Abstract:
The present invention consists of methods for joining DNA molecules (parts) together to form larger DNA molecules (assemblies) of specified sequence and organization. The invention exhibits three necessary characteristics. Firstly, the invention enables 2 or more parts to be joined in a single reaction. Secondly, the seam between joined parts is scarless, producing no residual sequence dependencies like restriction enzyme recognition sites. Thirdly, parts are modular and can easily be reused in novel assemblies without modification. Prior technologies have exhibited no more than two of the three necessary characteristics, limiting their utility in synthesizing and editing DNA molecules of arbitrary sequence.

Description:
PRIORITY 
       [0001]    The present application claims priority to, and the benefit of, U.S. Provisional Application No. 61/670,061 filed Jul. 10, 2012, and U.S. Provisional Application No. 61/789,032 filed Mar. 15, 2013, each of which is hereby incorporated by reference in its entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to methods for multipart, modular and scarless assembly of nucleic acids, including for high-throughput, automated, and/or large scale engineering of biological systems. 
       BACKGROUND 
       [0003]    A key concept within synthetic biology is that biological DNA parts can be standardized, abstracted, and combined to produce complex, engineered systems. Parts are routinely generated via cloning from the DNA of organisms or using DNA synthesis. However, assembling parts into complex systems remains a key bottleneck in the synthetic biology workflow. 
         [0004]    Numerous technologies have been developed to facilitate DNA assembly, yet none provide a robust solution. (See, e.g., U.S. Patent Application No. 2010/0035768, U.S. Patent Application No. US 2012/0040870; Engler C. et al.,  A One Pot, One Step, Precision Cloning Method with High Throughput Capability , PLoS ONE 3(11):e3647 (2008), doi:10.1371/journal.pone.0003647; Weber E., et al., A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE 6(2):e16765 (2011), doi:10.1371/journal.pone.0016765; and Ellis, T., et al. DNA assembly for synthetic biology: from parts to pathways and beyond, Integr. Biol., 3:109-118 (2011), DOI: 10.1039/C01B00070A 2; and information found on the World Wide Web at j5.jbei.org/j5manual/pages/1.html; all of which are incorporated by reference herein in their entireties.) 
         [0005]    There remains a need in the synthetic biology field by which biological DNA parts can be routinely combined, and at high-throughput, to produce complex, engineered systems. The present invention meets these objectives. 
       SUMMARY OF THE INVENTION 
       [0006]    The present invention provides for multipart, modular and scarless nucleic acid assembly in vitro. In some embodiments, the DNA assembly reactions, which can proceed in parallel and series, are designed computationally based on a desired sequence. For example, the nucleic acid assembly may involve a plurality of reactions in parallel and/or in series that are designed in silico for accurate, cost-effective engineering of biological systems. In some embodiments the methods and kits described herein can be employed with high-throughput, automated processing systems. 
         [0007]    In some embodiments, the invention provides a method for constructing a scarless nucleic acid molecule comprising a plurality of heterologous parts. Nucleases and nucleic acid staples or adaptors are selected, as described herein, to assemble the heterologous parts into a scarless nucleic acid molecule. Nuclease and ligation reactions can take place in parallel and/or in series, as needed for optimum control of the process. The process can be controlled computationally by user inputs, with reaction assembly and processing taking place by automation. 
         [0008]    In some embodiments, the method comprises generating a first nucleic acid molecule having a single stranded terminus, generating a second nucleic acid molecule having a single stranded terminus, and then ligating the first and second nucleic acid molecules with the aid of an intervening linker molecule such that the ligation product corresponds to the combined sequence of the first and second nucleic acid molecules. In some embodiments, the nucleic acid molecule is a DNA molecule. An algorithm can be employed to computationally determine, identify and/or optimize any of the parts, enzymes and/or other reagents to be employed with the present methods. 
         [0009]    Scarless nucleic acid assembly according to the methods of the present invention requires two classes of enzymes. The first enzyme catalyzes the formation of short (about 1 bp to 8 bp), single stranded 5′-overhangs on a nucleic acid. The second enzyme catalyzes the formation of short (about 1 bp to 8 bp), single stranded 3′-overhangs on a nucleic acid. Each of these enzymes and overhang size can be independently selected, and can be a Type II restriction enzyme in some embodiments. 
         [0010]    In some embodiments, the linker is a staple. A staple may be single stranded and can be DNA or RNA. In some embodiments, the staple is a defined sequence capable of binding with perfect complementarity to the single stranded DNA termini generated on the first and second DNA molecules. In some embodiments, the staple binds to a single stranded DNA terminus with a 3′-overhang on a first DNA molecule and a single stranded DNA terminus with a 5′-overhang on a second DNA molecule. In some embodiments, the staple binds to a single stranded DNA terminus with a 5′-overhang on a first DNA molecule and a single stranded DNA terminus with a 3′-overhang on a second DNA molecule. 
         [0011]    In some aspects, the present invention provides a plurality of reaction mixtures for performing one and/or a series of reaction mixtures for scarless nucleic acid assembly. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0012]      FIG. 1 : Schematic diagram showing Staple Implementation and Adapter Implementation for multipart, modular and scarless assembly (MMS). 
           [0013]      FIG. 2 : Assembly of two DNA parts using a “staple” linker. Two input DNA parts each with a size of 250 bp and 400 bp are ligated together to form a 650 bp product. Lane 1: 100 bp NEB DNA ladder. Lane 2: Input DNA only. Lane 3: Input DNA (without oligonucleotide “staple”) after ligation reaction. Lane 4: Input DNA+oligonucleotide staple after ligation reaction. 
           [0014]      FIG. 3 : Assembly of two DNA parts using an “adapter” linker. Lane 1: 1 kb NEB ladder. Lane 2: Two input DNA parts of sizes 1800 bp and 300 bp are assembled to form a 2100 bp product. Lane 3: Two input DNA parts of sizes 700 bp and 1800 bp are assembled to form a 2500 bp product. 
           [0015]      FIG. 4 : Isothermal Scarless Subcloning. Reaction mixture containing T4 ligase buffer, T7 DNA ligase, BsaI and BsaXI, and DNA parts. Isothermal reaction was performed at 37° C. for 1 hr. Colony PCRs and sequencing show 11 of 12 clones assembled correctly. 
           [0016]      FIG. 5 : Scarless Assembly of Multiple Parts. Reaction mixture containing T4 ligase buffer, T7 DNA ligase, BsaI and BstXI, and DNA parts. Isothermal reaction was performed at 37° C. for 8 hr. Colony PCRs and sequencing show 6 of 12 clones assembled correctly. 
           [0017]      FIG. 6 : Multiplex Assembly in One Tube. Reaction mixture containing T4 ligase buffer, T7 DNA ligase, BsaI and BstXI, and DNA parts. Isothermal reaction was performed at 37° C. for 8 hr. Colony PCRs and sequencing show 23 of 24 clones assembled correctly. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0018]    The present invention provides for multipart, modular and scarless nucleic acid assembly in vitro. In some embodiments, the DNA assembly reactions, which can proceed in parallel and series, are designed computationally based on a desired sequence. For example, the nucleic acid assembly may involve a plurality of reactions in parallel and/or in series that are designed in silico for accurate, cost-effective engineering of biological systems. In some embodiments the methods and kits described herein can be employed with high-throughput, automated processing systems. 
         [0019]    The term “scarless” refers to the fact that no changes or undesired sequences are introduced into assembled DNA by the reactions. The combined sequence will correspond to the exact sequence desired with no changes being introduced by the restriction enzyme/ligation procedure. The combined sequence can correspond exactly to a natural sequence, an engineered sequence, a synthetic sequence or any other desired reference sequence. 
         [0020]    The term “modular” refers to the fact that prepared nucleic acid parts can be ligated with any other prepared nucleic acid parts without dependencies on the nucleic acid sequence of the two parts. 
         [0021]    The term “multipart” refers to the fact that two or more nucleic acid parts can be ligated in a single in vitro reaction. 
         [0022]    The term “reagent” can include any component of a reaction described herein. Reagents can include but are not limited to buffers, enzymes (e.g., nucleases, ligases) and nucleic acids (e.g., parts, linkers, staples). Nucleic acid reagents can include one or more chemically modified bases, including for example but not limited to phosphorothioates, locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2′-0Me nucleotides, methylphosphonates or morpholinos, as well as any other modifications known in the art and that one of skill would find useful for the present methods. 
         [0023]    Perfect or near perfect complementarity occurs when two nucleic acids regions of interest share about 100%, about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 85%, about 80%, about 75%, or about 70% sequence identity, homology or complementarity to one another. 
         [0024]    In some embodiments, the method provides for assembly of any desired nucleic acid molecule, including DNA or RNA, as well as modified DNA and RNA molecules (e.g., nucleic acids containing chemically modified bases, such as but not limited to phosphorothioates, locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2′-0Me nucleotides, methylphosphonates or morpholinos). In some embodiments, assembly is via high-throughput methods and in some embodiments, said high-throughput methods are automated. The resulting DNA molecules can be at least 1 kb in length, at least 10 kb in length, at least 100 kb in length, or over 500 kb in length, or over 1000 kb in length. 
         [0025]    In some embodiments, the invention involves computational selection of the desired DNA parts, and/or desired reagents, as well as design of optimal parallel and/or series reactions for generating the desired DNA product. 
         [0026]    In some embodiments, the invention provides a method for constructing a scarless nucleic acid molecule comprising 2 or more heterologous parts, such as 5 or more, 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more heterologous parts. Nucleases and nucleic acid staples and/or adaptors are selected, as described herein, to assemble the heterologous parts into a scarless nucleic acid molecule by ligation. The restriction and ligation reactions can take place in parallel and/or in series, as needed for optimum control of the process. The process can be controlled computationally by user inputs, with reaction assembly and processing taking place by automation. 
         [0027]    In some embodiments, the method comprises generating a first nucleic acid molecule having a single stranded terminus, generating a second nucleic acid molecule having a single stranded terminus, and then ligating the first and second nucleic acid molecules with the aid of an intervening linker molecule such that the ligation product corresponds to the combined sequence of the first and second nucleic acid molecules. In some embodiments, the nucleic acid molecule is a DNA molecule. In some embodiments, an algorithm can be employed to computationally determine, identify and/or optimize any of the parts, enzymes and/or other reagents to be employed with the present methods. Ligation methods are well known in the art and any of these known ligation methods can be employed with the present invention. 
         [0028]    In some embodiments, the first nucleic acid molecule or the second nucleic acid molecule have single stranded termini generated with a restriction enzyme. In some embodiments, the nucleic acid molecule is a DNA molecule. 
         [0029]    Scarless nucleic acid assembly according to the methods of the present invention requires two classes of enzymes. The first enzyme catalyzes the formation of short (about 1 bp to 8 bp), single stranded 5′-overhangs on a nucleic acid. The second enzyme catalyzes the formation of short (about 1 bp to 8 bp), single stranded 3′-overhangs on a nucleic acid. Each of these enzymes and overhang size can be independently selected. In some embodiments, such restriction enzymes are selected from Type IIs, Type IIb, or Type IIp family enzymes. In some embodiments, in part in order to bypass constraints on nucleic acid sequences, the enzymes are selected from types that cleave the nucleic acid sequence at a position distal (about 1 bp to 25 bp) to the recognition site. 
         [0030]    In some embodiments, the single stranded termini can include 5′-overhangs, 3′-overhangs which are independently selected. In some embodiments, the overhangs are independently selected from the following ranges: about 1 bp to 8 bp, about 2 bp to 8 bp, about 2 bp to 6 bp, about 3 bp to 6 bp, about 3 bp to 5 bp, about 2 bp to 6 bp, about 2 bp to 5 bp, about 1 bp to 5 bp, about 2 bp to 4 bp, about 1 bp to about 4 bp, about 1 bp to 3 bp or about 1 bp to 2 bp. In some embodiments, the overhangs are about 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, or 8 bp or more in length. 
         [0031]    In some embodiments, the restriction enzyme is a Type IIs restriction enzyme. The Type II restriction enzymes that find use with the methods of the present invention can generate a single stranded nucleic acid terminus with a 3′-overhang or a 5′-overhang. Enzyme properties can also be found on the World Wide Web at rebase.neb.com. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1  
               
