC, polyT, or polyG sequence. The homopolymeric sequence may be 3–200 nucleotides in length. The homopolymeric sequence may be used to facilitate cassette purification, in which case the homopolymeric sequence may be 4–12 nucleotides in length, or 5–10 nucleotides in length. The homopolymeric sequence may be used to enhance mRNA expression, in which case the homopolymeric sequence may be 10–200 nucleotides in length, preferably 80–150 nucleotides in length.The homopolymeric sequence may be at least 10, at least 20, 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 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides long. Preferably, the homopolymeric sequence is at least 100 nucleotides long. More preferably, the homopolymeric sequence is at least 120 nucleotides long. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides. The linear DNA product may include a spacer. The spacer may be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 base pairs long. The spacer may improve the ligation efficiency of the first and second terminal adapter molecules to the linear double-stranded region. The spacer may improve cell transfection yields. The linear DNA product may comprise an inverted terminal repeat sequence. The linear DNA product may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the linear DNA product is at least 50 base pairs long. The double-stranded DNA molecule can be circular, or branched. The double-stranded DNA molecule may not comprise an adapter molecule. The double-stranded DNA molecule may not comprise a hairpin, loop, or stem-loop structure. The double-stranded DNA molecule may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example, a gene that encodes a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosome binding site, and a translation termination sequence. The cassette may further comprise sequences that aid protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas-mediated homology-directed repair (HDR). The cassette may encode the CRISPR guide RNA.The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, polyC, polyT, or polyG sequence. The homopolymeric sequence may be 3–200 nucleotides in length. The homopolymeric sequence may be used to facilitate cassette purification, in which case the homopolymeric sequence may be 4–12 nucleotides in length, or 5–10 nucleotides in length. The homopolymeric sequence may be used to enhance mRNA expression, in which case the homopolymeric sequence may be 10–200 nucleotides in length, preferably 80–150 nucleotides in length.The homopolymeric sequence may be at least 10, at least 20, 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 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides long. Preferably, the homopolymeric sequence is at least 100 nucleotides long. More preferably, the homopolymeric sequence is at least 120 nucleotides long. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides. The double-stranded DNA molecule may include a spacer. The spacer may be at least 10, at least 20, 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 125, at least 150, at least 175, or at least 200 base pairs long. The spacer may improve the amplification performance of the double-stranded DNA molecule. The spacer may improve the ligation efficiency of the first and second terminal adapter molecules to the linear double-stranded region. The spacer may improve cell transfection yields. The double-stranded DNA molecule can be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long. The double-stranded DNA molecule may comprise one or more cleavable target sequences (e.g., endonuclease). The double-stranded DNA molecule may comprise two cleavable target sequences (e.g., endonuclease). The one or more endonuclease target sequences may be type IIS endonuclease target sequences.The one or more diana sequences of the endonuclease may be diana sequences of BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AARI, Acc36I, AclWI, ACUI, AjuI, AloI, Alw26I, BabI, AlwI, BarsI, Ars BCCI, BCEAI, BCGI, BCI, BCODI, BFUAI, BFUI, BMRI, BMSI, BMUI, BPI, BPMI, BPUEI, BSAXI, BSE1I, BSE3DI, BSEGI, BSEMI, BSEMI, BSENI, BSERI,BSAI, BSEI, BSEXI BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BtsCI, BtsI-vSe, BtsMut, BtsMut EAM1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, GsuI, HGAI, HphI, HpyAV, LGUI, LmnI, Lsp1109I, LweI, MboII, MlyI,MMEQI,M1me1,Mn2III,MnIII,M PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI and / or TspGWI. The bicatenary DNA molecule may be an amplification product. Preferably, the amplification is amplification by rolling circle. The linear double-stranded region can be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long. The linear double-stranded region may comprise a 3-OH group at the first and / or second ends. The 3-OH group may facilitate bonding to the first and / or second terminal adapter molecule(s) (which may comprise a 5-phosphate). The linear double-stranded region may comprise a 5-phosphate at the first and / or second ends. The 5-phosphate may facilitate bonding to the first and / or second terminal adapter molecule(s) (which may comprise a 3-OH group). The linear double-stranded region (e.g., the linear portion of the double-stranded molecule) may include a protrusion. For example, the linear double-stranded region may include a 5-protrusion or a 3-protrusion. The linear double-stranded region may include one or more blunt ends. The linear double-stranded region may include: one 5-protrusion and one blunt end, two 5-protrusions, one 3-protrusion and one blunt end, two 3-protrusions, or one 5-protrusion and one 3-protrusion. The protrusion may be at least 3 nucleotides long (preferably 4 to 8 nucleotides). The protrusion may be on the sense strand or the antisense strand of the linear double-stranded region. The linear portion of the double-stranded DNA molecule (e.g., the linear portion of the double-stranded molecule) can be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may be a synthetic adapter molecule. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more n+m intermediate adapter molecules may not be a plasmid or a vector DNA. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more n+m intermediate adapter molecules may comprise a single-stranded portion. The single-stranded portion may comprise less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides. Preferably, the single-stranded portion comprises 5 nucleotides. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise a double-stranded portion. The double-stranded portion may comprise less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, or less than 10 base pairs. The double-stranded portion may comprise 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, or at least 15 base pairs. The first and / or second terminal adapter molecule may comprise or consist of the sequences of SEQ ID NO:13 and / or SEQ ID NO:14. The first and / or second terminal adapter molecule may comprise or at least 15, 14, 13, 12, 11, 10, 19, 8, 7, 6, 5 contiguous nucleotides thereof. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise a 5-phosphate. The 5-phosphate may facilitate ligation to the linear double-stranded region or to adjacent terminal or intermediate adapter molecules (which may comprise a 3-OH group at the first and / or second end). The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise a 3-OH group. The 3-OH group may facilitate ligation to the linear double-stranded region and / or to adjacent intermediate adapter molecules (which may comprise a 5-phosphate at the first and / or second end). The first terminal adapter molecule may comprise a portion that is complementary to the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second terminal adapter molecule may comprise a portion that is complementary to the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The first terminal adapter molecule may comprise a portion that hybridizes to the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second terminal adapter molecule may comprise a portion that hybridizes to the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule).The first terminal adapter molecule may comprise a portion that is complementary to and hybridizes with the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second terminal adapter molecule may comprise a portion that is complementary to and hybridizes with the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The portion that is complementary to or hybridizes with the first or second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule) can be a 5' or 3' protrusion of the first and / or second terminal adapter molecule. The protrusion of the first terminal adapter molecule can be complementary to the first end of the linear double-stranded region, and / or the protrusion of the second terminal adapter molecule can be complementary to the second end of the double-stranded region. The protrusion of the first terminal adapter molecule can hybridize to the first end of the linear double-stranded region, and / or the protrusion of the second terminal adapter molecule can hybridize to the second end of the linear double-stranded region.The protrusion of the first terminal adapter molecule can be complementary and hybridize with the first end of the linear double-stranded region and / or the protrusion of the second terminal adapter molecule can be complementary and hybridize with the second end of the linear double-stranded region. The first terminal adapter molecule may comprise a portion that is complementary to the first end of the nth intermediate adapter molecule. The second terminal adapter molecule may comprise a portion that is complementary to the first end of the mth intermediate adapter molecule. The first terminal adapter molecule may comprise a portion that hybridizes to the first end of the nth intermediate adapter molecule. The second terminal adapter molecule may comprise a portion that hybridizes to the first end of the mth intermediate adapter molecule. The first terminal adapter molecule may comprise a portion that is complementary to and hybridizes to the first end of the nth intermediate adapter molecule. The second terminal adapter molecule may comprise a portion that is complementary to and hybridizes to the first end of the mth intermediate adapter molecule.n can be 0, 1, 2, 3, 4, 5, 6, 7 or 8. m can be 0, 1, 2, 3, 4, 5, 6, 7 or 8. Preferably, n is 0, 1, 2, 3 or 4, and m is 0, 1, 2, 3 or 4. The portion that is complementary to or hybridizes with the first end of the nth or mth intermediate adapter molecule can be a 5-protrusion or a 3-protrusion of the first and / or second terminal adapter molecule. The protrusion of the first terminal adapter molecule can be complementary to the first end of the nth intermediate adapter molecule, and / or the protrusion of the second terminal adapter molecule can be complementary to the first end of the mth intermediate adapter molecule. The protrusion of the first terminal adapter molecule can hybridize with the first end of the nth intermediate adapter molecule, and / or the protrusion of the second terminal adapter molecule can hybridize with the first end of the mth intermediate adapter molecule.The protrusion of the first terminal adapter molecule can be complementary and hybridize with the first end of the nth intermediate adapter molecule and / or the protrusion of the second terminal adapter molecule can be complementary and hybridize with the first end of the mth intermediate adapter molecule. The first terminal adapter molecule and / or the second terminal adapter molecule may not comprise an IIS-type endonuclease target sequence. The first terminal adapter molecule and / or the second terminal adapter molecule may not comprise target sequences of Bbsl, Bsal, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, Acul, Ajul, Alol, Alw26, Alw26, Al, Awl, Bael, Arsul, Awl, A Barl, Bbvl, Bccl, BceAl, Bcgl, BciVI, BcoDI, BfuAl, Bful, Bmrl, Bmsl, Bmul, Bpil, Bpml, BpuEl, BsaXI, Bse1l, Bse3DI, BseGI, BseMI, BseMII, BseNI, BceRI, BseLX, Bcl, Bcl, Bcl, Bcl BsmFI, Bsml, Bso31l, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, Bsrl, Bst6I, BstF5I, BstMAl, BstV1I, BstV2I, Bsul, BtgZI, BcCI, Bcl-v2, BspCi, CspCI, Ccel, C Eam1104l, Earl, Ecil, Eco31l, Eco57l, Esp3I, Faql, Faul, Fokl, Gsul, Hgal, Hphl, HpyAV, Lgul, Lmnl, Lsp1109l, Lwel, Mboll, Mlyl, Mmel, Mnll, Mva1269l, Nmecl, PCI, PCI, PCI, PC Plel, Ppsl, Psrl, Schl, SfaNI, Taqll, TspDTI y / o TspGWI Sapl. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise a functional portion. The functional portion may be a binding molecule, a targeting sequence, a signal sequence, or a probe. The functional portion may be a cassette, an open reading frame, or a coding sequence. The functional portion may be a promoter and enhancer, an NLS sequence, a UTR, ITR, or other repeats, a termination sequence, modified nucleotides, or a fluorophore. The functional portion may be a probe. As used herein, the term "probe" refers to a fragment of DNA, RNA, or DNA / RNA chimera of variable length (e.g., 3–1000 bases), used to detect the presence of target nucleotide sequences that are complementary to the sequence in the probe. Typically, the probe hybridizes to single-stranded nucleic acid whose base sequence allows probe-target base pairing due to the complementarity between the probe and the target. Therefore, the functional portion may be a DNA sequence, an RNA sequence, or a DNA / RNA chimera sequence. As used herein, the term "complementary" refers to the pairing of nucleotide sequences according to the Watson / Crick pairing rules. For example, a sequence 5-GCGGTCCCA-3 has the complementary sequence 5-TGGGACCGC-3.A complementary sequence can also be an RNA sequence complementary to the DNA sequence. The functional portion may be a binding molecule. The term "binding molecule" refers to any molecule capable of binding to the linear DNA product described herein and / or capable of binding to an additional molecule or target. The binding molecule may be a protein, a polypeptide, or a peptide. The binding molecule may be an antibody, such as a monoclonal or polyclonal antibody. The binding molecule may be an antibody fragment. The functional portion can facilitate detection of the DNA product by binding to capture molecules (e.g., capture antibodies bound by protein-protein interactions). The functional portion can bind to a cellular target, for example, a cell receptor. The functional portion can be a tag. The tag can be any chemical entity that allows the detection of the double-stranded nucleic acid molecule through physical, chemical, and / or biological means. The tag can be a chromophore, a fluorophore, and / or a radioactive molecule. The functional portion may be a targeting sequence. The targeting sequence can be a fragment of DNA or RNA of variable length, used to direct the DNA product to a specific location within a cell. The targeting sequence can be used to increase the transfection efficiency of non-viral gene delivery by enhancing the nuclear import of the linear DNA product. For example, the targeting sequence may be a nuclear DNA targeting sequence (i.e., a recognition sequence for endogenous DNA-binding proteins), such as the SV40 enhancer sequence (preferably in the 3' direction of the cassette). The targeting sequence may be a proteomerase targeting sequence or a truncated variant thereof. To facilitate the detection and / or quantification of the DNA product, the functional portion may comprise a fluorophore, a radioactive compound, or a barcode. A signal corresponding to the presence, absence, and / or level of the linear DNA product can be measured using a barcode. The barcode may comprise at least one binding residue attached to a barcode portion, wherein the barcode portion comprises at least one nucleotide (i.e., the barcode portion comprises a nucleotide sequence at least one nucleotide in length), and wherein the binding residue is capable of binding to the 3', 5', or blunt end of the linear DNA product. The signal can be measured by determining the presence, absence, and / or level of the barcode portion of the barcode (e.g., by sequencing or PCR). The barcode portion may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides.The barcode may comprise at least two binding residues (e.g., a first binding residue and a second binding residue). For example, the first binding residue attached to the first barcode portion may be attached to the 3' end of the linear DNA product, and the second binding residue attached to the second barcode portion may be attached to the 5' end of the linear DNA product. The 3' and 5' ends may comprise a 3' protrusion, a 5' protrusion, or a blunt end. A signal corresponding to the presence, absence, and / or level of the linear DNA product can be measured using a fluorophore (i.e., a fluorescently labeled molecule) attached to or connected to protrusion 3, protrusion 5, or the blunt end of the linear DNA product. The signal can be measured by flow cytometry and / or fluorescence-activated cell sorting. The functional portion can also facilitate DNA sequencing. For example, the functional portion can be a sequencing adapter.The term "sequencing adapter" is intended to encompass one or more nucleic acid domains that include at least a portion of a nucleic acid sequence (or complement thereof) used by a sequencing platform of interest, such as a sequencing platform provided by Illumina® (e.g., the HiSeq™, MiSeq™ and / or Genome Analyzer™ sequencing systems), Oxford Nanopore™ Technologies (e.g., the MinION sequencing system), Ion Torrent™ (e.g., the Ion PGMTM and / or Ion Proton™ sequencing systems), Pacific Biosciences (e.g., the PACBIO RS II sequencing system); Life Technologies™ (e.g., a SOLiD sequencing system), Roche (e.g., the 454 GS FLX+ and / or GS Junior sequencing systems), or any other sequencing platform of interest. One or more of the n+m intermediate adapter molecules (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 intermediate adapter molecules) may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, e.g., a gene that encodes a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosome binding site, and a translation termination sequence. The cassette may further comprise sequences that aid protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas-mediated homology-directed repair (HDR).The cassette may encode the CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, polyC, polyT, or polyG sequence. The homopolymeric sequence may be 3–200 nucleotides in length. The homopolymeric sequence may be used to facilitate cassette purification, in which case the homopolymeric sequence may be 4–12 nucleotides in length, or 5–10 nucleotides in length. The homopolymeric sequence may be used to enhance mRNA expression, in which case the homopolymeric sequence may be 10–200 nucleotides in length, preferably 80–150 nucleotides in length.The homopolymeric sequence may be at least 10, at least 20, 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 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides long. Preferably, the homopolymeric sequence is at least 100 nucleotides long. More preferably, the homopolymeric sequence is at least 120 nucleotides long. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides. One or more of the n+m intermediate adapter molecules (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 intermediate adapter molecules) may comprise a spacer. The spacer may be at least 10, at least 20, 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 125, at least 150, at least 175, at least 200, or at least 300 base pairs in length. One or more of the n+m intermediate adapter molecules (for example, 1, 2, 3, 4, 5, 6, 7, or 8 intermediate adapter molecules) can be 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 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs in length. Preferably, one or more of the n+m intermediate adapter molecules (for example, 1, 2, 3, 4, 5, 6, 7, or 8 intermediate adapter molecules) are at least 10 base pairs in length. A termination adapter molecule can be 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 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100 base pairs in length. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise a polyA signal sequence. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise an aptamer. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules can confer resistance to nuclease digestion, such as exonuclease digestion (e.g., exonuclease I and / or exonuclease III digestion). 3. Methods for producing a partially closed linear DNA product comprising nuclease-resistant nucleotides The methods described herein can be used to produce a partially closed linear DNA product comprising nuclease resistance (i.e., protected nucleotides). The invention provides a method for producing a partially closed deoxyribonucleic acid (DNA) product, wherein the method comprises: (a) contacting a double-stranded DNA molecule with an endonuclease and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (b) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,and wherein the second terminal adapter molecule is a nucleic acid molecule comprising one or more nuclease-resistant nucleotides. The step of bringing the double-stranded DNA molecule into contact with the endonuclease and the first and second terminal adapter molecules is preferably performed in the presence of a ligase. Therefore, the method for producing a partially closed linear DNA product may comprise the following steps: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (b) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,and wherein the second terminal adapter molecule is a nucleic acid molecule comprising one or more nuclease-resistant nucleotides. The partially closed linear DNA product produced by the methods described herein has improved resistance to nuclease digestion (e.g., exonuclease). The addition of the first terminal adapter molecule and / or the second terminal adapter molecule to the first and / or second end of the linear double-stranded region (directly or indirectly through one or more intermediate adapter molecules) can be accomplished by hybridization or ligation of the adapter molecules to the ends of the linear double-stranded region and / or to sequential adapter molecules. Therefore, n intermediate adapter molecules can hybridize sequentially to the first end of the linear double-stranded region. m intermediate adapter molecules can hybridize sequentially to the second end of the linear double-stranded region. n intermediate adapter molecules can ligate sequentially to the first end of the linear double-stranded region. m intermediate adapter molecules can ligate sequentially to the second end of the linear double-stranded region.The addition of the n+m intermediate adapter molecules, the first terminal adapter molecule, and the second terminal adapter molecule can occur through either hybridization or ligation of the adapter molecules to the ends of the linear double-stranded region and / or adjacent adapter molecules. Therefore, n intermediate adapter molecules can hybridize and ligate sequentially to the first end of the linear double-stranded region. The m intermediate adapter molecules can hybridize and ligate sequentially to the second end of the linear double-stranded region. The addition can be facilitated by a ligand or spacer molecule that enables the adapter molecule to bind to the first and / or second end of the linear double-stranded region or to an adjacent adapter molecule.The first terminal adapter molecules can hybridize, ligate, or hybridize and ligate to the nth intermediate adapter molecule or to the first end of the double-stranded DNA molecule. The second terminal adapter molecule can hybridize, ligate, or hybridize and ligate to the mth intermediate adapter molecule or to the second end of the double-stranded DNA molecule. The method for producing a partially closed linear DNA product may comprise the following steps: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (b) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,and wherein the second terminal adapter molecule is a nucleic acid molecule comprising no or more nuclease-resistant nucleotides (i.e., protected nucleotides). n can be 0, 1, 2, 3, 4, 5, 6, 7, or 8. m can be 0, 1, 2, 3, 4, 5, 6, 7, or 8. Preferably, n is 0, 1, 2, 3, or 4, and m is 0, 1, 2, 3, or 4. The method for producing a partially closed linear DNA product may comprise the following steps: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (b) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,and wherein the second terminal adapter molecule is a nucleic acid molecule comprising one or more nuclease-resistant nucleotides (i.e., protected nucleotides). As used herein, the term "complementary" refers to the pairing of nucleotide sequences according to the Watson / Crick pairing rules. For example, a sequence 5-GCGGTCCCA-3 has the complementary sequence 5-TGGGACCGC-3. A complementary sequence can also be an RNA sequence complementary to the DNA sequence. Preferably, the step of bringing the double-stranded DNA molecule into contact with the endonuclease, ligase, and first and second terminal adapter molecules is carried out in a single reaction (i.e., a single step). The step of incubating the single contiguous aqueous volume to generate the partially closed linear DNA product may comprise generating the linear portion of the double-stranded DNA molecule by subjecting the double-stranded DNA molecule to digestion with the endonuclease. The addition of intermediate adapter molecules allows for additional flexibility in the production of the partially closed linear DNA products of the invention. Intermediate adapter molecules can enable the production of longer partially closed linear DNA products. These molecules allow for the incorporation of barcodes, promoter sequences, tags, poly-A signals, and / or open reading frames into the partially closed linear DNA product of the invention. An intermediate adapter molecule may comprise one cassette. For example, if it is advantageous to include several genes of interest, or open reading frames, in the partially closed linear DNA product, the intermediate adapter molecules may comprise one or more cassettes, and the linear DNA region may comprise one or more cassettes.An intermediate adapter molecule may comprise a tag, a signaling sequence, a targeting sequence, or a binding residue. An intermediate adapter molecule may comprise a promoter, a UTR, or a termination sequence. In addition to providing nuclease resistance to the partially closed linear DNA product, terminal adapter molecules can also provide additional functional features. A termination adapter molecule can enable the production of longer partially closed linear DNA products. Termination adapter molecules can allow barcodes, promoter sequences, tags, poly-A signals, and / or open reading frames to be incorporated into the partially closed linear DNA product of the invention. A terminal adapter molecule can comprise a tag, a signaling sequence, a targeting sequence, or a binding residue. A terminal adapter molecule can comprise a UTR or a termination sequence. The step of incubating the single contiguous aqueous volume can be carried out under conditions that promote the addition (or binding) of the first and second terminal adapter molecules and / or intermediate adapter molecules to the linear double-stranded region to produce the partially closed linear DNA product. The addition can be achieved by creating a covalent bond between the first and / or second terminal adapter molecule and / or n+m intermediate adapter molecules and the first and / or second end of the linear double-stranded region. The addition can also be achieved by creating a covalent bond between the first and / or second terminal adapter molecule and an nth and / or mth intermediate adapter molecule. Finally, the addition can be achieved by creating a covalent bond between an intermediate adapter molecule and the first or second end of the linear double-stranded region.The addition can be accomplished by creating a covalent bond between an intermediate adapter molecule and an adjacent adapter molecule. The incubation step of the single contiguous aqueous volume can be carried out under conditions that promote the digestion of the double-stranded DNA molecule to produce the linear portion. The digestion of the double-stranded DNA molecule to produce the linear portion can be performed at an initial temperature of 1°C–100°C, 1°C–80°C, 5°C–70°C, 10°C–60°C, 15°C–55°C, 20°C–50°C, 25°C–45°C, 30°C–40°C, 35°C–39°C, 36°C–38°C, or at approximately 37°C. The digestion can be by endonuclease digestion, preferably by type IIS endonuclease digestion. The step of incubating the single contiguous aqueous volume can be carried out under conditions that promote the ligation of the linear double-stranded region to the first and second terminal adapter molecules. The ligation can have an efficiency of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95%.For example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 90%, or at least 95% of the linear double-stranded regions (or portions of double-stranded DNA molecules) can be incorporated into closed linear DNA products. Preferably, the ligation has an efficiency of at least 15%. The step of linking the linear double-stranded region to the first and second terminal adapter molecules can be performed at a second temperature of 1 °C-90 °C, 2 °C-70 °C, 5 °C-60 °C, 8 °C-55 °C, 9 °C-50 °C, 10 °C-45 °C, 11 °C-40 °C, 12 °C-37 °C, 13 °C-30 °C, 14 °C-25 °C, 15 °C-20 °C or approximately 16 °C. The step of incubating the single contiguous aqueous volume may comprise incubating at a first temperature and then incubating at a second temperature. The first temperature may be 1°C–100°C, 1°C–80°C, 5°C–70°C, 10°C–60°C, 15°C–55°C, 20°C–50°C, 25°C–45°C, 30°C–40°C, 35°C–39°C, 36°C–38°C, or approximately 37°C. The second temperature can be 1°C–90°C, 2°C–70°C, 5°C–60°C, 8°C–55°C, 9°C–50°C, 10°C–45°C, 11°C–40°C, 12°C–37°C, 13°C–30°C, 14°C–25°C, 15°C–20°C, or approximately 16°C. Preferably, the first temperature is 35°C–39°C. Preferably, the second temperature is 14°C–18°C. The step of incubating the single contiguous aqueous volume may comprise subjecting it to cycling between the first temperature and the second temperature. The step of incubating the single contiguous aqueous volume may comprise subjecting it to cycling between the first temperature and the second temperature at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 80, at least 90, or at least 100 times, preferably at least 20 times. The incubation stage of the single contiguous aqueous volume may comprise subjection to cycles between the first temperature and the second temperature less than 40, less than 35, less than 30 times, less than 29, less than 25 times.The incubation stage of the single contiguous aqueous volume may comprise subjection to cycling between the first and second temperatures 2-100, 5-80, 10-70, 20-60, or 30-60 times. The incubation stage of the single contiguous aqueous volume may comprise subjection to cycling between the first and second temperatures 2-20, 5-29, 61-100, or 65-80 times. The step of incubating the single contiguous aqueous volume can be performed isothermally. This step can also involve incubation at a constant temperature. The constant temperature promotes the simultaneous digestion of the double-stranded DNA molecule to produce the linear portion and the ligation of the linear double-stranded region to the first and second terminal adapter molecules. For example, the constant temperature could be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. Preferably, the constant temperature is 30 °C. The constant temperature is intended to mean that the temperature does not change significantly during the reaction.Constant temperature is intended to mean that the temperature variation during the incubation stage of the single contiguous aqueous volume is less than 10°C, less than 9°C, less than 8°C, less than 7°C, less than 6°C, less than 5°C, less than 4°C, less than 3°C, less than 2°C, or less than 1°C. In a preferred embodiment, the temperature during the incubation stage of the single contiguous aqueous volume does not deviate by more than 5°C, preferably by no more than 3°C, and even more preferably by no more than 1°C. Therefore, the constant temperature can be a temperature in the range of 20°C–30°C, 22°C–32°C, 24°C–34°C, 26°C–36°C, 28°C–38°C, 30°C–40°C, 22°C–28°C, 32°C–38°C, 25°C–35°C, 26°C–34°C, 27°C–33°C, 27.5°C–32.5°C, 28°C–32°C, 28.5°C–31.5°C, 29°C–31°C, or 29.5°C–30.5°C. Preferably, the constant temperature is a temperature in the range of 27.5°C–32.5°C.Alternatively, the constant temperature can be a temperature in the range of 32°C–42°C, 33°C–41°C, 34°C–40°C, 35°C–39°C, or 36°C–38°C. Preferably, the constant temperature is a temperature in the range of 34.5°C–39.5°C. The first and second terminal adapter molecules may comprise one or more phosphorothioate nucleotides, such that, once the adapter molecules (e.g., ligated) are added to the linear double-stranded region, the partially closed linear DNA product is resistant to nuclease digestion or has enhanced or potentiated resistance to nuclease digestion. The partially closed linear DNA product may be resistant to 3-end exonuclease digestion (e.g., by exonuclease III) and / or 5-end exonuclease digestion (e.g., by exonuclease VIII). A terminal or intermediate adapter molecule may comprise a plurality of phosphorothioate nucleotides. For example, the terminal or intermediate adapter molecule may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 phosphorothioate nucleotides in each chain. A terminal or intermediate adapter molecule may be a nucleic acid adapter molecule. The terminal or intermediate adapter molecule may be double-stranded. The terminal or intermediate adapter molecule may comprise a portion that is double-stranded. The first and / or second terminal adapter molecules and / or one or more n+m intermediate adapter molecules may comprise 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, or at least 16 base pairs. The first and / or second terminal adapter molecules and / or one or more n+m intermediate adapter molecules may comprise at least 1000 base pairs, at least 750 base pairs, at least 500 base pairs, at least 250 base pairs, at least 200 base pairs, at least 150 base pairs, at least 100 base pairs, or at least 75 base pairs. The terminal or intermediate adapter molecule may comprise a plurality of nucleotides with phosphorothioate in each chain. For example, the terminal or intermediate adapter molecules may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 nucleotides with phosphorothioate in each chain. The terminal or intermediate adapter molecule may comprise a plurality of nucleotides with phosphorothioate in internal positions on each chain. For example, the terminal or intermediate adapter molecules may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 nucleotides with phosphorothioate in internal positions on each chain. Preferably, the terminal or intermediate adapter molecule comprises at least 2 nucleotides with phosphorothioate in internal positions on each chain. Internal positions may not be located between the second and penultimate nucleotides of the adapter molecule. Internal positions can be any position in the adapter molecules other than the last nucleotide at the end of each chain. The terminal or intermediate adapter molecule may comprise at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% protected nucleotides. Once the adapter molecules are added to the linear double-stranded region, the partially closed linear DNA product may comprise a protected nucleotide (e.g., a phosphorothioate nucleotide) at the 5' end (or in the 5' end region) of one or both strands. Preferably, the partially closed linear DNA product comprises a phosphorothioate nucleotide at the 5' end (or in the 5' end region) of one strand. Since most exonucleases, e.g., exonuclease III, remove nucleotides from the 3' end of the polynucleotide chain, the linear DNA product may comprise a protected nucleotide at the 3' end (or in the 3' end region) of one strand.Preferably, the partially closed linear DNA product comprises a nucleotide with phosphorothioate at the 3' end (or 3' end region) of one strand. The partially closed linear DNA product may comprise at least one nucleotide with phosphorothioate at the 3' end (or 3' end region) and at least one nucleotide with phosphorothioate at the 5' end (or 5' end region) of one strand. The partially closed linear DNA product may comprise at least one nucleotide with phosphorothioate at the 3' end (or 3' end region) of the sense strand and the 5' end (or 5' end region) of the antisense strand. The partially closed linear DNA product may comprise at least one nucleotide with phosphorothioate at the 5' end (or 5' end region) of the sense strand and the 3' end (or 3' end region) of the antisense strand.Therefore, both the sense and antisense strands in the partially closed double-stranded linear DNA product can be protected from nuclease digestion by using nuclease-resistant