Design, synthesis and assembly of synthetic nucleic acids

Abstract

Methods of synthesizing oligonucleotides with high coupling efficiency (>99.5%) are provided. Methods for purification of synthetic oligonucleotides are also provided. Instrumentation configurations for oligonucleotide synthesis are also provided. Methods of designing and synthesizing polynucleotides are also provided. Polynucleotide design is optimized for subsequent assembly from shorter oligonucleotides. Modifications of phosphoramidite chemistry to improve the subsequent assembly of polynucleotides are provided. The design process also incorporates codon biases into polynucleotides that favor expression in defined hosts. Design and assembly methods are also provided for the efficient synthesis of sets of polynucleotide variants. Software to automate the design and assembly process is also provided.

What is claimed: 
     
         1 . A method comprising:
 a) dividing a sequence of a polynucleotide into a plurality of non-overlapping polynucleotide sub-sequences using a division scheme;   b) adding a first restriction site for a first restriction enzyme that cleaves outside its recognition sequence to a 3′ end of a first polynucleotide sub-sequence in said plurality of polynucleotide sub-sequences to thereby form a first modified polynucleotide sub-sequence;   c) adding a second restriction site for a second restriction enzyme that cleaves outside its recognition sequence to a 5′ end of a second polynucleotide sub-sequence in said plurality of polynucleotide sub-sequences to thereby form a second modified polynucleotide sub-sequence, such that cleavage of said first restriction site and said second restriction site would cause a terminal portion of said first modified polynucleotide sub-sequence to become complementary with a terminal portion of said second modified polynucleotide sub-sequence;   d) synthesizing said first and second modified polynucleotide sub-sequences; and   e) digesting said first and second modified polynucleotide sub-sequences with said first and second restriction enzymes that cleave outside their recognition sequences, thereby producing two DNA fragments that can be joined together to form a single contiguous part of said single designed polynucleotide.   
     
     
         2 . The method of  claim 1 , wherein the first or second restriction site is a typeIIs site. 
     
     
         3 . The method of  claim 1  wherein the corresponding first or second recognition sequence is at least six contiguous base pairs in length. 
     
     
         4 . The method of  claim 1 , wherein the first or second restriction enzyme is Bbs1, Bsa1, BsmB1, BspM1, BseR1, Bpm1, Bsm1, Bsr1, or BsrD1. 
     
     
         5 . The method of  claim 1 , wherein the first or second polynucleotide sub-sequence is modified without changing the polypeptide sequence encoded by the polynucleotide subsequence so that each overhang resulting from digestion of the polynucleotide with one or more type IIs restriction endonuclease is unique. 
     
     
         6 . The method of  claim 1 , wherein the first and second recognition sites are between 10 and 500 bases apart in the sequence. 
     
     
         7 . The method of  claim 1 , wherein the first and second recognition sites are between 15 and 200 bases apart in the sequence. 
     
     
         8 . The method of  claim 1 , wherein the first and second recognition sites are between 25 and 100 bases apart in the sequence 
     
     
         9 . The method of  claim 1 , further comprising adding one or more additional restriction sites to the polynucleotide to produce one or more additional modified polynucleotide sub-sequences. 
     
     
         10 . The method of  claim 9 , wherein the single designed polynucleotide is assembled from three or more modified polynucleotide sub-sequences. 
     
     
         11 . The method of  claim 1 , wherein the plurality of polynucleotide sub-sequences is evaluated against one or more design criteria prior to the adding step b), adding step c) or synthesizing step d) and, when the plurality of polynucleotide sub-sequences is deemed to not satisfy the one or more design criteria, the dividing step a) is repeated with a different division scheme prior to final execution of the adding step b), adding step c), or synthesizing step d), thereby replacing the plurality of non-overlapping polynucleotide sub-sequences with a replacement plurality of non-overlapping polynucleotide sub-sequences. 
     
     
         12 . The method of  claim 11 , wherein the division scheme is based, at least in part, on a polynucleotide sub-sequence annealing temperature, a separation of incorrect annealing partners, random generation, or avoidance of a repeat sequence. 
     