             
             
               
                   
               
               
                 Type II restrictions enzymes producing  
               
               
                 5′-overhangs 
               
             
          
           
               
                 Length 
                 Overhang 
                 Enzymes 
                 Recognition Sequence 
               
               
                   
               
               
                 1 
                 N 
                 BccI 
                 CCATC (4/5) 
               
               
                   
               
               
                 1 
                 N 
                 BcefI 
                 ACGGC (12/13) 
               
               
                   
               
               
                 1 
                 N 
                 BinI 
                 GGATC (4/5) 
               
               
                   
               
               
                 1 
                 N 
                 EcoNI 
                 CCTNN↓NNNAGG 
               
               
                   
               
               
                 1 
                 N 
                 Fnu4HI 
                 GC↓NGC 
               
               
                   
               
               
                 1 
                 N 
                 PleI 
                 GAGTC (4/5) 
               
               
                   
               
               
                 1 
                 N 
                 ScrFI 
                 CC↓NGG 
               
               
                   
               
               
                 1 
                 N 
                 Tth111I 
                 GACN↓NNGTC 
               
               
                   
               
               
                 1 
                 S 
                 CauII 
                 CC↓SGG 
               
               
                   
               
               
                 1 
                 W 
                 BstNI 
                 CC↓WGG 
               
               
                   
               
               
                 2 
                 AT 
                 Asi256I 
                 G↓ATC 
               
               
                   
               
               
                 2 
                 AT 
                 CviAII 
                 C↓ATG 
               
               
                   
               
               
                 2 
                 CG 
                 AciI 
                 CCGC (−3/−1) 
               
               
                   
               
               
                 2 
                 CG 
                 AcII 
                 AA↓CGTT 
               
               
                   
               
               
                 2 
                 CG 
                 AcyI 
                 GR↓CGYC 
               
               
                   
               
               
                 2 
                 CG 
                 AsuII 
                 TT↓CGAA 
               
               
                   
               
               
                 2 
                 CG 
                 ClaI 
                 AT↓CGAT 
               
               
                   
               
               
