Abstract:
A method of assembling multi-fragment DNA molecule, the method including: 1) constructing a vector system including an acceptor vector A1, a first donor vector B1, and a second donor vector B2, and each of the acceptor vector A1, the second donor vector B2, and the second donor vector B2 including a multiple cloning site; 2) introducing a first target sequence L m  to the first donor vector B1, m being an odd number starting from 1, and introducing a second target sequence L n  to the second donor vector B2, n being an even number starting from 2; and 3) repeating 2) to assemble multiple fragments to yield a multi-fragment DNA molecule.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a continuation-in-part of International Patent Application No. PCT/CN2014/072894 with an international filing date of Mar. 5, 2014, designating the United States, now pending, and further claims priority benefits to Chinese Patent Application No. 201310094572.6 filed Mar. 22, 2013. The contents of all of the aforementioned applications, including any intervening amendments thereto, are incorporated herein by reference. Inquiries from the public to applicants or assignees concerning this document or the related applications should be directed to: Matthias Scholl P.C., Attn.: Dr. Matthias Scholl Esq., 245 First Street, 18th Floor, Cambridge, Mass. 02142. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    The invention relates to a method of assembling multi-fragment DNA molecule and use thereof. 
         [0004]    2. Description of the Related Art 
         [0005]    Typically, a DNA polymerase is able to amplify a DNA sequence to be no more than 10 kb, and the amplified DNA sequence has high mutation rate and requires sequencing and reverse mutation, which is time and labor consuming. The synthesis of a complete gene having long DNA sequences requires multiple cycles of PCR and specific splicing, which also involves multiple mutations. In addition, the construction of a multiple gene vector is complicate, the introduction of each single gene is a multi-step process, and the introduced genes are spliced at intervals. 
       SUMMARY OF THE INVENTION 
       [0006]    In view of the above-described problems, it is one objective of the invention to provide a method of assembling multi-fragment DNA molecule and use thereof. The method is adapted to seamlessly assemble multiple DNA fragments into an integral large DNA fragment and the can be applied in gene fragment synthesis, construction of transgenic vector for multiple genes, and synthetic biology. 
         [0007]    To achieve the above objective, in accordance with one embodiment of the invention, there is provided a method of assembling a multi-fragment DNA molecule. The method comprises: 
         [0008]    1) constructing a vector system comprising an acceptor vector A1, a first donor vector B1, and a second donor vector B2, and each of the acceptor vector A1, the second donor vector B2, and the second donor vector B2 comprising a multiple cloning site; 
         [0009]    where, the multiple cloning site of the acceptor vector A1 comprises one group of restriction sites of a restriction enzyme and has a sequence of “a vector backbone—a reverse restriction site of a restriction enzyme at an odd number site—arbitrary bases—a forward restriction site of the restriction enzyme at the odd number site—the vector backbone”; and the vector backbone is in the absence of any restriction site of the restriction enzyme; 
         [0010]    the multiple cloning site of the first donor vector B1 comprises three groups of restriction sites of restriction enzymes and the first target sequence L m  is added to a left side of the multiple cloning site; the sequence of the multiple cloning site of the first donor vector B1 is represented by “a vector backbone—the forward restriction site of the restriction enzyme at the odd number site—a reverse restriction site of a cloning restriction enzyme—arbitrary bases—a forward restriction site of the cloning restriction enzyme—a reverse restriction site of a restriction enzyme at an even number site—arbitrary bases—a forward restriction site of the restriction enzyme at the even number site—the reverse restriction site of the restriction enzyme at the odd number site—the vector backbone”; all restriction sites of the cloning restriction enzyme in the vector backbone of the first donor vector B1 are removed, and m represents an odd number, such as 1, 3, 5, 7, and so on; and 
         [0011]    the multiple cloning site of the second donor vector B2 comprises three groups of restriction sites of restriction enzymes and the second target sequence L n  is added to a left side of the multiple cloning site; the sequence of the multiple cloning site of the second donor vector B2 is represented by “a vector backbone—the forward restriction site of the restriction enzyme at the even number site—the reverse restriction site of the cloning restriction enzyme—arbitrary bases—the forward restriction site of the cloning restriction enzyme—the reverse restriction site of the restriction enzyme at the odd number site—arbitrary bases—the forward restriction site of the restriction enzyme at the odd number site—the reverse restriction site of the restriction enzyme at the even number site—the vector backbone”; the vector backbone of the second donor vector B2 is in the absence of any restriction site of the cloning restriction enzyme, and n represents an even number, such as 2, 4, 6, 8, and so on; 