     
         13 . The method of  claim 11 , wherein the one or more design criteria includes a design criterion selected from the group consisting of presence or absence of a repeat sequence in a sub-sequence in the plurality of sub-sequences, a length of a sub-sequence in the plurality of sub-sequences, a melting temperature between a pair of sub-sequences in the plurality of sub-sequences, a percent GC content of a sub-sequence in the plurality of subsequences, presence of a forbidden restriction site in a sub-sequence in the plurality of sub-sequences, and a codon frequency in a sub-sequence in the plurality of subsequences. 
     
     
         14 . The method of  claim 1 , wherein the sequence of the polynucleotide is generated based, at least in part, on the amino acid sequence of a predetermined protein at a time before the dividing a). 
     
     
         15 . The method of  claim 14 , wherein the sequence of the polynucleotide is also generated based, at least in part, on
 (i) exclusion of a restriction site sequence in the sequence of the polynucleotide;   (ii) incorporation of a restriction site sequence in the sequence of the polynucleotide;   (iii) a designation of a target G+C content in the sequence of the polynucleotide;   (iv) an allowable length of a sub-sequence that can be exactly repeated within either strand of the sequence of the polynucleotide;   (v) an allowable annealing temperature of any sub-sequence to any other subsequence within either strand of the sequence of the polynucleotide;   (vi) exclusion of a hairpin turn in the sequence of the polynucleotide;   (vii) exclusion of a repeat element in the sequence of the polynucleotide;   (viii) exclusion of a ribosome binding site in the sequence of the polynucleotide;   (ix) exclusion of a polyadenylation signal in the sequence of the polynucleotide;   (x) exclusion of a splice site in the sequence of the polynucleotide;   (xi) exclusion of an open reading frame in each possible 5′ reading frame in the sequence of the polynucleotide;   (xii) exclusion of a polynucleotide sequence that facilitates RNA degradation in the sequence of the polynucleotide;   (xiii) exclusion of an RNA polymerase termination signal in the sequence of the polynucleotide;   (xiv) exclusion of a transcriptional promoter in the sequence of the polynucleotide;   (xv) exclusion of an immunostimulatory sequence in the sequence of the polynucleotide;   (xvi) incorporation of an immunostimulatory sequence in the sequence of the polynucleotide;   (xvii) exclusion of an RNA methylation signal in the sequence of the polynucleotide;   (xviii) exclusion of a selenocysteine incorporation signal in the sequence of the polynucleotide;   (xix) exclusion of an RNA editing sequence in the sequence of the polynucleotide;   (xx) exclusion of an RNAi-targeted sequence in the sequence of the polynucleotide; or   (xxi) exclusion of an inverted repeat within the first 45 nucleotides encoding the predetermined protein in the sequence of the polynucleotide.   
     
     
         16 . A method comprising:
 a) dividing a sequence of a polynucleotide into a plurality of non-overlapping polynucleotide sub-sequences using a division scheme;   b) adding a first restriction site for a first restriction enzyme that cleaves outside its recognition sequence to a 3′ end of a first polynucleotide sub-sequence in said plurality of polynucleotide sub-sequences to thereby form a first modified polynucleotide sub-sequence;   c) adding a second restriction site for a second restriction enzyme that cleaves outside its recognition sequence to a 5′ end of a second polynucleotide sub-sequence in said plurality of polynucleotide sub-sequences to thereby form a second modified polynucleotide sub-sequence, such that cleavage of said first restriction site and said second restriction site would cause a terminal portion of said first modified polynucleotide sub-sequence to become complementary with a terminal portion of said second modified polynucleotide sub-sequence;   d) sub-cloning the first modified polynucleotide sub-sequence into a first cloning vector;   e) sub-cloning the second modified polynucleotide sub-sequence into a second cloning vector;   f) obtaining the first modified polynucleotide sub-sequence from the first cloning vector;   g) obtaining the second modified polynucleotide sub-sequence from the second cloning vector;   h) digesting the first modified polynucleotide sub-sequence with a first enzyme that recognizes the first restriction site thereby forming a digested product of the first modified polynucleotide sub-sequence;   i) digesting the second modified polynucleotide sub-sequence with the a second enzyme that recognizes the second restriction site thereby forming a digested product of the second modified polynucleotide sub-sequence; and   j) ligating the first digested product of the first modified polynucleotide sub-sequence with the digested product of the second modified polynucleotide sub-sequence to form the single designed polynucleotide.   
     