                 2 
                 CG 
                 HinP1I 
                 G↓CGC 
               
               
                   
               
               
                 2 
                 CG 
                 HpaII 
                 C↓CGG 
               
               
                   
               
               
                 2 
                 CG 
                 MaeII 
                 A↓CGT 
               
               
                   
               
               
                 2 
                 CG 
                 NarI 
                 GG↓CGCC 
               
               
                   
               
               
                 2 
                 CG 
                 TaqI 
                 T↓CGA 
               
               
                   
               
               
                 2 
                 MK 
                 AccI 
                 GT↓MKAC 
               
               
                   
               
               
                 2 
                 NN 
                 BceAI 
                 ACGGC (12/14) 
               
               
                   
               
               
                 2 
                 NN 
                 BscAI 
                 GCATC (4/6) 
               
               
                   
               
               
                 2 
                 NN 
                 BspD6I 
                 GACTC (4/6) 
               
               
                   
               
               
                 2 
                 NN 
                 FauI 
                 CCCGC (4/6) 
               
               
                   
               
               
                 2 
                 NN 
                 Hpy178III 
                 TC↓NNGA 
               
               
                   
               
               
                 2 
                 TA 
                 CviQI 
                 G↓TAC 
               
               
                   
               
               
                 2 
                 TA 
                 MaeI 
                 C↓TAG 
               
               
                   
               
               
                 2 
                 TA 
                 MseI 
                 T↓TAA 
               
               
                   
               
               
                 2 
                 TA 
                 NdeI 
                 CA↓TATG 
               
               
                   
               
               
                 2 
                 TA 
                 VspI 
                 AT↓TAAT 
               
               
                   
               
               
                 3 
                 ANT 
                 HinfI 
                 G↓ANTC 
               
               
                   
               
               
                 3 
                 AWT 
                 TfiI 
                 G↓AWTC 
               
               
                   
               
               
                 3 
                 CWG 
                 PasI 
                 CC↓CWGGG 
               
               
                   
               
               
                 3 
                 CWG 
                 TseI 
                 G↓CWGC 
               
               
                   
               
               
                 3 
                 GNC 
                 AsuI 
                 G↓GNCC 
               
               
                   
               
               
                 3 
                 GNC 
                 DraII 
                 RG↓GNCCY 
               
               
                   
               
               
                 3 
                 GTC 
                 SimI 
                 GGGTC (−3/0) 
               
               
                   
               
               
                 3 
                 GWC 
                 AvaII 
                 G↓GWCC 
               
               
                   
               
               
                 3 
                 GWC 
                 PpuMI 
                 RG↓GWCCY 
               
               
                   
               
               
                 3 
                 GWC 
                 RsrII 
                 CG↓GWCCG 
               
               
                   
               
               
                 3 
                 GWC 
                 SanDI 
                 GG↓GWCCC 
               
               
                   
               
               
                 3 
                 GWC 
                 Sse8647I 
                 AG↓GWCCT 
               
               
                   
               
               
                 3 
                 NNN 
                 Ksp632I 
                 CTCTTC (1/4) 
               
               
                   
               
               
                 3 
                 NNN 
                 SapI 
                 GCTCTTC (1/4) 
               
               
                   
               
               
                 3 
                 TCA 
                 BbvCI 
                 CCTCAGC (−5/−2) 
               
               
                   
               
               
                 3 
                 TNA 
                 Bpu10I 
                 CCTNAGC (−5/−2) 
               
               
                   
               
               
                 3 
                 TNA 
                 DdeI 
                 C↓TNAG 
               
               
                   
               
               
                 3 
                 TNA 
                 EspI 
                 GC↓TNAGC 
               
               
                   
               
               
                 3 
                 TNA 
                 SauI 
                 CC↓TNAGG 
               
               
                   
               
               
                 4 
                 AATT 
                 ApoI 
                 R↓AATTY 
               
               
                   
               
               
                 4 
                 AATT 
                 EcoRI 
                 G↓AATTC 
               
               
                   
               
               
                 4 
                 AATT 
                 MfeI 
                 C↓AATTG 
               
               
                   
               
               
                 4 
                 AATT 
                 TspEI 
                 ↓AATT 
               
               
                   
               
               
                 4 
                 ACGA 
                 BsiI 
                 CACGAG (−5/−1) 
               
               
                   
               
               
                 4 
                 AGCT 
                 HindIII 
                 A↓AGCTT 
               
               
                   
               
               
                 4 
                 CATG 
                 BspHI 
                 T↓CATGA 
               
               
                   
               
               
                 4 
                 CATG 
                 BspLU11I 
                 A↓CATGT 
               
               
                   
               
               
                 4 
                 CATG 
                 FatI 
                 ↓CATG 
               
               
                   
               
               
                 4 
                 CATG 
                 NcoI 
                 C↓CATGG 
               
               
                   
               
               
                 4 
                 CCAG 
                 BseYI 
                 CCCAGC (−5/−1) 
               
               
                   
               
               
                 4 
                 CCGG 
                 AgeI 
                 A↓CCGGT 
               
               
                   
               
               
                 4 
                 CCGG 
                 BetI 
                 W↓CCGGW 
               
               
                   
               
               
                 4 
                 CCGG 
                 BspMII 
                 T↓CCGGA 
               
               
                   
               
               
                 4 
                 CCGG 
                 Cfr10I 
                 R↓CCGGY 
               
               
                   
               
               
                 4 
                 CCGG 
                 Eco56I 
                 G↓CCGGC 
               
               
                   
               
               
                 4 
                 CCGG 
                 SgrAI 
                 CR↓CCGGYG 
               
               
                   
               
               
                 4 
                 CCGG 
                 Sse232I 
                 CG↓CCGGCG 
               
               
                   
               
               
                 4 
                 CCGG 
                 XmaI 
                 C↓CCGGG 
               
               
                   
               
               
                 4 
                 CGCG 
                 AscI 
                 GG↓CGCGCC 
               
               
                   
               
               
                 4 
                 CGCG 
                 BsePI 
                 G↓CGCGC 
               
               
                   
               
               
                 4 
                 CGCG 
                 MauBI 
                 CG↓CGCGCG 
               
               
                   
               
               
                 4 
                 CGCG 
                 MluI 
                 A↓CGCGT 
               
               
                   
               
               
                 4 
                 CGCG 
                 SeII 
                 ↓CGCG 
               
               
                   
               
               
                 4 
                 CNNG 
                 SecI 
                 C↓CNNGG 
               
               
                   
               
               
                 4 
                 CRYG 
                 AfIIII 
                 A↓CRYGT 
               
               
                   
               
               
                 4 
                 CRYG 
                 DsaI 
                 C↓CRYGG 
               
               
                   
               
               
                 4 
                 CTAG 
                 AvrII 
                 C↓CTAGG 
               
               
                   
               
               
                 4 
                 CTAG 
                 NheI 
                 G↓CTAGC 
               
               
                   
               
               
                 4 
                 CTAG 
                 SpeI 
                 A↓CTAGT 
               
               
                   
               
               
                 4 
                 CTAG 
                 XbaI 
                 T↓CTAGA 
               
               
                   
               
               
                 4 
                 CWWG 
                 StyI 
                 C↓CWWGG 
               
               
                   