         [0012]    2) introducing the first target sequence L m  to the first donor vector B1, the step comprising: amplifying the first target sequence L m  by PCR while adding restriction sites of restriction enzymes to two ends of the target sequence respectively via PCR primers, so that sticky ends of the target sequence after cleavage are the same as sticky ends of the first donor vector B1 after being cleaved by the cloning restriction enzyme, and cloning the first target sequence L m  into the first donor vector B1 to replace the sequence of “—the reverse restriction site of the cloning restriction enzyme—arbitrary bases—the forward restriction site of the cloning restriction enzyme—” on the first donor vector B1, to yield a vector B1-L m ; and introducing the second target sequence L n  to the second donor vector B2 comprising: amplifying the second target sequence L n  by PCR while adding restriction sites of restriction enzymes to two ends of the target sequence respectively via PCR primers, so that sticky ends of the target sequence after cleavage are the same as sticky ends of the second donor vector B2 after being cleaved by the cloning restriction enzyme, and cloning the second target sequence L n  into the second donor vector B2 to replace the sequence of “—the reverse restriction site of the cloning restriction enzyme—arbitrary bases—the forward restriction site of the cloning restriction enzyme—” on the second donor vector B2, to yield a vector B2-L n , in which m represents an odd number, such as 1, 3, 5, 7, and so on, and n represents an even number, such as 2, 4, 6, 8, and so on; and 
         [0013]    3) repeating 2) to assemble multiple fragments to yield a multi-fragment DNA molecule, the step comprising: 
         [0014]    a) digesting the acceptor vector A1 and a vector B1-L 1  by a first restriction enzyme, and ligating the digested acceptor vector A1 and the digested vector B1-L 1  to transfer the target sequence L 1  from the vector B1-L 1  to the acceptor vector A1, to yield a vector A1-L 1 ; 
         [0015]    b) digesting the vector A1-L 1  and a vector B2-L 2  by a second restriction enzyme, and ligating the vector A1-L 1  with the target sequence L 2  by a ligase, to yield a vector A1-L 1 -L 2 ; 
         [0016]    c) digesting the vector A1-L 1 -L 2  and a vector B1-L 3  by a third restriction enzyme, and ligating the vector A1-L 1 -L 2  with the target sequence L 3  by the ligase, to yield a vector A1-L 1 -L 2 -L 3 ; and 
         [0017]    d) continuing ligating the newly formed vector with another target sequence until a vector A1-L 1 -L 2 -L 3 -L 4 -L 5  . . . -L m -L n  is assembled, in which m represents an odd number, such as 1, 3, 5, 7, and so on, and n represents an even number, such as 2, 4, 6, 8, and so on. 
         [0018]    In a class of this embodiment, a resistance of the acceptor vector A1 is different from both a resistance of the first donor vector B1 and a resistance of the second donor vector B2. The resistance of the first donor vector B1 is the same as or different from the resistance of the second donor vector B2. 
         [0019]    The restriction enzymes are those having the restriction site and the restriction site not coincident with each other. The restriction enzyme features that the restriction site and the restriction site are not coincident, so that arbitrary sticky ends are produced. For a given DNA sequence, arbitrary specific sticky ends are produced. 
         [0020]    The restriction enzymes belong to a typells restriction enzyme or an artificially synthetic zinc-finger nuclease. The typells restriction enzyme is one or several selected from BsmBI, BsaI, SapI, and BbsI. 
         [0021]    The vector system in 1) is a Bio-Walk system. The acceptor vector A1 is an acceptor vector pBWA, the first donor vector B1 is a donor vector pBWD(a), and the second donor vector B2 is a donor vector pBWD(b). 
         [0022]    The ligase is a T4 DNA ligase. 
         [0023]    A method for gene synthesis, or a method for cloning a DNA fragment, or a method for multiple gene vector, comprises applying the method of assembling a multi-fragment DNA molecule. 
         [0024]    Advantages of the method of assembling a multi-fragment DNA molecule according to embodiments of the invention are summarized as follows: the method realizes seamless linkage of multiple DNA fragments to generate a long DNA fragment. The method not only largely decreases mutations that easily occur in the large DNA fragment synthesis but also largely shortens the synthesis time. In the aspect of the large DNA fragment cloning, the mutations easily occurring in one-time cloning of the large DNA fragment are prevented. Small fragments are first cloned and sequenced, the correct fragments are quickly assembled into large fragments and then successively ligated with the acceptor vector, thus realizing the accumulation of the fragments and generating the integral DNA fragment in a short time. In the aspect of construction of the multi-gene vector, multiple genes can be constructed on the same vector and the one-step transformation is realized. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0025]    The invention is described hereinbelow with reference to the accompanying drawings, in which: 
           [0026]      FIG. 1  illustrates a map and a multiple cloning site of pBWA in accordance with one embodiment of the invention; 
           [0027]      FIG. 2  illustrates a map and a multiple cloning site of pBWD(a) in accordance with one embodiment of the invention; and 
           [0028]      FIG. 3  illustrates a map and a multiple cloning site of pBWD(b) in accordance with one embodiment of the invention. 
       
