Methods, systems, and computer readable media for generating codon optimized nucleotide sequences

1 . A method of synthesizing a polynucleotide using a computer, the method comprising:

receiving, by the computer, a target amino acid sequence, at least one problematic nucleotide sequence, and a selected host expression system and/or a selected codon table, wherein the problematic nucleotide sequence comprises a guanine-cytosine content (GC content), a sequence repeat, a splice site, and/or a restriction enzyme recognition site that negatively affects transcription and/or translation in the selected host expression system;

reverse-translating, by the computer, the target amino acid sequence multiple times to generate a plurality of candidate codon optimized nucleotide sequences that each encode the target amino acid sequence, wherein a given candidate codon optimized nucleotide sequence in the plurality of candidate codon optimized nucleotide sequences comprises about 250 or fewer codons, wherein the plurality of candidate codon optimized nucleotide sequences comprises at least 100, 1000, 10000, 100000, or more nucleotide sequences, and wherein for an amino acid residue in the target amino acid sequence that is encoded by synonymous codons, a given codon is randomly selected from the synonymous codons encoding the amino acid residue and incorporated into a given candidate codon optimized nucleotide sequence in the plurality of candidate codon optimized nucleotide sequences;

determining, by the computer, a total number of occurrences of the problematic nucleotide sequence in each of the candidate codon optimized nucleotide sequences in the plurality of candidate codon optimized nucleotide sequences to generate a problematic nucleotide sequence count for each of the candidate codon optimized nucleotide sequences;

identifying, by the computer, one or more of the candidate codon optimized nucleotide sequences in the plurality of candidate codon optimized nucleotide sequences that comprise a lowest problematic nucleotide sequence count, thereby generating the codon optimized nucleotide sequence;

identifying, by the computer, a start position of the problematic nucleotide sequence, a length of the problematic nucleotide sequence, and/or a number of problematic codons in the problematic nucleotide sequence at each occurrence of the problematic nucleotide sequence in the codon optimized nucleotide sequence to generate problematic nucleotide sequence data;

replacing, by the computer, at least a sub-sequences of any occurrences of the problematic nucleotide sequence in the codon optimized nucleotide sequence with an unproblematic nucleotide sequence that is randomly selected from a list of weighted unproblematic nucleotide sequences using the problematic nucleotide sequence data, thereby generating a further codon optimized nucleotide sequence, wherein the unproblematic nucleotide sequence does not negatively affect transcription and/or translation in the selected host expression system; and,

synthesizing a polynucleotide comprising the further codon optimized nucleotide sequence encoding the target amino acid sequence.

2 . The method of claim 1 , further comprising:

determining, by the computer, a total number of codons, a number of unfavorable codons, a CAI, and/or a number of occurrences of the problematic nucleotide sequence in the codon optimized nucleotide sequence.

3 . The method of claim 1 , wherein the target amino acid sequence corresponds to at least one protein.

4 . The method of claim 1 , wherein the at least one problematic nucleotide sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 500, or more problematic nucleotide sequences.

5 . The method of claim 1 , wherein the at least one problematic nucleotide sequence corresponds to a splice site or a restriction enzyme recognition site.

6 . The method of claim 1 , wherein the selected host expression system comprises a viral species, a fungal species, a bacterial species, or a mammalian species.

7 . The method of claim 1 , wherein the selected codon table is a user specified codon table.

8 . The method of claim 1 , wherein the selected codon table is automatically received when the selected host expression system is received by the computer.

9 . The method of claim 1 , wherein the synonymous codons each comprise a corresponding weight.

10 . The method of claim 9 , wherein the corresponding weight is user specified.

11 . The method of claim 1 , wherein the codon optimized nucleotide sequence comprises about 250 or more codons.

12 . The method of claim 1 , further comprising:

inserting the synthesized polynucleotide comprising the further codon optimized nucleotide sequence encoding the target amino acid sequence into the selected host expression system;

expressing a polypeptide that comprises the target amino acid sequence; and,

purifying the polypeptide.

13 . A method of synthesizing a polynucleotide using a computer, the method comprising:

receiving, by the computer, a target amino acid sequence, at least one problematic nucleotide sequence, and a selected host expression system and/or a selected codon table, wherein the at least one problematic nucleotide sequence corresponds to a splice site or a restriction enzyme recognition site that negatively affects transcription and/or translation in the selected host expression system;

reverse-translating, by the computer, the target amino acid sequence multiple times to generate a plurality of candidate codon optimized nucleotide sequences that each encode the target amino acid sequence, wherein the plurality of candidate codon optimized nucleotide sequences comprises at least 100, 1000, 10000, 100000, or more nucleotide sequences, and wherein for an amino acid residue in the target amino acid sequence that is encoded by synonymous codons, a given codon is randomly selected from the synonymous codons encoding the amino acid residue and incorporated into a given candidate codon optimized nucleotide sequence in the plurality of candidate codon optimized nucleotide sequences;

determining, by the computer, a total number of occurrences of the problematic nucleotide sequence in each of the candidate codon optimized nucleotide sequences in the plurality of candidate codon optimized nucleotide sequences to generate a problematic nucleotide sequence count for each of the candidate codon optimized nucleotide sequences;

identifying, by the computer, one or more of the candidate codon optimized nucleotide sequences in the plurality of candidate codon optimized nucleotide sequences that comprise a lowest problematic nucleotide sequence count, thereby generating the codon optimized nucleotide sequence;

identifying, by the computer, a start position of the problematic nucleotide sequence, a length of the problematic nucleotide sequence, and/or a number of problematic codons in the problematic nucleotide sequence at each occurrence of the problematic nucleotide sequence in the codon optimized nucleotide sequence to generate problematic nucleotide sequence data;

replacing, by the computer, at least a sub-sequences of any occurrences of the problematic nucleotide sequence in the codon optimized nucleotide sequence with an unproblematic nucleotide sequence that is randomly selected from a list of weighted unproblematic nucleotide sequences using the problematic nucleotide sequence data, thereby generating a further codon optimized nucleotide sequence, wherein the unproblematic nucleotide sequence does not negatively affect transcription and/or translation in the selected host expression system; and,

synthesizing a polynucleotide comprising the further codon optimized nucleotide sequence encoding the target amino acid sequence.