               
               
                 4 
                 GATC 
                 BamHI 
                 G↓GATCC 
               
               
                   
               
               
                 4 
                 GATC 
                 BcII 
                 T↓GATCA 
               
               
                   
               
               
                 4 
                 GATC 
                 BgIII 
                 A↓GATCT 
               
               
                   
               
               
                 4 
                 GATC 
                 MboI 
                 ↓GATC 
               
               
                   
               
               
                 4 
                 GATC 
                 XhoII 
                 R↓GATCY 
               
               
                   
               
               
                 4 
                 GCGC 
                 KasI 
                 G↓GCGCC 
               
               
                   
               
               
                 4 
                 GGCC 
                 Bsp120I 
                 G↓GGCCC 
               
               
                   
               
               
                 4 
                 GGCC 
                 CfrI 
                 Y↓GGCCR 
               
               
                   
               
               
                 4 
                 GGCC 
                 GdiII 
                 CGGCCR (−5/−1) 
               
               
                   
               
               
                 4 
                 GGCC 
                 NotI 
                 GC↓GGCCGC 
               
               
                   
               
               
                 4 
                 GGCC 
                 XmaIII 
                 C↓GGCCG 
               
               
                   
               
               
                 4 
                 GTAC 
                 Asp718I 
                 G↓GTACC 
               
               
                   
               
               
                 4 
                 GTAC 
                 Bsp1407I 
                 T↓GTACA 
               
               
                   
               
               
                 4 
                 GTAC 
                 SpII 
                 C↓GTACG 
               
               
                   
               
               
                 4 
                 GTAC 
                 TatI 
                 W↓GTACW 
               
               
                   
               
               
                 4 
                 GYRC 
                 HgiCI 
                 G↓GYRCC 
               
               
                   
               
               
                 4 
                 NNNN 
                 AarI 
                 CACCTGC (4/8) 
               
               
                   
               
               
                 4 
                 NNNN 
                 AceIII 
                 CAGCTC (7/11) 
               
               
                   
               
               
                 4 
                 NNNN 
                 Bbr7I 
                 GAAGAC (7/11) 
               
               
                   
               
               
                 4 
                 NNNN 
                 BbvI 
                 GCAGC (8/12) 
               
               
                   
               
               
                 4 
                 NNNN 
                 BbvII 
                 GAAGAC (2/6) 
               
               
                   
               
               
                 4 
                 NNNN 
                 BsmAI 
                 GTCTC (1/5) 
               
               
                   
               
               
                 4 
                 NNNN 
                 BsmFI 
                 GGGAC (10/14) 
               
               
                   
               
               
                 4 
                 NNNN 
                 BspMI 
                 ACCTGC (4/8) 
               
               
                   
               
               
                 4 
                 NNNN 
                 BtgZI 
                 GCGATG (10/14) 
               
               
                   
               
               
                 4 
                 NNNN 
                 Eco31I 
                 GGTCTC (1/5) 
               
               
                   
               
               
                 4 
                 NNNN 
                 Esp3I 
                 CGTCTC (1/5) 
               
               
                   
               
               
                 4 
                 NNNN 
                 FokI 
                 GGATG (9/13) 
               
               
                   
               
               
                 4 
                 NNNN 
                 SfaNI 
                 GCATC (5/9) 
               
               
                   
               
               
                 4 
                 NNNN 
                 Sth132I 
                 CCCG (4/8) 
               
               
                   
               
               
                 4 
                 NNNN 
                 StsI 
                 GGATG (10/14) 
               
               
                   
               
               
                 4 
                 TCGA 
                 AbsI 
                 CC↓TCGAGG 
               
               
                   
               
               
                 4 
                 TCGA 
                 PspXI 
                 VC↓TCGAGB 
               
               
                   
               
               
                 4 
                 TCGA 
                 SaII 
                 G↓TCGAC 
               
               
                   
               
               
                 4 
                 TCGA 
                 SgrDI 
                 CG↓TCGACG 
               
               
                   
               
               
                 4 
                 TCGA 
                 XhoI 
                 C↓TCGAG 
               
               
                   
               
               
                 4 
                 TGCA 
                 ApaLI 
                 G↓TGCAC 
               
               
                   
               
               
                 4 
                 TGCA 
                 Ppu10I 
                 A↓TGCAT 
               
               
                   
               
               
                 4 
                 TRYA 
                 SfeI 
                 C↓TRYAG 
               
               
                   
               
               
                 4 
                 TTAA 
                 AfIII 
                 C↓TTAAG 
               
               
                   
               
               
                 4 
                 TYRA 
                 SmII 
                 C↓TYRAG 
               
               
                   
               
               
                 4 
                 YCGR 
                 AvaI 
                 C↓YCGRG 
               
               
                   
               
               
                 5 
                 CCNGG 
                 PfoI 
                 T↓CCNGGA 
               
               
                   
               
               
                 5 
                 CCNGG 
                 SsoII 
                 ↓CCNGG 
               
               
                   
               
               
                 5 
                 CCSGG 
                 EcoHI 
                 ↓CCSGG 
               
               
                   
               
               
                 5 
                 CCWGG 
                 EcoRII 
                 ↓CCWGG 
               
               
                   
               
               
                 5 
                 CCWGG 
                 SexAI 
                 A↓CCWGGT 
               
               
                   
               
               
                 5 
                 GGNCC 
                 UnbI 
                 ↓GGNCC 
               
               
                   
               
               
                 5 
                 GGWCC 
                 VpaK11AI 
                 ↓GGWCC 
               
               
                   
               
               
                 5 
                 GTNAC 
                 BstEII 
                 G↓GTNACC 
               
               
                   
               
               
                 5 
                 GTNAC 
                 MaeIII 
                 ↓GTNAC 
               
               
                   
               
               
                 5 
                 GTSAC 
                 Tsp45I 
                 ↓GTSAC 
               
               
                   
               
               
                 5 
                 NNNNN 
                 HgaI 
                 GACGC (5/10) 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 2  
               
             
             
               
                   
               
               
                 Type II restriction enzymes 
               
               
                 producing 3′-overhangs 
               
             
          
           
               
                 Length 
                 Overhang 
                 Enzymes 
                 Recognition Sequence 
               
               
                   
               
               
                 1 
                 N 
                 BciVI 
                 GTATCC (6/5) 
               
               
                   
               
               
                 1 
                 N 
                 BfiI 
                 ACTGGG (5/4) 
               
               
                   
               
               
                 1 
                 N 
                 Eam1105I 
                 GACNNN↓NNGTC 
               
               
                   
               
               
                 1 
                 N 
                 Hin4II 
                 CCTTC (6/5) 
               
               
                   
               
               
                 1 
                 N 
                 HphI 
                 GGTGA (8/7) 
               
               
                   
               
               
                 1 
                 N 
                 Hpy188I 
                 TCN↓GA 
               
               
                   
               
               
                 1 
                 N 
                 MboII 
                 GAAGA (8/7) 
               
               
                   
               
               
                 1 
                 N 
                 MnII 
                 CCTC (7/6) 
               
               
                   
               
               
                 1 
                 N 
                 Tsp4CI 
                 ACN↓GT 
               
               
                   
               
               