nucleotides at one end of the partially closed linear DNA product. One of the terminal adapter molecules used in the methods described herein may comprise a self-complementary element that creates a loop, such as a hairpin loop or a stem-loop. Therefore, one of the terminal adapter molecules may comprise a hairpin or a stem-loop. The terminal adapter molecule may comprise a double-stranded portion comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are joined together in a hairpin such that the sense strand hybridizes with the antisense strand. The double-stranded portion of a terminal adapter may comprise a 3- or 5-prong of at least 1, at least 2, at least 3, at least 4, or at least 5 nucleotides. Preferably, the 3- or 5-prong is 4–8 nucleotides long. Each end of the linear double-stranded region (or linear portion of the double-stranded DNA molecule) may comprise a 3- or 5-prong.A portion of the terminal adapter molecule (e.g., the protrusion) can be complementary to either the first or second end of the linear double-stranded region. The method described herein may use a first terminal adapter molecule comprising a hairpin loop or stem-loop, or any other structure capable of closing one end of the linear DNA molecule, and a second terminal adapter molecule that is a linear nucleic acid molecule comprising nuclease-resistant nucleotides to produce a partially closed linear DNA product. The partially closed linear DNA product can be a covalently partially closed DNA product. Therefore, in embodiments where the first terminal adapter molecule comprises a loop (e.g., a hairpin), the terminal adapter molecule closes one end of the linear double-stranded region, forming a covalently partially closed DNA product. The first terminal adapter molecule may comprise a single-stranded portion. The single-stranded portion may form a hairpin or a stem-loop. The single-stranded portion may comprise less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides. Preferably, the single-stranded portion comprises 5 nucleotides. The first terminal adapter molecule may comprise or consist of the sequence SEQ ID NO: 9 or a portion thereof. The first terminal adapter molecule may comprise at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides of SEQ ID NO: 9. The double-stranded portion of the first terminal adapter molecule may comprise the sequence SEQ ID NO: 10 or a portion thereof. The double-stranded portion of the first and / or second terminal adapter molecule may comprise at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 contiguous nucleotides of SEQ ID NO: 10. The single-stranded portion of the first terminal adapter molecule may comprise an ACTCA sequence.The single-stranded portion of the first terminal adapter molecule may comprise at least 1, at least 2, at least 3, at least 4, or at least 5 contiguous nucleotides of the ACTCA sequence. The first terminal adapter molecule may comprise the sequence SEQ ID NO: 12. The first terminal adapter molecule may comprise at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides of SEQ ID NO: 12. The second terminal adapter molecule may comprise or consist of the sequences of SEQ ID NO:13 and / or SEQ ID NO:14. The second terminal adapter molecule may comprise or at least 15, 14, 13, 12, 11, 10, 19, 8, 7, 6, 5 contiguous nucleotides thereof. The partially closed linear DNA product may further comprise a plurality of protected nucleotides (e.g., phosphorothioate nucleotides) in internal positions on each strand. For example, the linear DNA product may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, 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 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g., phosphorothioate-containing nucleotides) in internal positions on each strand. Preferably, the partially closed linear DNA product comprises at least 2 protected nucleotides (e.g., phosphorothioate nucleotides) in internal positions on each strand. Internal positions may not be located between the second and penultimate nucleotides of the partially closed linear DNA product. Internal positions may be any position in the adapter molecules other than the last nucleotide at the end of each strand. The linear double-stranded region (or linear portion of the double-stranded molecule) may comprise a plurality of nucleotides with phosphorothioate in internal positions in each chain. For example, the linear double-stranded region (or linear portion of the double-stranded molecule) may comprise at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, 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 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 protected nucleotides (e.g., phosphorothioate-containing nucleotides) in internal positions in each strand. Preferably, the linear double-stranded region (or linear portion of the double-stranded molecule) comprises at least 2 protected nucleotides (e.g., phosphorothioate-containing nucleotides) in internal positions on each strand.Internal positions may not be located between the second and penultimate nucleotide of the linear double-stranded region (or linear portion of the double-stranded molecule). Nucleotides resistant to exonuclease digestion (i.e., protected nucleotides) can be nucleotides containing phosphorothioate of at least one type. For example, at least one type of nucleotide containing phosphorothioate is aS-dATP (i.e., 2-deoxyadenosine-5-(a-thio)-triphosphate), aS-dCTP (i.e., 2-deoxycytidine-5-(a-thio)-triphosphate), aS-dGTP (i.e., 2-deoxyguanosine-5-(a-thio)-triphosphate), aS-dTTP (i.e., 2-deoxythymidine-5-(a-thio)-triphosphate), aS-dUTP (i.e., 2-deoxyuridine-5-(a-thio)-triphosphate), and / or uridine 2,3-cyclophosphorothioate. Terminal or intermediate adapter molecules may comprise at least two types of phosphorothioate nucleotides. For example, the at least two types of phosphorothioate nucleotides are: aS-dATP and aS-dCTP, aS-dATP and aS-dGTP, aS-dATP and aS-dTTP, aS-dCTP and aS-dGTP, aS-dCTP and aS-dTTP, or aS-dGTP and aS-dTTP. Terminal or intermediate adapter molecules may comprise at least three types of phosphorothioate nucleotides. For example, the at least three types of phosphorothioate nucleotides are: (i) aS-dATP, aS-dCTP and aS-dGTP; (j) aS-dATP, aS-dCTP and aS-dTTP; (k) aS-dATP, aS-dGTP and aS-dTTP; either (l) aS-dCTP, aS-dGTP and aS-dTTP. Adapter molecules can comprise at least four types of nucleotides with phosphorothioate. For example, the at least four types of protected nucleotides are aS-dATP, aS-dCTP, aS-dGTP, and aS-dTTP. Nucleotides containing phosphorothioate can be Sp isomers, Rp isomers, or a mixture of both Sp and Rp isomers. Nucleotides resistant to exonuclease digestion (i.e., protected nucleotides) can be MOE nucleotides of at least one type, or at least two, three, or four types. For example, MOE nucleotides can be 2-O-methoxyethylguanosine, 2-O-methoxyethylcytidine, 2-O-methoxyethyladenosine, and / or 2-O-methoxyethylthymidine. The method may further comprise, prior to step (a) (i.e., the step of contacting the double-stranded DNA molecule with the endonuclease, ligase, and the first and second terminal adapter molecules), a step of amplifying a DNA template molecule to produce the double-stranded DNA molecule. Therefore, the invention provides a method for producing a partially closed linear DNA product, the method comprising: (a) amplifying a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (c) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,and wherein the second terminal adapter molecule is a nucleic acid molecule comprising one or more nuclease-resistant nucleotides (i.e., protected nucleotides). The amplification step can be performed by in vitro or in vivo amplification. Preferably, the amplification step is performed by in vitro amplification. For example, the amplification step can be performed by rolling circle amplification (RCA), the MALBAC method, traditional polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), and polymerase recombinase amplification (RPA). Preferably, the amplification step is performed by rolling circle amplification. Therefore, the invention provides a method for producing a partially closed linear DNA product, the method comprising: (a) amplifying a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule, wherein the DNA template molecule is amplified by rolling circle amplification; (b) contacting the double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (c) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,and wherein the second terminal adapter molecule is a nucleic acid molecule comprising one or more nuclease-resistant nucleotides (i.e., protected nucleotides). Rolling circle amplification can be performed without a primer, or with one or more primers. For example, the primer can be a synthetic primer. The primers can be random primers. Rolling circle amplification can be performed in the presence of a primase. The primase can be TtfiPrimPol. Preferably, if rolling circle amplification is performed without a primer, it is performed in the presence of a primase, such as TtfiPrimPol. Similarly, if a primer is used during the amplification reaction, a primase is not used. The double-stranded DNA product can be generated by rolling circle amplification in vitro under isothermal conditions using a suitable nucleic acid polymerase, such as Phi29 DNA polymerase. In the methods described herein, the DNA template molecule may comprise at least one cleavable target sequence. The cleavable target sequence may be an endonuclease target sequence. Preferably, the DNA template molecule comprises at least two endonuclease target sequences. The endonuclease target sequences may be the same or different. Preferably, at least one endonuclease target sequence is a restriction endonuclease target sequence. Different restriction endonuclease target sequences shall be known to the person skilled in the art. The cleavable target sequence may be a type IIS restriction endonuclease target sequence.For example, the target sequence of restriction endonuclease can be a target sequence of BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuHPI, BaeI, BarI, BbvI, BccI, 1999; BceAI, BcgI, BciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, BsgI, BslFI, BsmAI, BsmFI, . BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BtsCI, BtsI-v2, BtsMutI, BveI, CseI, CspCI, Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, GsuI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, Mva1269I, NmeAIII, PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI and / or TspGWI. The at least one cleavable sequence (e.g., endonuclease target sequence) can be a native cleavable sequence (that is, a cleavable sequence present in the mold molecule) .Alternatively, at least one cleavable sequence (e.g., endonuclease target sequence) can be introduced into the DNA template molecule prior to the production of the partially closed linear DNA product. An endonuclease can be a restriction endonuclease enzyme. An endonuclease can be a type IIS restriction enzyme. An endonuclease can be any enzyme that recognizes a DNA sequence and cleaves it outside the recognition sequence.For example, the endonuclease can be a restriction enzyme BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuI, BaccP, Bacc, Bib, Bib BceAI, BcgI, BciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, BsgI, BsgFI, BsmFIA, BsmFII BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BtsCI, BtsI-v2, BtsMut, BspCI, CIS, CIS Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, GsuI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, MnIII, PaqI, NCIA PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI y / o TspGWI. The ligase can be a DNA ligase, such as a DNA T4 ligase, DNA T7 ligase, mammalian DNA ligase I, III and IV; Taq DNA ligase, Tth DNA ligase, or E. coli DNA ligase. The DNA template molecule used in the methods described herein may be single-stranded or double-stranded. Preferably, the DNA template molecule is double-stranded. The DNA template molecule may be a naturally occurring circular DNA molecule. For example, the DNA template molecule may be (i) a plasmid, (ii) a minicircle, (iii) a cosmid, (iv) a bacterial artificial chromosome (BAC), or (v) a molecular inversion probe (MIP). The DNA template molecule may be an enzymatically produced circular DNA molecule. For example, the DNA template molecule may be (i) a circular DNA molecule obtained from a recombinase reaction, preferably the Cre recombinase reaction, or (ii) a circular DNA molecule obtained from a ligase reaction, preferably using the Golden Gate assembly. The DNA template molecule may be an enzymatically produced covalently closed linear DNA molecule.For example, the DNA template molecule can be (i) a DNA molecule processed with TelN proteomerase; or (ii) a DNA molecule generated by ligating the DNA ends with an adapter. The DNA template molecule can comprise a double-stranded element and a single-stranded element. For example, the DNA template molecule can comprise a double-stranded DNA molecule and a single-stranded hairpin loop. The DNA template molecule can be linear. If the DNA template molecule is linear, prior to amplification (e.g., rolling circle amplification), a DNA template molecule can be circularized to produce a DNA template molecule suitable for use in the methods described herein. The DNA template molecule may comprise a cassette. The cassette may be a mammalian expression cassette. The cassette may further comprise a promoter. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence. The cassette may further comprise a LoxP sequence, preferably two LoxP sequences. If the two LoxP sequences are oriented in the same direction, the DNA sequence between the two LoxP sequences is cut as a circular loop of DNA. If the two LoxP sequences are oriented in opposite directions, the DNA sequence between the two LoxP sequences is inverted. Therefore, preferably, the two LoxP sequences are in the same orientation in the DNA template molecule. The DNA template molecule may comprise a homopolymeric sequence at a 5' end, a 3' end, or both. The homopolymeric sequence may be added to the DNA template molecule before circularization. The homopolymeric sequence may be a polyA, polyC, polyG, or polyT sequence. The homopolymeric sequence may be 3–200 nucleotides in length. The homopolymeric sequence may be used to facilitate purification of the partially closed linear DNA product, in which case the homopolymeric sequence may be 4–12 nucleotides in length, or 5–10 nucleotides in length. The homopolymeric sequence may be used to enhance mRNA expression, in which case the homopolymeric sequence may be 10–200 nucleotides in length, preferably 80–150 nucleotides in length.The homopolymeric sequence may be at least 10, at least 20, 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 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides long. Preferably, the homopolymeric sequence is at least 100 nucleotides long. More preferably, the homopolymeric sequence is at least 120 nucleotides long. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides. The method may further comprise, after the incubation stage of the single contiguous aqueous volume, a purification stage of the partially enclosed linear DNA product. The method may further comprise, after the incubation step in the single contiguous aqueous volume, a nuclease digestion step. The nuclease digestion may be exonuclease digestion, such as exonuclease I and / or exonuclease III. The nuclease digestion step may take place before or after the purification step. This step allows for the removal of any double-stranded DNA and / or adapter molecules that were not used during the execution of the method. Therefore, the method may comprise the following steps: (a) amplifying a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) bringing the double-stranded DNA molecule into contact with an endonuclease, a ligase and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; (c) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,and wherein the second terminal adapter molecule is a nucleic acid molecule comprising one or more nuclease-resistant nucleotides (i.e., protected nucleotides); and, (d) incubate the single contiguous aqueous volume with a nuclease (e.g., exonuclease). The method may include the following stages: (a) amplifying a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) bringing the double-stranded DNA molecule into contact with an endonuclease, a ligase and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; (c) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,and wherein the second terminal adapter molecule is a nucleic acid molecule comprising no or more nuclease-resistant nucleotides (i.e., protected nucleotides); (d) purify the closed linear DNA product; and (e) incubate the purified product from step (d) with a nuclease (e.g., exonuclease). The method may include the following stages: (a) amplifying a DNA template molecule comprising at least one cleavable target sequence (e.g., endonuclease) to generate a double-stranded DNA molecule; (b) bringing the double-stranded DNA molecule into contact with an endonuclease, a ligase and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; (c) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,and wherein the second terminal adapter molecule is a nucleic acid molecule comprising one or more nuclease-resistant nucleotides (i.e., protected nucleotides); (d) incubate the single contiguous aqueous volume with a nuclease (for example, exonuclease); and (e) purify the closed linear DNA product. The step of incubating the single contiguous aqueous volume (or the purified product from step (d)) with a nuclease may be carried out at a temperature of 5–90 °C, 10–80 °C, 15–70 °C, 20–60 °C, 25–50 °C, 30–45 °C, or 35–40 °C. The step of incubating the single contiguous aqueous volume (or the purified product from step (d)) with a nuclease may be carried out for at least 10, at least 20, at least 30, at least 40, at least 50, or at least 60 min. The step of incubating the single contiguous aqueous volume (or the purified product from step (d)) may be carried out at two different temperatures. For example, the step of incubating the single contiguous aqueous volume (or the purified product from step (d)) can be carried out at 15-40 °C for 10-60 minutes followed by a temperature of 60-90 °C for 10-30 minutes. The higher temperature normally inactivates the nuclease (e.g., exonuclease).Therefore, the method also provides a nuclease (e.g., exonuclease) inactivation step. The step of incubating the single contiguous aqueous volume (or the purified product from step (d)) can be carried out at 37 °C for 30 min and 80 °C for 20 min. Preferably, the nuclease (e.g., exonuclease) inactivation step is carried out at a temperature of 70–80 °C. The nuclease (e.g., exonuclease) inactivation step can be carried out for at least 1, at least 5, at least 10, at least 20, or at least 30 min. Preferably, the nuclease (e.g., exonuclease) inactivation step is carried out for at least 5 min. The first terminal adapter molecule and / or the second terminal adapter molecule may comprise a protrusion. The end of the linear double-stranded region may comprise a protrusion 3 or 5. A portion of the first terminal adapter molecule (e.g., the protrusion) may be complementary to the first end of the linear double-stranded region. A portion of the second terminal adapter molecule may be complementary to the second end of the linear double-stranded region. The first and second ends of the linear double-stranded region may be resistant to nuclease digestion. Preferably, the first and second ends of the linear double-stranded region are resistant to exonuclease digestion, such as exonuclease III and / or exonuclease I. The linear DNA product can be partially double-stranded and / or partially single-stranded. The linear DNA product can comprise a portion that is double-stranded and a portion that is single-stranded. The partially closed linear DNA product may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example, a gene that encodes a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosome binding site, and a translation termination sequence. The cassette may further comprise sequences that aid protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas-mediated homology-directed repair (HDR). The cassette may encode the CRISPR guide RNA.The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, polyC, polyT, or polyG sequence. The homopolymeric sequence may be 3–200 nucleotides in length. The homopolymeric sequence may be used to facilitate cassette purification, in which case the homopolymeric sequence may be 4–12 nucleotides in length, or 5–10 nucleotides in length. The homopolymeric sequence may be used to enhance mRNA expression, in which case the homopolymeric sequence may be 10–200 nucleotides in length, preferably 80–150 nucleotides in length.The homopolymeric sequence may be at least 10, at least 20, 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 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides long. Preferably, the homopolymeric sequence is at least 100 nucleotides long. More preferably, the homopolymeric sequence is at least 120 nucleotides long. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides. The partially closed linear DNA product may include a spacer. The spacer may be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 base pairs long. The spacer may improve the ligation efficiency of the first and second terminal adapter molecules to the linear double-stranded region. The spacer may improve cell transfection yields. The partially closed linear DNA product may comprise an inverted terminal repeat sequence. The partially closed linear DNA product may be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs in length. Preferably, the partially closed linear DNA product is at least 50 base pairs in length. The double-stranded DNA molecule can be circular or branched. The double-stranded DNA molecule may not comprise an adapter molecule. The double-stranded DNA molecule may not comprise a hairpin, loop, or stem-loop structure. The double-stranded DNA molecule may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, for example, a gene that encodes a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosome binding site, and a translation termination sequence. The cassette may further comprise sequences that aid protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas-mediated homology-directed repair (HDR). The cassette may encode the CRISPR guide RNA.The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, polyC, polyT, or polyG sequence. The homopolymeric sequence may be 3–200 nucleotides in length. The homopolymeric sequence may be used to facilitate cassette purification, in which case the homopolymeric sequence may be 4–12 nucleotides in length, or 5–10 nucleotides in length. The homopolymeric sequence may be used to enhance mRNA expression, in which case the homopolymeric sequence may be 10–200 nucleotides in length, preferably 80–150 nucleotides in length.The homopolymeric sequence may be at least 10, at least 20, 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 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides long. Preferably, the homopolymeric sequence is at least 100 nucleotides long. More preferably, the homopolymeric sequence is at least 120 nucleotides long. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides. The double-stranded DNA molecule may include a spacer. The spacer may be at least 10, at least 20, 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 125, at least 150, at least 175, or at least 200 base pairs long. The spacer may improve the amplification performance of the double-stranded DNA molecule. The spacer may improve the ligation efficiency of the first and second terminal adapter molecules to the linear double-stranded region. The spacer may improve cell transfection yields. The double-stranded DNA molecule can be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long. The double-stranded DNA molecule may comprise one or more cleavable target sequences (e.g., endonuclease). The double-stranded DNA molecule may comprise two cleavable target sequences (e.g., endonuclease). The one or more endonuclease target sequences may be type IIS endonuclease target sequences.The one or more target sequences of endonuclease can be target sequences of BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw26I, AlwI, ArsI, AsuI, BaeHPI, BaeHPI, BaeHPI, Bae BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMII, BseNI, BseRI, BseXI, BseXI, BsFI, BsFI, BseI BsmFI, BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BcCI, BcI-v2, BcCI, BcCi, CI, CI, CI, Mu Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, GsuI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, MlyI, MmeI, MnII, MnIII, PaqI, NCIA PciSI, PctI, PleI, PpsI, Psrl, SchI, SfaNI, TaqlI, TspDTI y / o TspGWI. The double-stranded DNA molecule can be an amplification product. Preferably, the amplification is rolling circle amplification. The linear double-stranded region can be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long. The linear double-stranded region may comprise a 3-OH group at the first and / or second ends. The 3-OH group may facilitate bonding to the first and / or second terminal adapter molecule(s) and / or intermediate n+M adapter molecules (which may comprise a 5-phosphate). The linear double-stranded region may comprise a 5-phosphate at the first and / or second ends. The 5-phosphate may facilitate bonding to the first and / or second terminal adapter molecule(s) and / or intermediate adapter molecules (which may comprise a 3-OH group). The linear double-stranded region (e.g., the linear portion of the double-stranded molecule) may include a protrusion. For example, the linear double-stranded region may include a 5-protrusion or a 3-protrusion. The linear double-stranded region may include one or more blunt ends. The linear double-stranded region may include: one 5-protrusion and one blunt end, two 5-protrusions, one 3-protrusion and one blunt end, two 3-protrusions, or one 5-protrusion and one 3-protrusion. The protrusion may be at least 3 nucleotides long (preferably 4 to 8 nucleotides). The protrusion may be on the sense strand or the antisense strand of the linear double-stranded region. The linear portion of the double-stranded DNA molecule (e.g., the linear portion of the double-stranded molecule) can be at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs long. Preferably, the double-stranded DNA molecule is at least 50 base pairs long. The first terminal adapter molecule and / or the second terminal adapter molecule or one or more n+m intermediate adapter molecules may be a synthetic adapter molecule. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more n+m intermediate adapter molecules may not be a plasmid or a vector DNA. The adapter molecule may comprise a double-stranded portion. The double-stranded portion may comprise less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, or less than 10 base pairs. The double-stranded portion may comprise 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, or at least 15 base pairs. The adapter molecule may comprise a 5-phosphate. The 5-phosphate may facilitate binding to the linear double-stranded region. The first terminal adapter molecule may comprise a portion that is complementary to the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second terminal adapter molecule may comprise a portion that is complementary to the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The first terminal adapter molecule may comprise a portion that hybridizes to the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second terminal adapter molecule may comprise a portion that hybridizes to the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule).The first terminal adapter molecule may comprise a portion that is complementary to and hybridizes with the first end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The second terminal adapter molecule may comprise a portion that is complementary to and hybridizes with the second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule). The portion that is complementary to or hybridizes with the first or second end of the linear double-stranded region (or the linear portion of the double-stranded DNA molecule) can be a 5' or 3' protrusion of the first and / or second terminal adapter molecule. The protrusion of the first terminal adapter molecule can be complementary to the first end of the linear double-stranded region, and / or the protrusion of the second terminal adapter molecule can be complementary to the second end of the double-stranded region. The protrusion of the first terminal adapter molecule can hybridize to the first end of the linear double-stranded region, and / or the protrusion of the second terminal adapter molecule can hybridize to the second end of the linear double-stranded region.The protrusion of the first terminal adapter molecule can be complementary and hybridize with the first end of the linear double-stranded region and / or the protrusion of the second terminal adapter molecule can be complementary and hybridize with the second end of the linear double-stranded region. The first terminal adapter molecule may comprise a portion that is complementary to the first end of the nth intermediate adapter molecule. The second terminal adapter molecule may comprise a portion that is complementary to the first end of the mth intermediate adapter molecule. The first terminal adapter molecule may comprise a portion that hybridizes to the first end of the nth intermediate adapter molecule. The second terminal adapter molecule may comprise a portion that hybridizes to the first end of the mth intermediate adapter molecule. The first terminal adapter molecule may comprise a portion that is complementary to and hybridizes to the first end of the nth intermediate adapter molecule. The second terminal adapter molecule may comprise a portion that is complementary to and hybridizes to the first end of the mth intermediate adapter molecule. The portion that is complementary to or hybridizes with the first end of the nth or mth intermediate adapter molecule can be a 5-protrusion or a 3-protrusion of the first and / or second terminal adapter molecule. The protrusion of the first terminal adapter molecule can be complementary to the first end of the nth intermediate adapter molecule, and / or the protrusion of the second terminal adapter molecule can be complementary to the first end of the mth intermediate adapter molecule. The protrusion of the first terminal adapter molecule can hybridize with the first end of the nth intermediate adapter molecule, and / or the protrusion of the second terminal adapter molecule can hybridize with the first end of the mth intermediate adapter molecule.The protrusion of the first terminal adapter molecule can be complementary and hybridize with the first end of the nth intermediate adapter molecule and / or the protrusion of the second terminal adapter molecule can be complementary and hybridize with the first end of the mth intermediate adapter molecule. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise a 5-phosphate. The 5-phosphate may facilitate ligation to the linear double-stranded region or to adjacent terminal or intermediate adapter molecules (which may comprise a 3-OH group at the first and / or second end). The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise a 3-OH group. The 3-OH group may facilitate ligation to the linear double-stranded region and / or to adjacent intermediate adapter molecules (which may comprise a 5-phosphate at the first and / or second end). A portion of the first terminal adapter molecule (e.g., the protruding portion) may be complementary to the first end of the linear double-stranded region or to a first end of an intermediate adapter molecule, preferably the nth intermediate adapter molecule. A portion of the second terminal adapter molecule (e.g., the protruding portion) may be complementary to the second end of the linear double-stranded region or to a first end of an intermediate adapter molecule, preferably the mth adapter molecule. A portion of the first end of the linear double-stranded region may be complementary to a first end of an intermediate adapter molecule, preferably an n-(n-1) intermediate adapter molecule. A portion of the second end of the linear double-stranded region may be complementary to a first end of an intermediate adapter molecule, preferably an m-(m-1) intermediate adapter molecule.A portion of a second end of an intermediate adapter molecule may be complementary to a first end of an adjacent intermediate adapter molecule; for example, a portion of the second end of the nth intermediate adapter molecule may be complementary to a portion of the first end of the n-1st intermediate adapter molecule, and a portion of the second end of the n-1st adapter molecule may be complementary to a portion of the first end of the n-2nd intermediate adapter molecule, and so on.A portion of the second end of an intermediate adapter molecule may be complementary to a first end of an adjacent intermediate adapter molecule; for example, a portion of the second end of the m-th intermediate adapter molecule may be complementary to a portion of the first end of the m-1st intermediate adapter molecule, and a portion of the second end of the m-1st adapter molecule may be complementary to a portion of the first end of the m-2nd intermediate adapter molecule, and so on. n may be 0, 1, 2, 3, 4, 5, 6, 7, or 8. m may be 0, 1, 2, 3, 4, 5, 6, 7, or 8. Preferably, n is 0, 1, 2, 3, or 4, and m is 0, 1, 2, 3, or 4. The first terminal adapter molecule and / or the second terminal adapter molecule may not comprise an IIS-type endonuclease target sequence. The first terminal adapter molecule and / or the second terminal adapter molecule may not comprise target sequences of BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AloI, Alw2I, ArwI, ASUI, ASUI BaeI, BarI, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseGI, BseMI, BseMIX, BseMIX, BsegI, BsegI, BseI, BseMI BslFI, BsmAI, BsmFI, BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsrDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BsuI, BtgZI, BcCI, BcI-Mut-I, Bsp-MutI BveI, CseI, CspCI, Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, GsuI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109I, LweI, MboII, Mly, MnI, MnI, MnI, 1269 NmeAIII, PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI y / o TspGWI SapI. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise a functional portion. The functional portion may be a binding molecule, a targeting sequence, or a probe. The functional portion may be a cassette, an open reading frame, or a coding sequence. The functional portion may be a promoter and enhancer, an NLS sequence, a UTR, ITR, or other repeats, a termination sequence, modified nucleotides, or a fluorophore. The functional portion may be a probe. As used herein, the term "probe" refers to a fragment of DNA, RNA, or DNA / RNA chimera of variable length (e.g., 3–1000 bases), used to detect the presence of target nucleotide sequences that are complementary to the sequence in the probe. Typically, the probe hybridizes to single-stranded nucleic acid whose base sequence allows probe-target base pairing due to the complementarity between the probe and the target. Therefore, the functional portion may be a DNA sequence, an RNA sequence, or a DNA / RNA chimera sequence. As used herein, the term "complementary" refers to the pairing of nucleotide sequences according to the Watson / Crick pairing rules. For example, a sequence 5-GCGGTCCCA-3 has the complementary sequence 5-TGGGACCGC-3.A complementary sequence can also be an RNA sequence complementary to the DNA sequence. The functional portion may be a binding molecule. The term "binding molecule" refers to any molecule capable of binding to the linear DNA product described herein and / or capable of binding to an additional molecule or target. The binding molecule may be a protein, a polypeptide, or a peptide. The binding molecule may be an antibody, such as a monoclonal or polyclonal antibody. The binding molecule may be an antibody fragment. The functional portion can facilitate detection of the DNA product by binding to capture molecules (e.g., capture antibodies bound by protein-protein interactions). The functional portion can bind to a cellular target, for example, a cell receptor. The functional portion can be a tag. The tag can be any chemical entity that allows the detection of the double-stranded nucleic acid molecule through physical, chemical, and / or biological means. The tag can be a chromophore, a fluorophore, and / or a radioactive molecule. The functional portion may be a targeting sequence. The targeting sequence can be a fragment of DNA or RNA of variable length, used to direct the DNA product to a specific location within a cell. The targeting sequence can be used to increase the transfection efficiency of non-viral gene delivery by enhancing the nuclear import of the partially closed