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
       [0029]    For further illustrating the invention, experiments detailing a method of assembling multi-fragment DNA molecule and applications thereof are described below. It should be noted that the following examples are intended to describe and not to limit the invention. 
       Example 1 
     Construct of Bio-Walk System 
       [0030]    1) An acceptor vector pBWA adopts a pBR322 replicon and has kanamycin resistance. The restriction enzyme at an odd number site (firstly accepted) is BsmBI. As shown in  FIG. 1 , a sequence of a multiple cloning site is SEQ ID NO: 4 represented by 
         [0000]    
       
         
               
               
             
           
               
                   
                 tagacgagacgagactgtccgtctcagtcg. 
               
             
          
         
       
     
         [0031]    in which, gagacg and cgtctc are a reverse recognition sequence and a forward recognition sequence, respectively, and “taga” and “gtcg” are sticky ends produced by cleavage of BsmBI, respectively. MCS is a multiple cloning site, Kanar is Kanamycin resistance gene, ori is originated from a replication origin of the plasmid pBR322 and adapted to replicate in  Escherichia coli  cells, and bom is originated from a basis of mobility of the plasmid Pbr322. 
         [0032]    2) A first donor vector pBWD(a) contains an ampicillin resistance gene, and a map and a multiple cloning site thereof are illustrated in  FIG. 2 . A sequence of the multiple cloning site is SEQ ID NO: 5 represented by 
         [0000]    
       
         
               
             
           
               
                 cgtctcAGAGACCNN . . . NNNGGTCTCN gaagagcaGctcttcgg 
               
               
                   
               
               
                 tcgcGagacg 
               
             
          
         
       
     
         [0033]    In which, cgtctc and gagacg are the forward recognition sequence and the reverse recognition sequence of BsmbI, respectively. gagacc and ggtctc are a reverse recognition sequence and a forward recognition sequence of BsaI, respectively. gaagagc and gctcttc are a reverse recognition sequence and a forward recognition sequence of SapI, respectively. MCS is the multiple cloning site, Amp r  represents the ampicillin resistance gene, ori is originated from the replication origin of the plasmid pBR322 and adapted to replicate in  Escherichia coli  cells, and bom is originated from the basis of mobility of the plasmid Pbr322. “N” represents arbitrary bases, and “. . . ” represents abbreviated arbitrary bases. 
         [0034]    3) A second donor vector pBWD(b) contains a spectinomycin resistance gene, and a map and a multiple cloning site of the second donor vector pBWD(b) were illustrated in  FIG. 3 . A sequence of the multiple cloning site is SEQ ID NO: 6 represented by 
         [0000]    
       
         
               
             
           
               
                 GctcttcNGAGACCNN . . . NNGGTCTCNGagacgNNN . . . NNN 
               
               
                   
               
               
                 cgtctcggtcggaagagc. 
               
             
          
         
       