     
         17 . The method of  claim 16 , wherein steps h) and i) are performed sequentially. 
     
     
         18 . The method of  claim 16 , wherein steps h) and i) or steps h), i) and j) are performed in a single mixture. 
     
     
         19 . The method of  claim 16 , wherein the first or second restriction enzyme is Bbs1, Bsa1, BsmB1, BspM1, BseR1, Bpm1, Bsm1, Bsr1, or BsrD1. 
     
     
         20 . The method of  claim 16 , wherein the first or second polynucleotide sub-sequence is modified without changing the polypeptide sequence encoded by the polynucleotide subsequence so that each overhang resulting from digestion of the polynucleotide with one or more type IIs restriction endonuclease is unique. 
     
     
         21 . The method of  claim 16 , wherein the first and second recognition sites are between 10 and 500 bases apart in the sequence. 
     
     
         22 . The method of  claim 16 , wherein the first and second recognition sites are between 15 and 200 bases apart in the sequence. 
     
     
         23 . The method of  claim 16 , wherein the first and second recognition sites are between 25 and 100 bases apart in the sequence. 
     
     
         24 . The method of  claim 16 , further comprising adding one or more additional restriction sites to the polynucleotide to produce one or more additional modified polynucleotide sub-sequences. 
     
     
         25 . The method of  claim 24 , wherein the single designed polynucleotide is assembled from three or more modified polynucleotide sub-sequences. 
     
     
         26 . The method of  claim 16 , wherein the plurality of polynucleotide sub-sequences is evaluated against one or more design criteria prior to the adding step b), adding step c) or sub-cloning step d) and, when the plurality of polynucleotide sub-sequences is deemed to not satisfy the one or more design criteria, the dividing step a) is repeated with a different division scheme prior to final execution of the adding step b), adding step c), or sub-cloning step d), thereby replacing the plurality of non-overlapping polynucleotide sub-sequences with a replacement plurality of non-overlapping polynucleotide sub-sequences. 
     
     
         27 . The method of  claim 26 , wherein the division scheme is based, at least in part, on a polynucleotide sub-sequence annealing temperature, a separation of incorrect annealing partners, random generation, or avoidance of a repeat sequence. 
     
     
         28 . The method of  claim 26 , wherein the one or more design criteria includes a design criterion selected from the group consisting of presence or absence of a repeat sequence in a sub-sequence in the plurality of sub-sequences, a length of a sub-sequence in the plurality of sub-sequences, a melting temperature between a pair of sub-sequences in the plurality of sub-sequences, a percent GC content of a sub-sequence in the plurality of subsequences, presence of a forbidden restriction site in a sub-sequence in the plurality of sub-sequences, and a codon frequency in a sub-sequence in the plurality of subsequences. 
     
     
         29 . The method of  claim 16 , wherein the sequence of the polynucleotide is generated based, at least in part, on the amino acid sequence of a predetermined protein at a time before the dividing a). 
     
     
         30 . The method of  claim 29 , wherein the sequence of the polynucleotide is also generated based, at least in part, on
 (i) exclusion of a restriction site sequence in the sequence of the polynucleotide;   (ii) incorporation of a restriction site sequence in the sequence of the polynucleotide;   (iii) a designation of a target G+C content in the sequence of the polynucleotide;   (iv) an allowable length of a sub-sequence that can be exactly repeated within either strand of the sequence of the polynucleotide;   (v) an allowable annealing temperature of any sub-sequence to any other subsequence within either strand of the sequence of the polynucleotide;   (vi) exclusion of a hairpin turn in the sequence of the polynucleotide;   (vii) exclusion of a repeat element in the sequence of the polynucleotide;   (viii) exclusion of a ribosome binding site in the sequence of the polynucleotide;   (ix) exclusion of a polyadenylation signal in the sequence of the polynucleotide;   (x) exclusion of a splice site in the sequence of the polynucleotide;   (xi) exclusion of an open reading frame in each possible 5′ reading frame in the sequence of the polynucleotide;   (xii) exclusion of a polynucleotide sequence that facilitates RNA degradation in the sequence of the polynucleotide;   (xiii) exclusion of an RNA polymerase termination signal in the sequence of the polynucleotide;   (xiv) exclusion of a transcriptional promoter in the sequence of the polynucleotide;   (xv) exclusion of an immunostimulatory sequence in the sequence of the polynucleotide;   (xvi) incorporation of an immunostimulatory sequence in the sequence of the polynucleotide;   (xvii) exclusion of an RNA methylation signal in the sequence of the polynucleotide;   (xviii) exclusion of a selenocysteine incorporation signal in the sequence of the polynucleotide;   (xix) exclusion of an RNA editing sequence in the sequence of the polynucleotide;   (xx) exclusion of an RNAi-targeted sequence in the sequence of the polynucleotide; or   (xxi) exclusion of an inverted repeat within the first 45 nucleotides encoding the predetermined protein in the sequence of the polynucleotide.   
     