                 1 
                 N 
                 XcmI 
                 CCANNNNN↓NNNNTGG 
               
               
                   
               
               
                 1 
                 S 
                 AgsI 
                 TTS↓AA 
               
               
                   
               
               
                 2 
                 AT 
                 BspKT6I 
                 GAT↓C 
               
               
                   
               
               
                 2 
                 AT 
                 PacI 
                 TTAAT↓TAA 
               
               
                   
               
               
                 2 
                 AT 
                 PvuI 
                 CGAT↓CG 
               
               
                   
               
               
                 2 
                 AT 
                 SgfI 
                 GCGAT↓CGC 
               
               
                   
               
               
                 2 
                 CG 
                 HhaI 
                 GCG↓C 
               
               
                   
               
               
                 2 
                 CN 
                 BsmI 
                 GAATGC (1/−1) 
               
               
                   
               
               
                 2 
                 GC 
                 McaTI 
                 GCGC↓GC 
               
               
                   
               
               
                 2 
                 GC 
                 SacII 
                 CCGC↓GG 
               
               
                   
               
               
                 2 
                 GN 
                 BsrI 
                 ACTGG (1/−1) 
               
               
                   
               
               
                 2 
                 NN 
                 ApyPI 
                 ATCGAC (20/18) 
               
               
                   
               
               
                 2 
                 NN 
                 AquII 
                 GCCGNAC (20/18) 
               
               
                   
               
               
                 2 
                 NN 
                 AquIII 
                 GAGGAG (20/18) 
               
               
                   
               
               
                 2 
                 NN 
                 AquIV 
                 GRGGAAG (19/17) 
               
               
                   
               
               
                 2 
                 NN 
                 Bce83I 
                 CTTGAG (16/14) 
               
               
                   
               
               
                 2 
                 NN 
                 BsbI 
                 CAACAC (21/19) 
               
               
                   
               
               
                 2 
                 NN 
                 BseMII 
                 CTCAG (10/8) 
               
               
                   
               
               
                 2 
                 NN 
                 BseRI 
                 GAGGAG (10/8) 
               
               
                   
               
               
                 2 
                 NN 
                 BsgI 
                 GTGCAG (16/14) 
               
               
                   
               
               
                 2 
                 NN 
                 BspCNI 
                 CTCAG (9/7) 
               
               
                   
               
               
                 2 
                 NN 
                 BsrDI 
                 GCAATG (2/0) 
               
               
                   
               
               
                 2 
                 NN 
                 BstF5I 
                 GGATG (2/0) 
               
               
                   
               
               
                 2 
                 NN 
                 BtsI 
                 GCAGTG (2/0) 
               
               
                   
               
               
                 2 
                 NN 
                 BtsIMutI 
                 CAGTG (2/0) 
               
               
                   
               
               
                 2 
                 NN 
                 CchII 
                 GGARGA (11/9) 
               
               
                   
               
               
                 2 
                 NN 
                 CchIII 
                 CCCAAG (20/18) 
               
               
                   
               
               
                 2 
                 NN 
                 CdpI 
                 GCGGAG (20/18) 
               
               
                   
               
               
                 2 
                 NN 
                 CjeNIII 
                 GKAAYG (19/17) 
               
               
                   
               
               
                 2 
                 NN 
                 CstMI 
                 AAGGAG (20/18) 
               
               
                   
               
               
                 2 
                 NN 
                 DraRI 
                 CAAGNAC (20/18) 
               
               
                   
               
               
                 2 
                 NN 
                 DrdI 
                 GACNNNN↓NNGTC 
               
               
                   
               
               
                 2 
                 NN 
                 EciI 
                 GGCGGA (11/9) 
               
               
                   
               
               
                 2 
                 NN 
                 Eco57I 
                 CTGAAG (16/14) 
               
               
                   
               
               
                 2 
                 NN 
                 Eco57MI 
                 CTGRAG (16/14) 
               
               
                   
               
               
                 2 
                 NN 
                 GsuI 
                 CTGGAG (16/14) 
               
               
                   
               
               
                 2 
                 NN 
                 HauII 
                 TGGCCANNNNNNNNNNN↓ 
               
               
                   
               
               
                 2 
                 NN 
                 MaqI 
                 CRTTGAC (21/19) 
               
               
                   
               
               
                 2 
                 NN 
                 MmeI 
                 TCCRAC (20/18) 
               
               
                   
               
               
                 2 
                 NN 
                 NlaCI 
                 CATCAC (19/17) 
               
               
                   
               
               
                 2 
                 NN 
                 NmeAIII 
                 GCCGAG (21/19) 
               
               
                   
               
               
                 2 
                 NN 
                 PlaDI 
                 CATCAG (21/19) 
               
               
                   
               
               
                 2 
                 NN 
                 PspOMII 
                 CGCCCAR (20/18) 
               
               
                   
               
               
                 2 
                 NN 
                 PspPRI 
                 CCYCAG (15/13) 
               
               
                   
               
               
                 2 
                 NN 
                 RceI 
                 CATCGAC (20/18) 
               
               
                   
               
               
                 2 
                 NN 
                 RdeGBII 
                 ACCCAG (20/18) 
               
               
                   
               
               
                 2 
                 NN 
                 RpaI 
                 GTYGGAG (11/9) 
               
               
                   
               
               
                 2 
                 NN 
                 RpaBI 
                 CCCGCAG (20/18) 
               
               
                   
               
               
                 2 
                 NN 
                 RpaB5I 
                 CGRGGAC (20/18) 
               
               
                   
               
               
                 2 
                 NN 
                 SdeAI 
                 CAGRAG (21/19) 
               
               
                   
               
               
                 2 
                 NN 
                 SstE37I 
                 CGAAGAC (20/18) 
               
               
                   
               
               
                 2 
                 NN 
                 TagII 
                 GACCGA (11/9) 
               
               
                   
               
               
                 2 
                 NN 
                 TsoI 
                 TARCCA (11/9) 
               
               
                   
               
               
                 2 
                 NN 
                 TspDTI 
                 ATGAA (11/9) 
               
               
                   
               
               
                 2 
                 NN 
                 TspGWI 
                 ACGGA (11/9) 
               
               
                   
               
               
                 2 
                 NN 
                 Tth111II 
                 CAARCA (11/9) 
               
               
                   
               
               
                 2 
                 NN 
                 WviI 
                 CACRAG (21/19) 
               
               
                   
               
               
                 2 
                 RY 
                 McrI 
                 CGRY↓CG 
               
               
                   
               
               
                 2 
                 TA 
                 PabI 
                 GTA↓C 
               
               
                   
               
               
                 3 
                 CNG 
                 BthCI 
                 GCNG↓C 
               
               
                   
               
               
                 3 
                 CSG 
                 TauI 
                 GCSG↓C 
               
               
                   
               
               
                 3 
                 GNC 
                 FmuI 
                 GGNC↓C 
               
               
                   
               
               
                 3 
                 GNC 
                 PssI 
                 RGGNC↓CY 
               
               
                   
               
               
                 3 
                 GWC 
                 Psp03I 
                 GGWC↓C 
               
               
                   
               
               
                 3 
                 NNN 
                 AlwNI 
                 CAGNNN↓CTG 
               
               
                   
               
               