     
         [0035]    in which, cgtctc and gagacg are the forward recognition sequence and the reverse recognition sequence of BsmbI, respectively. gagacc and ggtctc are the reverse recognition sequence and the forward recognition sequence of BsaI, respectively. gaagagc and gctcttc are the reverse recognition sequence and the forward recognition sequence of SapI, respectively. MCS represents the multiple cloning site, and Sp r  represents the spectinomycin resistance gene. ori is originated from the replication origin of the plasmid pBR322 and adapted to replicate in  Escherichia coli  cells. bom is originated from the basis of mobility of the plasmid Pbr322. “N” represents arbitrary bases, and “. . . ” represents abbreviated arbitrary bases. 
       Example 2 
     Multi-DNA Assembly by Bio-Walk System 
       [0036]    First step, a target gene is cloned into the first donor vector pBWD(a). Five genes are adopted in this example. 
         [0037]    pBWD(a)-GENE1 
         [0038]    GENE1 is amplified using a forward primer “Gene1+” and a reverse primer “Gene1−” of GENE1. “catggagtcaaagattcaaatag” and “agcggatggcctaaaaaaaaaac” are sequences of the forward primer and the reverse primer of GENE1, respectively. Full sequences of the Gene1+ and Gene1− are as follows: 
         [0000]    
       
         
               
               
             
           
               
                   
                 Gene1 + (SEQ ID NO: 2): 
               
               
                   
                 ggtctcAtctcatagacatggagtcaaagattcaaatag 
               
               
                   
                   
               
               
                   
                 Gene1 − (SEQ ID NO: 3): 
               
               
                   
                 ggtctc t cttc tctagttttttttttaggccatccgct 
               
             
          
         
       
     
         [0039]    A new sequence obtained from gene amplification by the above primers is as follows: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0040]    The MCS sequence of pBWD(a) is as follows: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0041]    The amplified new sequence and the MCS sequence are cleaved by BsaI (restriction sites of restriction enzymes are underscored) and sticky ends thereof after the cleavage are completely complementary at sites indicated by boxes. The PCR product of GENE1 after digested by the BsaI and the vector backbone digested by the BsaI are connected by a T4 DNA ligase to generate pBWD(a)-GENE1, a sequence structure of which is as follows: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0042]    pBWD(a)-GENE3 and pBWD(a)-GENE5 are also generated by the same way, and sequence structures thereof are as follows: 
         [0043]    pBWD(a)-GENE3: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0044]    pBWD(a)-GENE5: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0045]    Similar to the GENE1, GENE3, GENES, Gene2 and Gene4 are amplified by adding different primer joints during amplification of the target genes (the forward primer joint is 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    and the reverse primer joint is 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    the amplified gene (Gene2 or Gene4) and the second donor vector pBWD(b) are then cleaved by BsaI, and the sticky ends of the amplified gene and the second donor vector are completely complementary at sites indicated by boxes and then lignated by the T4 DNA ligase to generate pBWD(b)-GENE2 or pBWD(b)-GENE4. The sequence structure of pBWD(b)-GENE2 and pBWD(b)-GENE4 are listed as follows: 
         [0046]    pBWD(b)-GENE2: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0047]    pBWD(b)-GENE4: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0048]    Second step: target genes on pBWD(a)-GENE1, pBWD(a)-GENE3, pBWD(a)-GENE5 and the second donor vector pBWD(b) are assembled to the acceptor vector pBWA. 
         [0049]    1. Construct of pBWA-1: 
         [0050]    The sequence of the multiple cloning site of the acceptor vector pBWA is as follows: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0051]    The pBWA and the pBWD(a)-GENE1 are digested by BsmbI, and sticky ends thereof after the cleavage are completely complementary at sites indicated by boxes. Since the produced sticky ends are completely complementary, the backbone of the acceptor vector pBWA and the fragment of GENE1 are then ligated together by the T4 DNA ligase, and a newly produced sequence is named pBWA-1. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0052]    2. Construct of pBWA-2: 
         [0053]    The pBWA-1 and the pBWD(b)-GENE2 are digested by SapI, and sticky ends thereof after the cleavage are completely complementary at sites indicated by boxes. Since the produced sticky ends are completely complementary, the backbone of the acceptor vector pBWA-1 and the fragment of GENE2 are then ligated together by the T4 DNA ligase, and a newly produced sequence is named pBWA-2. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0054]    3. Construct of pBWA-3: 
         [0055]    The pBWA-2 and the pBWD(a)-GENE3 are digested by BsmBI, since produced sticky ends are completely complementary, the backbone of the acceptor vector pBWA-2 and the fragment of GENE3 are then ligated by the T4 DNA ligase, and a newly produced sequence is named pBWA-3. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0056]    4. Construct of pBWA-4 
         [0057]    The pBWA-3 and the pBWD(b)-GENE4 are digested by SapI, since produced sticky ends are completely complementary, the backbone of the acceptor vector pBWA-3 and the fragment of GENE4 are then ligated by the T4 DNA ligase, and a newly produced sequence is named pBWA-4. 
         [0058]    pBWA-4: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0059]    5. Construct of pBWA-5 
         [0060]    The pBWA-4 and the pBWD(a)-GENE5 are digested by BsmbI, and sticky ends thereof after the cleavage are completely complementary at sites indicated by boxes. Since the produced sticky ends are completely complementary, the backbone of the acceptor vector pBWA-4 and the fragment of GENES are then ligated by the T4 DNA ligase, and a newly produced sequence is named pBWA-5. 
         [0061]    pBWA-5: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0062]    The operations are repeated likewise so as to assemble five or more genes to the acceptor vector. 
       Example 3 
     Gene Synthesis and Cloning of Large DNA Fragment 
       [0063]    Gene synthesis of Cos9. Gene Cos9 is an important mediated gene in the recently invented DNAi and originated from prokaryotes. Thus, the codon optimization is required in eukaryotes so as to realize a relative good expression of the Cos9, and the gene synthesis is necessitated. A total length of the sequence of Cos9 is 4147 bp, and a synthetic sequence (SEQ ID NO: 1) is as follows: 
         [0000]    
       