     
         31 . A computer system comprising:
 at least one processor;   memory; and   at least one program stored in the memory and executable by the at least one processor, the at least one program comprising instructions to:
 a) divide a sequence of a polynucleotide into a plurality of non-overlapping polynucleotide sub-sequences using a division scheme; 
 b) add a first restriction site for a first restriction enzyme that cleaves outside its recognition sequence to a 3′ end of a first polynucleotide sub-sequence in said plurality of polynucleotide sub-sequences to thereby form a first modified polynucleotide sub-sequence; and 
 c) add a second restriction site for a second restriction enzyme that cleaves outside its recognition sequence to a 5′ end of a second polynucleotide sub-sequence in said plurality of polynucleotide sub-sequences to thereby form a second modified polynucleotide sub-sequence, wherein the first modified polynucleotide sub-sequence and the second modified polynucleotide sub-sequence are configured so that cleavage of the first restriction site and the second restriction site cause a terminal portion of said first modified polynucleotide sub-sequence to become complementary with a terminal portion of said second modified polynucleotide sub-sequence, wherein the set of modified polynucleotide sub-sequences comprises the first modified polynucleotide sub-sequence and the second modified polynucleotide sub-sequence. 
   
     
     
         32 . The computer system of  claim 31 , wherein the at least one program further comprises instructions to make the first or second restriction site a typeIIs site. 
     
     
         33 . The computer system of  claim 31 , wherein the at least one program further comprises instructions to make the first or second recognition sequence at least six contiguous base pairs in length. 
     
     
         34 . The computer system of  claim 31 , wherein the first or second restriction enzyme is Bbs1, Bsa1, BsmB1, BspM1, BseR1, Bpm1, Bsm1, Bsr1, or BsrD1. 
     
     
         35 . The computer system of  claim 31 , wherein the at least one program further comprises instructions to modify the first or second polynucleotide sub-sequence without changing the polypeptide sequence encoded by the polynucleotide subsequence so that each overhang resulting from digestion of the polynucleotide with one or more type IIs restriction endonuclease is unique. 
     
     
         36 . The computer system of  claim 31 , wherein the at least one program further comprises instruction to make the first and second recognition sites between 10 and 500 bases apart in the sequence. 
     
     
         37 . The computer system of  claim 31 , wherein the at least one program further comprises instruction to make the first and second recognition sites between 15 and 200 bases apart in the sequence. 
     
     
         38 . The computer system of  claim 31 , wherein the at least one program further comprises instruction to make the first and second recognition sites between 25 and 100 bases apart in the sequence. 
     
     
         39 . The computer system of  claim 31 , wherein the at least one program further comprises instructions to add one or more additional restriction sites to the polynucleotide to produce one or more additional modified polynucleotide sub-sequences. 
     
     
         40 . The computer system of  claim 31 , wherein the at least one program further comprises instructions to assemble the single designed polynucleotide from three or more modified polynucleotide sub-sequences. 
     
     
         41 . The computer system of  claim 31 , wherein the plurality of polynucleotide sub-sequences is evaluated against one or more design criteria prior execution of the instructions to add b) or instructions to add c) and, when the plurality of polynucleotide sub-sequences is deemed to not satisfy the one or more design criteria, the instructions to divide a) are repeated with a different division scheme prior to final execution of the instructions to add b) or instructions to add c), thereby replacing the plurality of non-overlapping polynucleotide sub-sequences with a replacement plurality of non-overlapping polynucleotide sub-sequences. 
     