                 3 
                 NNN 
                 BgII 
                 GCCNNNN↓NGGC 
               
               
                   
               
               
                 3 
                 NNN 
                 BsiYI 
                 CCNNNNN↓NNGG 
               
               
                   
               
               
                 3 
                 NNN 
                 BstAPI 
                 GCANNNN↓NTGC 
               
               
                   
               
               
                 3 
                 NNN 
                 DraIII 
                 CACNNN↓GTG 
               
               
                   
               
               
                 3 
                 NNN 
                 MwoI 
                 GCNNNNN↓NNGC 
               
               
                   
               
               
                 3 
                 NNN 
                 PflMI 
                 CCANNNN↓NTGG 
               
               
                   
               
               
                 3 
                 NNN 
                 RleAI 
                 CCCACA (12/9) 
               
               
                   
               
               
                 3 
                 NNN 
                 SfiI 
                 GGCCNNNN↓NGGCC 
               
               
                   
               
               
                 4 
                 ACGT 
                 AatII 
                 GACGT↓C 
               
               
                   
               
               
                 4 
                 ACGT 
                 TaiI 
                 ACGT↓ 
               
               
                   
               
               
                 4 
                 AGCT 
                 SacI 
                 GAGCT↓C 
               
               
                   
               
               
                 4 
                 ASST 
                 SetI 
                 ASST↓ 
               
               
                   
               
               
                 4 
                 CATG 
                 NlaIII 
                 CATG↓ 
               
               
                   
               
               
                 4 
                 CATG 
                 NspI 
                 RCATG↓Y 
               
               
                   
               
               
                 4 
                 CATG 
                 SphI 
                 GCATG↓C 
               
               
                   
               
               
                 4 
                 CCAG 
                 GsaI 
                 CCCAGC (−1/−5) 
               
               
                   
               
               
                 4 
                 CCGG 
                 FseI 
                 GGCCGG↓CC 
               
               
                   
               
               
                 4 
                 CTAG 
                 AceII 
                 GCTAG↓C 
               
               
                   
               
               
                 4 
                 DGCH 
                 SduI 
                 GDGCH↓C 
               
               
                   
               
               
                 4 
                 GATC 
                 ChaI 
                 GATC↓ 
               
               
                   
               
               
                 4 
                 GCGC 
                 BbeI 
                 GGCGC↓C 
               
               
                   
               
               
                 4 
                 GCGC 
                 HaeII 
                 RGCGC↓Y 
               
               
                   
               
               
                 4 
                 GGCC 
                 ApaI 
                 GGGCC↓C 
               
               
                   
               
               
                 4 
                 GTAC 
                 KpnI 
                 GGTAC↓C 
               
               
                   
               
               
                 4 
                 KGCM 
                 BseSI 
                 GKGCM↓C 
               
               
                   
               
               
                 4 
                 NNNN 
                 BstXI 
                 CCANNNNN↓NTGG 
               
               
                   
               
               
                 4 
                 RGCY 
                 HgiJII 
                 GRGCY↓C 
               
               
                   
               
               
                 4 
                 TGCA 
                 EcoT22I 
                 ATGCA↓T 
               
               
                   
               
               
                 4 
                 TGCA 
                 PstI 
                 CTGCA↓G 
               
               
                   
               
               
                 4 
                 TGCA 
                 Sse8387I 
                 CCTGCA↓GG 
               
               
                   
               
               
                 4 
                 WGCW 
                 HgiAI 
                 GWGCW↓C 
               
               
                   
               
               
                 4 
                 YCGR 
                 Nli3877I 
                 CYCGR↓G 
               
               
                   
               
               
                 5 
                 CGWCG 
                 Hpy99I 
                 CGWCG↓ 
               
               
                   
               
               
                 5 
                 NNNNN 
                 ApaBI 
                 GCANNNNN↓TGC 
               
               
                   
               
               
                 9 
                 NNCASTGNN 
                 TspRI 
                 CASTGNN↓ 
               
               
                   
               
             
          
         
       
     
         [0032]    The standard IUPAC nucleic acid codes are shown in Table 3 below: 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 IUPAC nucleic acid codes 
               
             
          
           
               
                 IUPAC nucleotide code 
                 Base 
               
               
                   
               
               
                 A 
                 Adenine 
               
               
                 C 
                 Cytosine 
               
               
                 G 
                 Guanine 
               
               
                 T (or U) 
                 Thymine (or Uracil) 
               
               
                 R 
                 A or G 
               
               
                 Y 
                 C or T 
               
               
                 S 
                 G or C 
               
               
                 W 
                 A or T 
               
               
                 K 
                 G or T 
               
               
                 M 
                 A or C 
               
               
                 B 
                 C or G or T 
               
               
                 D 
                 A or G or T 
               
               
                 H 
                 A or C or T 
               
               
                 V 
                 A or C or G 
               
               
                 N 
                 any base 
               
               
                   
               
             
          
         
       
     