         
               
             
           
               
                 CCTAGGCCACCATGGACAAGAAGTACTCCATTGGGCTCGATATCG 
               
               
                   
               
               
                 GCACAAACAGCGTCGGCTGGGCCGTCATTACGGACGAGTACAAGG 
               
               
                   
               
               
                 TGCCGAGCAAAAAATTCAAAGTTCTGGGCAATACCGATCGCCACA 
               
               
                   
               
               
                 GCATAAAGAAGAACCTCATTGGCGCCCTCCTGTTCGACTCCGGGG 
               
               
                   
               
               
                 AAACGGCCGAAGCCACGCGGCTCAAAAGAACAGCACGGCGCAGAT 
               
               
                   
               
               
                 ATACCCGCAGAAGAATCGGATCTGCTACCTCCAGGAGATCTTTAG 
               
               
                   
               
               
                 TAATGAGATGGCTAAGGTGGATGACTCTTTCTTCCATAGGCTGGA 
               
               
                   
               
               
                 GGAGTCCTTTTTGGTGGAGGAGGATAAAAAGCACGAGCGCCACCC 
               
               
                   
               
               
                 AATCTTTGGCAATATCGTGGACGAGGTGGCGTACCATGAAAAGTA 
               
               
                   
               
               
                 CCCAACCATATATCATCTGAGGAAGAAGCTGGTAGACAGTACTGA 
               
               
                   
               
               
                 TAAGGCTGACTTGCGGTTGATCTATCTCGCGCTGGCGCACATGAT 
               
               
                   
               
               
                 CAAATTTCGGGGACACTTCCTCATCGAGGGGGACCTGAACCCAGA 
               
               
                   
               
               
                 CAACAGCGATGTGGACAAACTCTTTATCCAACTGGTTCAGACTTA 
               
               
                   
               
               
                 CAATCAGCTTTTCGAAGAGAACCCGATCAACGCATCCGGAGTTGA 
               
               
                   
               
               
                 CGCCAAAGCAATCCTGAGCGCTAGGCTGTCCAAATCCCGGCGGCT 
               
               
                   
               
               
                 CGAAAACCTCATCGCACAGCTCCCTGGGGAGAAGAAGAACGGCCT 
               
               
                   
               
               
                 GTTTGGTAATCTTATCGCCCTGTCACTCGGGCTGACCCCCAACTT 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                 CAAAGACACCTACGATGATGATCTCGACAATCTGCTGGCCCAGAT 
               
               
                   
               
               
                 CGGCGACCAGTACGCAGACCTTTTTTTGGCGGCAAAGAACCTGTC 
               
               
                   
               
               
                 AGACGCCATTCTGCTGAGTGATATTCTGCGAGTGAACACGGAGAT 
               
               
                   
               
               
                 CACCAAAGCTCCGCTGAGCGCTAGTATGATCAAGCGCTATGATGA 
               
               
                   
               
               
                 GCACCACCAAGACTTGACTTTGCTGAAGGCCCTTGTCAGACAGCA 
               
               
                   
               
               
                 ACTGCCTGAGAAGTACAAGGAAATTTTCTTCGATCAGTCTAAAAA 
               
               
                   
               
               
                 TGGCTACGCCGGATACATTGACGGCGGAGCAAGCCAGGAGGAATT 
               
               
                   