     
         42 . The computer system of  claim 41 , wherein the division scheme is based, at least in part, on a polynucleotide sub-sequence annealing temperature, a separation of incorrect annealing partners, random generation, or avoidance of a repeat sequence. 
     
     
         43 . The computer system of  claim 41 , wherein the one or more design criteria includes a design criterion selected from the group consisting of presence or absence of a repeat sequence in a sub-sequence in the plurality of sub-sequences, a length of a sub-sequence in the plurality of sub-sequences, a melting temperature between a pair of sub-sequences in the plurality of sub-sequences, a percent GC content of a sub-sequence in the plurality of subsequences, presence of a forbidden restriction site in a sub-sequence in the plurality of sub-sequences, and a codon frequency in a sub-sequence in the plurality of subsequences. 
     
     
         44 . The computer system of  claim 31 , wherein the sequence of the polynucleotide is generated based, at least in part, on the amino acid sequence of a predetermined protein at a time before execution of the instructions to divide a). 
     
     
         45 . The computer system of  claim 44 , wherein the sequence of the polynucleotide is also generated based, at least in part, on:
 (i) exclusion of a restriction site sequence in the sequence of the polynucleotide;   (ii) incorporation of a restriction site sequence in the sequence of the polynucleotide;   (iii) a designation of a target G+C content in the sequence of the polynucleotide;   (iv) an allowable length of a sub-sequence that can be exactly repeated within either strand of the sequence of the polynucleotide;   (v) an allowable annealing temperature of any sub-sequence to any other subsequence within either strand of the sequence of the polynucleotide;   (vi) exclusion of a hairpin turn in the sequence of the polynucleotide;   (vii) exclusion of a repeat element in the sequence of the polynucleotide;   (viii) exclusion of a ribosome binding site in the sequence of the polynucleotide;   (ix) exclusion of a polyadenylation signal in the sequence of the polynucleotide;   (x) exclusion of a splice site in the sequence of the polynucleotide;   (xi) exclusion of an open reading frame in each possible 5′ reading frame in the sequence of the polynucleotide;   (xii) exclusion of a polynucleotide sequence that facilitates RNA degradation in the sequence of the polynucleotide;   (xiii) exclusion of an RNA polymerase termination signal in the sequence of the polynucleotide;   (xiv) exclusion of a transcriptional promoter in the sequence of the polynucleotide;   (xv) exclusion of an immunostimulatory sequence in the sequence of the polynucleotide;   (xvi) incorporation of an immunostimulatory sequence in the sequence of the polynucleotide;   (xvii) exclusion of an RNA methylation signal in the sequence of the polynucleotide;   (xviii) exclusion of a selenocysteine incorporation signal in the sequence of the polynucleotide;   (xix) exclusion of an RNA editing sequence in the sequence of the polynucleotide;   (xx) exclusion of an RNAi-targeted sequence in the sequence of the polynucleotide; or   (xxi) exclusion of an inverted repeat within the first 45 nucleotides encoding the predetermined protein in the sequence of the polynucleotide.   
     
     
         46 . A non-transitory computer readable storage medium storing at least one program configured for execution by at least one processor of a computer system, the at least one program comprising instructions for:
 a) dividing a sequence of a polynucleotide into a plurality of non-overlapping polynucleotide sub-sequences using a division scheme;   b) adding a first restriction site for a first restriction enzyme that cleaves outside its recognition sequence to a 3′ end of a first polynucleotide sub-sequence in said plurality of polynucleotide sub-sequences to thereby form a first modified polynucleotide sub-sequence; and   c) adding a second restriction site for a second restriction enzyme that cleaves outside its recognition sequence to a 5′ end of a second polynucleotide sub-sequence in said plurality of polynucleotide sub-sequences to thereby form a second modified polynucleotide sub-sequence, wherein the first modified polynucleotide sub-sequence and the second modified polynucleotide sub-sequence are configured so that cleavage of the first restriction site and the second restriction site cause a terminal portion of said first modified polynucleotide sub-sequence to become complementary with a terminal portion of said second modified polynucleotide sub-sequence, wherein the set of modified polynucleotide sub-sequences comprises the first modified polynucleotide sub-sequence and the second modified polynucleotide sub-sequence.   
     