linear DNA product. For example, the targeting sequence may be a nuclear DNA targeting sequence (i.e., a recognition sequence for endogenous DNA-binding proteins), such as the SV40 enhancer sequence (preferably in the 3' direction of the cassette). The targeting sequence may be a proteomerase targeting sequence, or a truncated variant thereof. To facilitate the detection and / or quantification of the DNA product, the functional portion may comprise a fluorophore, a radioactive compound, or a barcode. A signal corresponding to the presence, absence, and / or level of the partially closed linear DNA product can be measured using a barcode. The barcode may comprise at least one binding residue attached to a barcode portion, wherein the barcode portion comprises at least one nucleotide (i.e., the barcode portion comprises a nucleotide sequence at least one nucleotide in length), and wherein the binding residue is capable of binding to the 3', 5', or blunt end of the partially closed linear DNA product. The signal can be measured by determining the presence, absence, and / or level of the barcode portion of the barcode (e.g., by sequencing or PCR).The barcode portion may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides. The barcode may comprise at least 2 linker residues (e.g., a first linker residue and a second linker residue). For example, the first linker residue attached to the first barcode portion may be attached to the 3' end of the partially closed linear DNA product, and / or the second linker residue attached to the second barcode portion may be attached to the 5' end of the partially closed linear DNA product. A signal corresponding to the presence, absence, and / or level of the partially closed linear DNA product can be measured using a fluorophore (i.e., a fluorescently labeled molecule) attached to or connected to protrusion 3, protrusion 5, or the blunt end of the partially closed linear DNA product. The signal can be measured by flow cytometry and / or fluorescence-activated cell sorting. The functional portion can also facilitate DNA sequencing. For example, the functional portion can be a sequencing adapter.The term "sequencing adapter" is intended to encompass one or more nucleic acid domains that include at least a portion of a nucleic acid sequence (or complement thereof) used by a sequencing platform of interest, such as a sequencing platform provided by Illumina® (e.g., the HiSeq™, MiSeq™ and / or Genome Analyzer™ sequencing systems), Oxford Nanopore™ Technologies (e.g., the MinlON sequencing system), Ion Torrent™ (e.g., the Ion PGMTM and / or Ion Proton™ sequencing systems), Pacific Biosciences (e.g., the PACBIO RS II sequencing system); Life Technologies™ (e.g., a SOLiD sequencing system), Roche (e.g., the 454 GS FLX+ and / or GS Junior sequencing systems), or any other sequencing platform of interest. One or more of the n+m intermediate adapter molecules (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 intermediate adapter molecules) may comprise a cassette. The cassette may comprise a coding sequence. The coding sequence may encode a gene of interest, e.g., a gene that encodes a protein. The cassette may comprise at least a portion of a promoter and a coding sequence. The cassette may comprise a promoter and a coding sequence. The cassette may comprise a promoter, a coding sequence, a ribosome binding site, and a translation termination sequence. The cassette may further comprise sequences that aid protein expression, such as a cap-independent translation element. The cassette may comprise (or encode) a repair template (or editing template). The repair template (or editing template) may be for use in CRISPR-Cas-mediated homology-directed repair (HDR).The cassette may encode the CRISPR guide RNA. The cassette may be a mammalian expression cassette. The promoter may be a CMV promoter. The cassette may further comprise an enhancer. The cassette may further comprise a reporter gene, such as an eGFP reporter gene or a luciferase reporter gene. The cassette may further comprise a homopolymeric sequence, such as a polyA, polyC, polyT, or polyG sequence. The homopolymeric sequence may be 3–200 nucleotides in length. The homopolymeric sequence may be used to facilitate cassette purification, in which case the homopolymeric sequence may be 4–12 nucleotides in length, or 5–10 nucleotides in length. The homopolymeric sequence may be used to enhance mRNA expression, in which case the homopolymeric sequence may be 10–200 nucleotides in length, preferably 80–150 nucleotides in length.The homopolymeric sequence may be at least 10, at least 20, 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 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides long. Preferably, the homopolymeric sequence is at least 100 nucleotides long. More preferably, the homopolymeric sequence is at least 120 nucleotides long. For example, the homopolymeric sequence may comprise a polyA sequence of at least 120 nucleotides. One or more of the n+m intermediate adapter molecules (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 intermediate adapter molecules) may comprise a spacer. The spacer may be at least 10, at least 20, 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 125, at least 150, at least 175, or at least 200 base pairs in length. One or more of the n+m intermediate adapter molecules (for example, 1, 2, 3, 4, 5, 6, 7, or 8 intermediate adapter molecules) can be 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 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12,000, at least 13,000, at least 14,000, or at least 15,000 base pairs in length. Preferably, one or more of the n+m intermediate adapter molecules (for example, 1, 2, 3, 4, 5, 6, 7, or 8 intermediate adapter molecules) are at least 10 base pairs in length. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise a polyA signal sequence. One or more of the n+m intermediate adapter molecules may comprise a polyA signal in the 3 direction of a barcode. The first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more of the n+m intermediate adapter molecules may comprise an aptamer. 4. Methods for protein transcription and expression The invention provides a method for the in vitro transcription of a linear DNA product (e.g., a closed linear DNA product or a partially closed linear DNA product), wherein the method comprises contacting the linear DNA product (e.g., a closed linear DNA product or a partially closed linear DNA product), produced by the methods described herein, with a polymerase and producing a transcription product encoded by the linear DNA product (e.g., the closed linear DNA product or a partially closed linear DNA product). The invention provides a method for the in vitro transcription of a linear DNA product (e.g., a closed linear DNA product or a partially closed linear DNA product), wherein the method comprises: (a) produce a linear DNA product (for example, a closed linear DNA product or a partially closed linear DNA product) by any of the methods described herein; (b) contacting the linear DNA product (for example, a closed linear DNA product or a partially closed linear DNA product) with a polymerase; and (c) produce a transcription product encoded by the linear DNA product (e.g., the closed linear DNA product or a partially closed linear DNA product). The invention provides a method for the in vitro transcription of a linear DNA product, wherein the method comprises: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region; and (c) contacting the linear DNA product with a polymerase; and (d) produce a transcription product encoded by the linear DNA product. The method may use adapter molecules that generate a closed linear DNA product, such as the adapter molecules described herein. A method for the in vitro transcription of a closed linear DNA product may comprise: (a) bringing a double-stranded DNA molecule into contact with an endonuclease, a ligase and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; (b) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and closed at a second end by the second terminal adapter molecule added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region; (c) contacting the closed linear DNA product with a polymerase; and (d) produce a transcription product encoded by the closed linear DNA product. The method may use adapter molecules comprising protected nucleotides, such as the adapter molecules described herein. A method for the in vitro transcription of a linear DNA product may comprise: (a) bringing a double-stranded DNA molecule into contact with an endonuclease, a ligase and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,and wherein the first terminal adapter molecule and the second terminal adapter molecules are nucleic acid molecules comprising one or more nuclease-resistant nucleotides (i.e., protected nucleotides); (c) contacting the linear DNA product with a polymerase; and (d) produce a transcription product encoded by the linear DNA product. The method may use adapter molecules comprising protected nucleotides and adapter molecules comprising a hairpin or stem-loop, such as the adapter molecules described herein. A method for the in vitro transcription of a partially closed linear DNA product may comprise: (a) bringing a double-stranded DNA molecule into contact with an endonuclease, a ligase and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; (b) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the partially closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,and wherein the second terminal adapter molecule is a nucleic acid molecule comprising one or more nuclease-resistant nucleotides; (c) contacting the partially closed linear DNA product with a polymerase; and (d) produce a transcription product encoded by the partially closed linear DNA product. The invention provides a method for producing a protein, wherein the method comprises introducing the linear DNA product (e.g., the closed linear DNA product or the partially closed linear DNA product), produced by the methods described herein, into a cell (e.g., a prokaryotic cell or a eukaryotic cell) or a cell-free expression system to generate a protein encoded by the linear DNA product (e.g., the closed linear DNA product or the partially closed linear DNA product). The invention provides a method for producing a protein, wherein the method comprises: (a) produce a linear DNA product (for example, a closed linear DNA product or a partially closed linear DNA product) by any of the methods described herein; and (b) introducing the linear DNA product (e.g., the closed linear DNA product or the partially closed linear DNA product) into a cell (e.g., a prokaryotic cell or a eukaryotic cell) or a cell-free expression system to generate a protein encoded by the linear DNA product (e.g., the closed linear DNA product or the partially closed linear DNA product). The invention provides a method for producing a protein comprising: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region; and (c) introducing the linear DNA product into a cell (e.g., a prokaryotic cell or a eukaryotic cell) or a cell-free expression system to generate a protein encoded by the linear DNA product. The method may use adapter molecules that generate a closed linear DNA product, such as the adapter molecules described herein. A method for producing a protein may comprise: (a) bringing a double-stranded DNA molecule into contact with an endonuclease, a ligase and first and second terminal adapter molecules and n+m intermediate adapter molecules, to form a single contiguous aqueous volume, wherein nym are each 0 or an integer of at least 1, and wherein n+m is at least 1; (b) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the closed linear DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and closed at a second end by the second terminal adapter molecule added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region; and (c) introducing the closed linear DNA product into a cell (e.g., a prokaryotic cell or a eukaryotic cell) or a cell-free expression system to generate a protein encoded by the closed linear DNA product. The method may use adapter molecules comprising protected nucleotides, such as the adapter molecules described herein. A method for producing a protein may comprise: (a) bringing a double-stranded DNA molecule into contact with an endonuclease, a ligase and first and...