         [0033]    In some embodiments, the restriction enzymes do not have a specific recognition sequence. 
         [0034]    In some embodiments, the Type II restriction enzyme that generates a single stranded DNA with a 3′-overhang can include but is not limited to BsaXI (Type IIb), BstXI (Type IIp), RleAI (Type IIs) or TstI (Type IIb). 
         [0035]    In some embodiments, the Type IIs restriction enzyme that generates a single stranded DNA with a 3′-overhang can include but is not limited to RleAI. 
         [0036]    In some embodiments, the Type IIb restriction enzyme that generates a single stranded DNA with a 3′-overhang can include but is not limited to BsaXI. 
         [0037]    In some embodiments, the Type IIp restriction enzyme that generates a single stranded DNA with a 3′-overhang can include but is not limited to BstXI. 
         [0038]    In some embodiments, the Type IIs restriction enzyme that generates a single stranded DNA with a 5′-overhang can include but is not limited to Earl, BspMI, BsaI, BbsI, or BsmBI. 
         [0039]    In some embodiments, the first DNA molecule or the second DNA molecule have single stranded termini generated with an exonuclease. 
         [0040]    In some embodiments, the exonuclease that generates single stranded DNA with a 3′-overhang can include but is not limited to T7 exonuclease, T5 exonuclease, or Lambda exonuclease. 
         [0041]    In some embodiments, the exonuclease acts on DNA parts that were created via PCR with primers containing phosphorothioate bonds. Primers can also contain other chemically modified bases, such as but not limited to phosphorothioates, locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2′-0Me nucleotides, methylphosphonates or morpholinos. 
         [0042]    In some embodiments, the first DNA molecule or the second DNA molecule have single stranded termini generated with an endonuclease and a second enzyme. 
         [0043]    In some embodiments, the endonuclease that generates single stranded DNA with a 3′-overhang can include but is not limited to DNA glycosylase-lyase endonuclease VIII. In some embodiments, the second enzyme used in concert with DNA glycosylase-lyase endonuclease VIII to generate single stranded termini can include but is not limited to uracil DNA glycosylase (UDG). 
         [0044]    In some embodiments, the single stranded terminus on one DNA molecule is a 3′-overhang and the single stranded terminus on the other DNA molecule is a 5′-overhang. In some embodiments, the first and second DNA molecules can be ligated using a single stranded DNA (ssDNA) linker (staple). 
         [0045]    In some embodiments, the linker is a staple. A staple may be single stranded and can be DNA or RNA. In some embodiments, the staple is a defined sequence capable of binding with perfect complementarity to the single stranded DNA termini generated on the first and second DNA molecules. In some embodiments, the staple binds to a single stranded DNA terminus with a 3′-overhang on a first DNA molecule and a single stranded DNA terminus with a 5′-overhang on a second DNA molecule. In some embodiments, the staple binds to a single stranded DNA terminus with a 5′-overhang on a first DNA molecule and a single stranded DNA terminus with a 3′-overhang on a second DNA molecule. 
         [0046]    In some embodiments, the staple is an oligonucleotide between about 4 and about 20 nucleotides in length, and in some embodiments between about 4 nucleotides and about 16 nucleotides, in some embodiments between about 4 nucleotides and about 12 nucleotides, and in some embodiments about 4 nucleotides to about 10 nucleotides in length. In some embodiments, the staple is single stranded DNA or RNA. 
         [0047]    In some embodiments, the present invention provides a plurality of reaction mixtures. The reaction mixtures include 1) a first reaction mixture comprising DNA molecules and a restriction enzyme capable of generating a 5′ single stranded DNA terminus for use with the methods of the present invention, 2) a second reaction mixture comprising DNA molecules and a restriction enzyme capable of generating a 3′ single stranded DNA terminus for use with the methods of the present invention, and 3) a third reaction in which the products of the first two reactions are pooled together with a staple linker and ligated. In some embodiments, the first reaction mixture generates a single stranded DNA terminus that is the opposite orientation of the single stranded terminus generated by the second reaction mixture (i.e., one reaction generates a terminus with a 3′-overhang and one reaction generates a terminus with a 5′-overhang). In some embodiments, the single stranded termini generated by both the first and second reaction mixtures are complementary to the staple. In some embodiments, the staple in the reaction mixture contains a defined sequence capable of binding with perfect complementarity to the single stranded terminus generated by the first and second reaction mixtures. In some embodiments, the reaction mixture can contain a staple that is an oligonucleotide between 4 and 10 nucleotides in length, between 4 and 8 nucleotides, or between 6 and 10 nucleotides. 
         [0048]    In some embodiments, the first DNA molecule has a single stranded terminus and the second DNA molecule has a single stranded terminus that are each ligated to an intervening double stranded DNA (dsDNA) linker (adapter). 
         [0049]    In some embodiments, the linker is an adapter. An adapter is double stranded and can be DNA or RNA. In some embodiments, the adapter contains at least one single stranded terminus containing a degenerate sequence. In some embodiments, the adapter is comprised of oligonucleotides between at least about 5 bp and about 500 bp in length or more, in some embodiments between about 5 bp and about 300 bp, in some embodiments between about 5 bp and about 200 bp and in some embodiments between about 5 bp and 100 bp. 
         [0050]    In some embodiments, the single stranded terminus of the adapter is ligated to a 3′ or 5′-overhang of one DNA molecule. In some embodiments, a second single stranded terminus of the adapter is ligated to the 3′ or 5′-overhang of a second DNA molecule. The second single stranded terminus of the adapter can be generated prior to or after ligation of the adapter to the first DNA molecule. 
         [0051]    In some embodiments, the present invention provides a plurality of reaction mixtures. The reaction mixtures include 1) a first reaction mixture comprising DNA molecules and enzyme(s) capable of generating a 3′ or 5′ single stranded DNA terminus for use with the methods of the present invention, 2) a second reaction mixture of the same nature as the first reaction but comprising different DNA molecules for use with the methods of the present invention, 3) a third reaction mixture in which the product of the first reaction is ligated to an adapter that contains a degenerate single stranded terminus, 4) a fourth reaction in which the product of the third reaction is pooled with the product of the second reaction and ligated. In some embodiments, the second reaction mixture generates a single stranded DNA terminus that is complementary to the single stranded terminus of the adapter. In some embodiments, the single stranded termini generated by the first reaction mixtures are complementary to the adapter. In some embodiments, the reaction mixture can contain an adapter with a single stranded terminus that contains a degenerate sequence capable of binding to a single stranded DNA terminus complementary to the single stranded DNA terminus generated by the first reaction mixture. In some embodiments, the reaction mixture can contain an adapter that is between 5 and 100 bp in length. 
         [0052]    The methods of the present invention can be repeated as tandem steps to assemble final ligation products that contain at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 70, 100 or more starting molecules, such as DNA or RNA molecules. 
         [0053]    The present invention can be employed to assemble, for example plasmids, cosmids, and genomes, of novel sequence. The utility of engineered and synthetic DNA can be found throughout life sciences. In some embodiments, the methods of the present invention generate nucleic acid molecules that are linear or circular. Molecules generated by the methods of the present invention can include but are not limited to plasmids, cosmids, operons, genes, synthetic genes, complete genes, partial genomes, complete genomes, partial synthetic genomes, and complete synthetic genomes. Molecules generated by the methods of the present invention can also include naturally occurring pathway components or synthetically derived pathway components. 
         [0054]    In some embodiments, the assembly of the desired nucleic acid molecule can be performed in a single step. In some embodiments, the step is a single isothermal step. According to the present methods, the nucleic acid portions of the invention desired to be assembled are combined with appropriate staples and an assembly buffer to form a reaction mixture. The assembly buffer can include for example, the desired restriction and ligase enzymes necessary to assemble the nucleic acid. In some embodiments, the assembly buffer includes restriction enzymes (at least one 5′-overhang-generating enzyme and at least one 3′-overhang-generating enzyme) and DNA ligase (e.g., T7 DNA ligase). The reaction mixture can then be incubated at a single temperature reaction (i.e., isothermal reaction) that allows for digestion, annealing and ligation steps. In some embodiments the temperature is about 30° C. to about 50° C., about 30° C. to about 40° C., about 37° C. to about 42° C., about 37° C. or about 42° C. In some embodiments, the reaction mixture is incubated at 37° C. and all necessary digestion, annealing and ligation steps occur to assemble DNA and/or RNA molecules together. In some embodiments, at least about 2 to 100 or more DNA and/or RNA molecules are assembled in a isothermal reaction. In some embodiments at least about 2 to about 100, about 2 to 70, about 2 to 50, about 2 to 20, about 2 to about 12, about 2 to about 10, about 2 to about 8, about 2 to 6, about 2 to 4 or about 2 DNA and/or RNA molecules are assembled in an isothermal reaction. In some embodiments, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 70, 100 DNA and/or RNA molecules are assembled in an isothermal reaction. 