     
         47 . The non-transistory computer readable storage medium of  claim 46 , wherein the plurality of polynucleotide sub-sequences is evaluated against one or more design criteria prior to execution of the instructions for adding b) or prior to execution of the instructions for adding c) and, when the plurality of polynucleotide sub-sequences is deemed to not satisfy the one or more design criteria, the instructions for dividing a) are repeated with a different division scheme prior to final execution of the instructions for adding b) or instructions for adding c), thereby replacing the plurality of non-overlapping polynucleotide sub-sequences with a replacement plurality of non-overlapping polynucleotide sub-sequences. 
     
     
         48 . The non-transistory computer readable storage medium of  claim 47 , wherein the division scheme is based, at least in part, on a polynucleotide sub-sequence annealing temperature, a separation of incorrect annealing partners, random generation, or avoidance of a repeat sequence. 
     
     
         49 . The non-transistory computer readable storage medium of  claim 47 , wherein the one or more design criteria includes a design criterion selected from the group consisting of presence or absence of a repeat sequence in a sub-sequence in the plurality of sub-sequences, a length of a sub-sequence in the plurality of sub-sequences, a melting temperature between a pair of sub-sequences in the plurality of sub-sequences, a percent GC content of a sub-sequence in the plurality of subsequences, presence of a forbidden restriction site in a sub-sequence in the plurality of sub-sequences, and a codon frequency in a sub-sequence in the plurality of subsequences. 
     
     
         50 . The non-transistory computer readable storage medium of  claim 46 , wherein the sequence of the polynucleotide is generated based, at least in part, on the amino acid sequence of a predetermined protein at a time before the dividing a). 
     
     
         51 . The non-transistory computer readable storage medium of  claim 50 , wherein the sequence of the polynucleotide is also generated based, at least in part, on:
 (i) exclusion of a restriction site sequence in the sequence of the polynucleotide;   (ii) incorporation of a restriction site sequence in the sequence of the polynucleotide;   (iii) a designation of a target G+C content in the sequence of the polynucleotide;   (iv) an allowable length of a sub-sequence that can be exactly repeated within either strand of the sequence of the polynucleotide;   (v) an allowable annealing temperature of any sub-sequence to any other subsequence within either strand of the sequence of the polynucleotide;   (vi) exclusion of a hairpin turn in the sequence of the polynucleotide;   (vii) exclusion of a repeat element in the sequence of the polynucleotide;   (viii) exclusion of a ribosome binding site in the sequence of the polynucleotide;   (ix) exclusion of a polyadenylation signal in the sequence of the polynucleotide;   (x) exclusion of a splice site in the sequence of the polynucleotide;   (xi) exclusion of an open reading frame in each possible 5′ reading frame in the sequence of the polynucleotide;   (xii) exclusion of a polynucleotide sequence that facilitates RNA degradation in the sequence of the polynucleotide;   (xiii) exclusion of an RNA polymerase termination signal in the sequence of the polynucleotide;   (xiv) exclusion of a transcriptional promoter in the sequence of the polynucleotide;   (xv) exclusion of an immunostimulatory sequence in the sequence of the polynucleotide;   (xvi) incorporation of an immunostimulatory sequence in the sequence of the polynucleotide;   (xvii) exclusion of an RNA methylation signal in the sequence of the polynucleotide;   (xviii) exclusion of a selenocysteine incorporation signal in the sequence of the polynucleotide;   (xix) exclusion of an RNA editing sequence in the sequence of the polynucleotide;   (xx) exclusion of an RNAi-targeted sequence in the sequence of the polynucleotide; or   (xxi) exclusion of an inverted repeat within the first 45 nucleotides encoding the predetermined protein in the sequence of the polynucleotide.   
     