               
               
                 TTACAAATTTATTAAGCCCATCTTGGAAAAAATGGACGGCACCGA 
               
               
                   
               
               
                 GGAGCTGCTGGTAAAGCTGAACAGAGAAGATCTGTTGCGCAAACA 
               
               
                   
               
               
                 GCGCACTTTCGACAATGGAAGCATCCCCCACCAGATTCACCTGGG 
               
               
                   
               
               
                 CGAACTGCACGCTATCCTCAGGCGGCAAGAGGATTTCTACCCCTT 
               
               
                   
               
               
                 TTTGAAAGATAACAGGGAAAAGATTGAGAAAATCCTCACATTTCG 
               
               
                   
               
               
                 GATACCCTACTATGTAGGCCCCCTCGCACGCGGAAATTCCAGATT 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                 CTTCGAGGAAGTCGTGGATAAGGGGGCCTCTGCCCAGTCCTTCAT 
               
               
                   
               
               
                 CGAAAGGATGACTAACTTTGATAAAAATCTGCCTAACGAAAAGGT 
               
               
                   
               
               
                 GCTTCCTAAACACTCTCTGCTGTACGAGTACTTCACAGTTTATAA 
               
               
                   
               
               
                 CGAACTCACCAAGGTCAAATACGTCACAGAAGGGATGAGAAAGCC 
               
               
                   
               
               
                 AGCATTCCTGTCTGGAGAGCAGAAGAAAGCTATCGTGGACCTCCT 
               
               
                   
               
               
                 CTTCAAGACGAACCGGAAAGTTACCGTGAAACAGCTCAAAGAGGA 
               
               
                   
               
               
                 CTATTTCAAAAAGATTGAATGTTTCGACTCTGTTGAAATCAGCGG 
               
               
                   
               
               
                 AGTGGAGGATCGCTTCAACGCATCCCTGGGAACGTATCACGATCT 
               
               
                   
               
               
                 CCTGAAAATCATTAAAGACAAGGACTTCCTGGACAATGAGGAGAA 
               
               
                   
               
               
                 CGAGGACATTCTTGAGGACATTGTCCTCACCCTTACGTTGTTTGA 
               
               
                   
               
               
                 AGATAGGGAGATGATTGAAGAACGCTTGAAAACTTACGCTCATCT 
               
               
                   
               
               
                 CTTCGACGACAAAGTCATGAAACAGCTCAAGAGGCGCCGATATAC 
               
               
                   
               
               
                 AGGATGGGGGCGGCTGTCAAGAAAACTGATCAATGGGATTCGAGA 
               
               
                   
               
               
                 CAAGCAGAGTGGAAAGACAATCCTGGATTTTCTTAAGTCCGATGG 
               
               
                   
               
               
                 ATTTGCCAACCGGAACTTCATGCAGTTGATCCATGATGACTCTCT 
               
               
                   
               
               
                 CACCTTTAAGGAGGACATCCAGAAAGCACAAGTTTCTGGCCAGGG 
               
               
                   
               
               
                 GGACAGTCTGCACGAGCACATCGCTAATCTTGCAGGTAGCCCAGC 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                 CGTCAAAGTAATGGGAAGGCATAAGCCCGAGAATATCGTTATCGA 
               
               
                   
               
               
                 GATGGCCCGAGAGAACCAAACTACCCAGAAGGGACAGAAGAACAG 
               
               
                   
               
               
                 TAGGGAAAGGATGAAGAGGATTGAAGAGGGTATAAAAGAACTGGG 
               
               
                   
               
               
                 GTCCCAAATCCTTAAGGAACACCCAGTTGAAAACACCCAGCTTCA 
               
               
                   
               
               
                 GAATGAGAAGCTCTACCTGTACTACCTGCAGAACGGCAGGGACAT 
               
               
                   
               
               
                 GTACGTGGATCAGGAACTGGACATCAATCGGCTCTCCGACTACGA 
               
               
                   
               
               
                 CGTGGATCATATCGTGCCCCAGTCTTTTCTCAAAGATGATTCTAT 
               
               
                   
               
               
                 TGATAATAAAGTGTTGACAAGATCCGATAAAAATAGAGGGAAGAG 
               
               
                   
               
               
                 TGATAACGTCCCCTCAGAAGAAGTTGTCAAGAAAATGAAAAATTA 
               
               
                   