Claims

1. A method for producing a closed linear deoxyribonucleic acid (DNA) product, wherein the method comprises: (a) contacting a double-stranded DNA molecule with an endonuclease, a first terminal adapter molecule, a second terminal adapter molecule, and n+m intermediate adapter molecules to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (b) incubating the single contiguous aqueous volume to generate the closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region.and wherein the closed linear DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and closed at a second end by the second terminal adapter molecule added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region.

2. The method of claim 1, wherein the first and / or second terminal adapter molecules comprise a hairpin.

3. A method for producing a linear deoxyribonucleic acid (DNA) product, wherein the method comprises: (a) contacting a double-stranded DNA molecule with an endonuclease, a first terminal adapter molecule, a second terminal adapter molecule, and n+m intermediate adapter molecules to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer less than or equal to 1,and wherein n+m is at least 1; and (b) incubating the single contiguous aqueous volume to generate the linear DNA product, wherein the linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule, and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the first terminal adapter molecule is added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region,and wherein the first terminal adapter molecule and the second terminal adapter molecules are nucleic acid molecules comprising one or more nuclease-resistant nucleotides.

4. A method for producing a partially closed deoxyribonucleic acid (DNA) product, wherein the method comprises: (a) contacting a double-stranded DNA molecule with an endonuclease, a first terminal adapter molecule, a second terminal adapter molecule, and n+m intermediate adapter molecules to form a single contiguous aqueous volume, wherein n and m are each 0 or an integer of at least 1, and wherein n+m is at least 1; and (b) incubating the single contiguous aqueous volume to generate the partially closed linear DNA product, wherein the closed linear DNA product comprises a linear double-stranded region, wherein the linear double-stranded region comprises a linear portion of the double-stranded DNA molecule,and wherein n intermediate adapter molecules are sequentially added to a first end of the linear double-stranded region and m intermediate adapter molecules are sequentially added to a second end of the linear double-stranded region, and wherein the partially closed DNA product is closed at a first end by the first terminal adapter molecule added to the nth intermediate adapter molecule or, when n is 0, to the first end of the linear double-stranded region, and the second terminal adapter molecule is added to the mth intermediate adapter molecule, or when m is 0, to the second end of the linear double-stranded region, and wherein the second terminal adapter molecule is a nucleic acid molecule comprising one or more nuclease-resistant nucleotides.

5. Method of claim 4, wherein the first terminal adapter molecule comprises a hairpin.

6. Method of any one of claims 1-5,wherein the endonuclease is a restriction endonuclease of type IIS, optionally wherein the endonuclease is a restriction endonuclease BbsI, BsaI, BsmBI, BspQI, BtgZI, Esp3I, SapI, AarI, Acc36I, AclWI, AcuI, AjuI, AjuI, Aju AlwI, ArsI, AsuHPI, BaeI, BarI, BbvI, BccI, BceAI, BcgI, BciVI, BcoDI, BfuAI, BfuI, BmrI, BmsI, BmuI, BpiI, BpmI, BpuEI, BsaXI, Bse1I, Bse3DI, BseI, BseMI, BseMI, BseMI, BseMI BseXI, BsgI, BslFI, BsmAI, BsmFI, BsmI, Bso31I, BspCNI, BspMI, BspPI, BspQI, BspTNI, BsprDI, BsrI, Bst6I, BstF5I, BstMAI, BstV1I, BstV2I, BtsmAI, BcgI, BspI, BtsI-v2, BtsMutI, BveI, CseI, CspCI, Eam1104I, EarI, EciI, Eco31I, Eco57I, Esp3I, FaqI, FauI, FokI, GsuI, HgaI, HphI, HpyAV, LguI, LmnI, Lsp1109, LweI, Lwe, MI, Mly MmeI, MnII, Mva1269I, NmeAIII, PaqCI, PciSI, PctI, PleI, PpsI, PsrI, SchI, SfaNI, TaqII, TspDTI y / o TspGWI.

7. Method of any one of claims 1-6,wherein the first terminal adapter molecule and / or the second terminal adapter molecule and / or one or more intermediate adapter molecules are nucleic acid adapter molecules.

8. The method of any one of claims 1-7, wherein step a) further comprises contacting the double-stranded DNA molecule with a ligase.

9. The method of any one of claims 1-8, wherein the first-end adapter molecule and / or the second-end adapter molecule and / or one or more intermediate adapter molecules comprise a double-stranded region with a protrusion.

10. The method of any one of claims 3-9, wherein the one or more nuclease-resistant nucleotides are one or more phosphorothioate nucleotides.

11. The method of any one of claims 1-10, wherein one or more of the n+m intermediate adapter molecules have 10-500, 50-400,100-300 or 200-250 base pairs in length.

12. A method of any one of claims 1-11, wherein one or more of the n+m intermediate adapter molecules comprise a fluorophore, a barcode, a polyA signal, a biotinylated nucleotide, protected nucleotides, spacers, a polyA sequence, a promoter, an open reading frame, a targeting sequence, or a localization signal.

13. A method of any one of claims 1-12, wherein n is 0, 1, 2, or 3.

14. A method of any one of claims 1-13, wherein m is 0, 1, 2, or 3.