     
         52 . A method of forming a plurality of chimeric polynucleotides, the method comprising:
 a) introducing a first plurality of type IIs restriction sites into a first polynucleotide thereby forming a first modified polynucleotide;   a) introducing a second plurality of type IIs restriction sites into a second polynucleotide, wherein the second polynucleotide is other than the first polynucleotide, thereby forming a second modified polynucleotide;   c) digesting the first and second modified polynucleotide with one or more type IIs restriction endonucleases to form a respective first and second plurality of digestion products; and   d) exposing the first and second plurality of digestion products to ligation conditions, thereby producing the plurality of chimeric polynucleotides, wherein each chimeric polynucleotide in the plurality of polynucleotides includes at least one digestion product from the first modified polynucleotide and at least one digestion product from the second modified polynucleotide.   
     
     
         53 . The method of  claim 52 , wherein
 the first polynucleotide and the first modified polynucleotide exclusively encode the same first polypeptide sequence; and   the second polynucleotide and the second modified polynucleotide exclusively encode the same second polypeptide sequence.   
     
     
         54 . The method of  claim 52 , wherein each respective overhang in the first plurality of digestion products is unique and pairs with a corresponding overhang in the second plurality of digestion products. 
     
     
         55 . The method of  claim 52 , wherein a first restriction site in the first plurality of type IIs restriction sites and a second restriction site in the second plurality of type IIs restrictions sites is selected from the group consisting of AlwI, BbsI, BbvI, BpmI, BsaI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, BspMI, BsrDI, EarI, FokI, HgaI, HphI, MboII, MnlI, PleI, SapI, SfaNI, BstF51, and FauI. 
     
     
         56 . The method of  claim 52 , wherein
 the first plurality of digestion products collectively encode the first modified polynucleotide, and   the second plurality of digestion products collectively encode the second modified polynucleotide.   
     
     
         57 . The method of  claim 52 , wherein
 each respective type IIs restriction site in the first plurality of type IIs restrictions sites is between 25 and 100 bases apart from a neighboring type IIs restriction site in the first plurality of type IIs restrictions sites in the first modified polynucleotide, and   each respective type IIs restriction site in the second plurality of type IIs restrictions sites is between 25 and 100 bases apart from a neighboring type IIs restriction site in the second plurality of type IIs restrictions sites in the second modified polynucleotide.   
     
     
         58 . A library of isolated chimeric polynucleotides, comprising:
 a) a first chimeric polynucleotide, wherein the first chimeric polynucleotide comprises a first plurality of subsequences and a second plurality of subsequences, wherein
 each subsequence in the first plurality of subsequences and each subsequence in the second plurality of subsequences is bounded by a unique pair of type IIs restriction sites from a plurality of type IIs restriction sites, 
 each subsequence in the first plurality of subsequences is from a first modified polynucleotide, wherein the first modified polynucleotide is formed by the introduction of a plurality of type IIs restriction sites into a first polynucleotide, 
 each subsequence in the second plurality of subsequences is from a second 
 modified polynucleotide, wherein the second modified polynucleotide is formed by the introduction of the first plurality of type IIs restrictions sites into a second polynucleotide, 
 the first polynucleotide is other than the second polynucleotide, and 
 the first modified polynucleotide is other than the second modified polynucleotide; and 
   b) a second chimeric polynucleotide in the library of chimeric polynucleotides comprises a third plurality of subsequences and a fourth plurality of subsequences, wherein
 the third plurality of subsequences and the fourth plurality of subsequences collectively encode the second chimeric polynucleotide, 
 each subsequence in the third plurality of subsequences and each subsequence in the fourth plurality of subsequences is bounded by a unique pair of type IIs restriction sites from the plurality of type IIs restriction sites, 
 each subsequence in the third plurality of subsequences is from the first modified polynucleotide, 
 each subsequence in the fourth plurality of subsequences is from the second modified polynucleotide, and 
   the first plurality of subsequences contain a subsequence that is not in the third plurality of subsequences.   
     
     
         59 . The library of  claim 58 , wherein the plurality of type IIs restriction sites includes AlwI, BbsI, BbvI, BpmI, BsaI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, BspMI, BsrDI, EarI, FokI, HgaI, HphI, MboII, MnlI, PleI, SapI, SfaNI, BstF51, or FauI restriction site. 
     
     
         60 . The library of  claim 58 , wherein
 the first polynucleotide and the first modified polynucleotide exclusively encode the same first polypeptide sequence, and   the second polynucleotide and the second modified polynucleotide exclusively encode the same second polypeptide sequence.