               
               
                 TTGGCGGCAGCTGCTGAACGCCAAACTGATCACACAACGGAAGTT 
               
               
                   
               
               
                 CGATAATCTGACTAAGGCTGAACGAGGTGGCCTGTCTGAGTTGGA 
               
               
                   
               
               
                 TAAAGCCGGCTTCATCAAAAGGCAGCTTGTTGAGACACGCCAGAT 
               
               
                   
               
               
                 CACCAAGCACGTGGCCCAAATTCTCGATTCACGCATGAACACCAA 
               
               
                   
               
               
                 GTACGATGAAAATGACAAACTGATTCGAGAGGTGAAAGTTATTAC 
               
               
                   
               
               
                 TCTGAAGTCTAAGCTGGTGTCAGATTTCAGAAAGGACTTTCAGTT 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                 CTACCTGACCACCATGCGCATGATGCCTACCTGAATGCAGTGGTA 
               
               
                   
               
               
                 GGCACTGCACTTATCAAAAAATATCCCAAGCTGGAATCTGAATTT 
               
               
                   
               
               
                 GTTTACGGAGACTATAAAGTGTACGATGTTAGGAAAATGATCGCA 
               
               
                   
               
               
                 AAGTCTGAGCAGGAAATAGGCAAGGCCACCGCTAAGTACTTCTTT 
               
               
                   
               
               
                 TACAGCAATATTATGAATTTTTTCAAGACCGAGATTACACTGGCC 
               
               
                   
               
               
                 AATGGAGAGATTCGGAAGCGACCACTTATCGAAACAAACGGAGAA 
               
               
                   
               
               
                 ACAGGAGAAATCGTGTGGGACAAGGGTAGGGATTTCGCGACAGTC 
               
               
                   
               
               
                 CGGAAGGTCCTGTCCATGCCGCAGGTGAACATCGTTAAAAAGACC 
               
               
                   
               
               
                 GAAGTACAGACCGGAGGCTTCTCCAAGGAAAGTATCCTCCCGAAA 
               
               
                   
               
               
                 AGGAACAGCGACAAGCTGATCGCACGCAAAAAAGATTGGGACCCC 
               
               
                   
               
               
                 AAGAAATACGGCGGATTCGATTCTCCTACAGTCGCTTACAGTGTA 
               
               
                   
               
               
                 CTGGTTGTGGCCAAAGTGGAGAAAGGGAAGTCTAAAAAACTCAAA 
               
               
                   
               
               
                 AGCGTCAAGGAACTGCTGGGCATCACAATCATGGAGCGATCAAGT 
               
               
                   
               
               
                 TTCGAAAAAAACCCCATCGACTTTCTGGAGGCGAAAGGATATAAA 
               
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                 TTTGAGCTTGAAAACGGCCGGAAACGAATGCTCGCTAGTGCGGGC 
               
               
                   
               
               
                 GAGCTGCAGAAAGGTAACGAGCTGGCACTGCCCTCTAAATACGTT 
               
               
                   
               
               
                 AATTTCTTGTATCTGGCCAGCCACTATGAAAAGCTCAAAGGGTCC 
               
               
                   
               
               
                 CCCGAAGATAATGAGCAGAAGCAGCTGTTCGTGGAACAACACAAA 
               
               
                   
               
               
                 CACTACCTTGATGAGATCATCGAGCAAATAAGCGAGTTCTCCAAA 
               
               
                   
               
               
                 AGAGTGATCCTCGCCGACGCTAACCTCGATAAGGTGCTTTCTGCT 
               
               
                   
               
               
                 TACAATAAGCACAGGGATAAGCCCATCAGGGAGCAGGCAGAAAAC 
               
               
                   
               
               
                 ATTATCCACTTGTTTACTCTGACCAACTTGGGCGCACCTGCAGCC 
               
               
                   
               
               
                 TTCAAGTACTTCGACACCACCATAGACAGAAAGCGGTACACCTCT 
               
               
                   
               
               
                 ACAAAGGAGGTCCTGGACGCCACACTGATTCATCAGTCAATTACG 
               
               
                   
               
               
                 GGGCTCTATGAAACAAGAATCGACCTCTCTCAGCTCGGTGGAGAC 
               
               
                   
               
               
                 AGCAGGGCTGACCCCAAGAAGAAGAGGAAGGTG 
               
             
          
         
       