         [0055]    The methods of the present invention further provide for the ability to multiplex different assemblies within the same reaction vessel. Multiple reactions can be carried out in the same buffer due to the specificity afforded by each staple. As a result, assembly reagents can be minimized while increasing the productivity of an assembly process. 
         [0056]    In some embodiments, the DNA molecules generated by the present invention can be transformed or transfected into a variety of cells, including but not limited to bacteria, insect and mammal cells. The DNA molecules of the present invention can also be inserted into viruses or virus-like particles. Transfection and transformation methods are well known in the art and any standard methods can be employed with the present invention. 
         [0057]    The selection of nucleic acid parts, restriction enzymes, and staples, as well as the individual reaction assemblies and/or reagents employed therein, can be determined computationally, taking into account a variety of parameters, including logistical, cost and biophysical parameters. For example, the reaction assemblies and assembly routes can be guided by limitations or parameters for enzymes or other reagents, as experimentally-derived or known from the literature, and/or guided by cost, availability, or compatibility of the various reagents. 
         [0058]    Parameters can include logistical parameters. In some embodiments, logistical parameters for designing the assembly route include logistical considerations such as part availability or historical performance metrics. Part availability can include availability of nucleic acid sequences, restriction enzymes, buffers, or any other reagent employed with the multipart, modular and scarless assembly described herein. Historical performance can include but is not limited to compatibility of reagents, efficiency of reagents, and/or specificity of reagents. 
         [0059]    Parameters can include financial parameters. In some embodiments, financial parameters may address part cost, manipulation, reagents, and/or overhead. Consideration of financial parameters may determine that certain optimal parts should be synthesized by de novo nucleic acid synthesis (rather than scarless assembly). 
         [0060]    Parameters can also include functional or biophysical parameters. Ligation conditions and/or enzymatic digestion conditions are exemplary functional parameters. In some embodiments, an algorithm selects nucleic acid parts based on desired functional properties of the desired sequence. For instance, the algorithm can select DNA parts that encode promoters, ribosome binding sites, terminators, or other regulatory elements to elicit designed levels of gene expression. 
         [0061]    In some embodiments, the method utilizes an algorithm to determine and/or optimize the steps for assembling a complex nucleic acid molecule, i.e., for assembly of a multipart, modular and scarless nucleic acid sequence. In some embodiments, the algorithm selects reaction reagents to ensure sufficient reaction efficiency and fidelity during multiplex reactions and across multiple rounds of nucleic acid assembly. Reaction efficiency and fidelity can be predicted from empirical and biophysical data, and can include selecting the number and composition of nucleic acid parts in each reaction. For example, empirical data might suggest a maximum of 5 nucleic acid parts per reaction based on ligation efficiency. In some exemplary embodiments, the algorithm would determine that a 10 part nucleic acid assembly be split into 3 reactions spanning 2 iterative rounds of assembly to produce the final nucleic acid molecule. 
         [0062]    In some embodiments, ssDNA overhangs generated during assembly must be specific to ensure correct assembly. In some embodiments, the algorithm identifies incompatible ssDNA overhangs and separates component parts into different reactions in order to ensure specificity of assembly. 
         [0063]    In some embodiments, the algorithm considers specifications and limitations of automation hardware when determining the required and/or optimal assembly steps. Such specifications and/or limitations can include, for example, but are not limited to volume tolerances of a liquid handling robot, speed of execution, and throughput of the system. 
         [0064]    The present invention also provides for kits. Kits contemplated by the methods of the of the present invention can include 1) a single stranded staple or a double stranded terminus adapter, 2) enzymes capable of generating single stranded DNA termini and 3) an instruction for use. In some embodiments, the kit comprises a DNA ligase, a 5′-overhang-generating enzyme, and a 3′-overhang-generating enzyme. In some embodiments, the kit comprises the enzymes capable of generating single stranded DNA termini and an appropriate buffer for enzyme function. In some embodiments, the kit comprises a standard set of staples. In some embodiments, the staples are not part of the kit. In some embodiments, the kit comprise a plurality of reaction mixtures. In some embodiments, the kit comprises a plurality of adapters and enzymes for performing a plurality of reactions. 
         [0065]    In some embodiments, the kit further comprises an implementation of an algorithm as described herein, i.e. software for use according to the present methods. 
         [0066]    In some embodiments, the enzyme in the kit for generating the 5′-overhang is selected from Type IIs, Type IIb or Type IIp restriction enzymes or combinations thereof, including those listed in Table 1. In some embodiments, the enzymes in the kit for generating the 5′-overhang is selected from EarI, BspMI, BsaI, BbsI, and BsmBI, or combinations thereof. In some embodiments, the enzyme in the kit for generating the 3′-overhang is selected from Type IIs, Type IIb or Type IIp restriction enzymes or combinations thereof, including those listed in Table 2. In some embodiments, the enzymes in the kit for generating the 3′-overhang is selected from BsaXI, RleAI, and TstI and combinations thereof. 
       EXAMPLES 
     Example 1 
     Staple Method 
       [0067]    One example of the methods is the “Staple Method.” DNA parts are prepared by digestion with Type IIs restriction enzymes to generate termini with 5′ and 3′ single stranded DNA overhangs. Most Type IIs enzymes create short single stranded DNA overhangs (about 2 bp to 6 bp). This results in a relatively small “gap” at the junction between two DNA parts. This “gap” can be filled by a defined oligonucleotide (i.e., staple linker) that is perfectly complementary to the generated single stranded DNA overhangs. The oligonucleotide spans the junction and anneals to both the 5′ single stranded DNA overhang of one part and the 3′ single stranded DNA overhang of the other part. More than two DNA parts can be simultaneously joined together, and the order of assembly will be dictated by the sequence of the oligonucleotides provided in the reaction. See, for example,  FIGS. 1 and 2 . 
         [0068]    The staple method can also be employed in performing isothermal scarless subcloning. For isothermal scarless subcloning, the reaction mixture contained T4 ligase buffer, T7 DNA ligase, BsaI and BsaXI, and DNA parts. The isothermal reaction was performed at 37° C. for 1 hr. Colony PCRs and sequencing show that 11 of 12 clones assembled correctly. See, for example,  FIG. 4 . 
         [0069]    In another example, the reaction mixture contained T4 ligase buffer, T7 DNA ligase, BsaI and BstXI, and DNA parts. The isothermal reaction was performed at 37° C. for 8 hr. Colony PCRs and sequencing show that 6 of 12 clones assembled correctly. 
         [0070]    In a further example, performing the multiplex assembly in one tube, the reaction mixture contained T4 ligase buffer, T7 DNA ligase, BsaI and BstXI, and DNA parts. The isothermal reaction was performed at 37° C. for 8 hr. Colony PCRs and sequencing show that 23 of 24 clones assembled correctly. 
       Example 2 
     Adapter Method 
       [0071]    A second example of the methods is the “Adapter Method.” A dsDNA adapter (i.e., single stranded terminus adapter) is created for each part (linker paired part or LPP) such that it contains a single stranded DNA termini comprising degenerate bases, e.g. NNNN. The dsDNA sequence in the adapter can either duplicate the terminal sequence of the LPP, or it can serve as a replacement for the terminal sequence of the LPP. In the latter case, the LPP would be reconstructed to be a smaller size. In the “Adapter Method,” DNA parts are modified with restriction enzymes to generate single stranded DNA termini. The adapter corresponding to the desired neighboring part is then ligated to the single stranded DNA termini. Finally, the adapter is joined to its LPP. In the accompanying example, we utilized the second class of assembly (exonuclease based) to ligate the adapter to its LPP. See, for example,  FIGS. 1 and 3 . 
         [0072]    It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. It is understood that the disclosed invention is not limited to the particular methodology, protocols and materials described as these can vary. It is also understood that the terminology used herein is for the purposes of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the appended claims. 
         [0073]    All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.