     
         [0064]    The synthesis strategy is as follows: Cos9 is synthesized by dividing the gene into six fragments (the divided position is indicated by boxes in the above sequence), and each fragment containing between 600 and 700 bp. Each fragment is synthesized by asymmetric PCR method because the asymmetric PCR can only synthesize a DNA sequence containing less than 1000 bp. The six synthetic fragments are then cloned to pBWD(a) and pBWD(b) and are named as follows: pBWD(a)-cos91, pBWD(a)-cos93, pBWD(a)-cos95, pBWD(b)-cos92, pBWD(b)-cos94, pBWD(b)-cos96. 
         [0065]    The synthesis process is as follows: 
         [0066]    First step: the six fragments synthesized by the asymmetric PCR are cloned into the first donor vector pBWD(a) and the second donor vector pBWD(b) and are correctly sequenced. Sticky ends after cleavage are completely complementary at positions indicated by the boxes. 
         [0067]    Structures of the vectors after cloning are as follows: 
         [0068]    pBWD(a)-cos91: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0069]    pBWD(b)-cos92: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0070]    pBWD(a)-cos93: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0071]    pBWD(b)-cos94: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0072]    pBWD(a)-cos95: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0073]    pBWD(b)-cos96: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0074]    Second step: assembly of the DNA fragments. The sticky ends after the cleavage are completely complementary at positions indicated by boxes. 
         [0075]    The multiple cloning site of the pBWA is as follows: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0076]    1. Construct of pBWA-cos9(1): 
         [0077]    pBWA and pBWD(a)-cos91 are digested by BsmbI. Because produced sticky ends are completely complementary, the backbone of pBWA and the fragment cos91 are then ligated together by the T4 DNA ligase to generate pBWA-cos9(1). 
         [0000]    
       
                 
         
             
             
         
       
     
         [0078]    2. Construct of pBWA-cos9(2): 
         [0079]    pBWA-cos9(1) and pBWD(b)-cos92 are digested by SapI. Because produced sticky ends are completely complementary, the backbone of pBWA-cos9(1) and the fragment cos92 are then ligated together by the T4 DNA ligase to generate pBWA-cos9(2). 
         [0000]    
       
                 
         
             
             
         
       
     
         [0080]    3. Construct of pBWA-cos9(3): 
         [0081]    pBWA-cos9(2) and pBWD(a)-cos93 are digested by BsmbI. Because produced sticky ends are completely complementary, the backbone of pBWA-cos9(2) and the fragment cos93 are then ligated together by the T4 DNA ligase to produce pBWA-cos9(3). 
         [0082]    pBWA-cos9(3): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0083]    4. Construct of pBWA-cos9(4): 
         [0084]    pBWA-cos9(3) and pBWD(b)-cos94 are digested by SapI. Because produced sticky ends are completely complementary, the backbone of pBWA-cos9(3) and the fragment cos94 are then ligated together by the T4 DNA ligase to form pBWA-cos9(4). 
         [0085]    pBWA-cos9(4): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0086]    5. Construct of pBWA-cos9(5): 
         [0087]    pBWA-cos9(4) and pBWD(a)-cos95 are digested by BsmbI. Because produced sticky ends are completely complementary, the backbone of pBWA-cos9(4) and the fragment cos95 are then ligated together by the T4 DNA ligase to form pBWA-cos9(5). 
         [0088]    pBWA-cos9(5): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0089]    6. Construct of pBWA-cos9(6): 
         [0090]    pBWA-cos9(5) and pBWD(b)-cos96 are digested by SapI. Because produced sticky ends are completely complementary, the backbone of pBWA-cos9(5) and the fragment cos96 are then ligated together by the T4 DNA ligase to form pBWA-cos9(6). 
         [0091]    pBWA-cos9(6): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0092]    Thus, the six fragments are seamlessly linked into an integral DNA fragment. 
         [0093]    The long fragment DNA can be cloned by the method similarly to the long sequence synthesis method. The long sequence is firstly divided into small sequences which are then cloned and sequenced and finally seamlessly assembled into the long sequence by the Bio-Walk system. 
         [0094]    Unless otherwise indicated, the numerical ranges involved in the invention include the end values. While particular embodiments of the invention have been shown and described, it will be obvious to those skilled in the art that changes and modifications may be made without departing from the invention in its broader aspects, and therefore, the aim in the appended claims is to cover all such changes and modifications as fall within the true spirit and scope of the invention.