Method of constructing a synthetic polynucleotide
A method of constructing a synthetic polynucleotide, the method including selecting a first codon of a parent polynucleotide that encodes a polypeptide for replacement with a synonymous codon, wherein the synonymous codon is selected on the basis that it exhibits a higher translational efficiency in an epithelial cell of a mammal than the first codon in a comparison of translational efficiencies of codons in test cells of the same type as the epithelial cell; and replacing the first codon with the synonymous codon to construct the synthetic polynucleotide.
1. A method of constructing a synthetic polynucleotide, the method comprising:
selecting a first codon of a parent polynucleotide that encodes a polypeptide for replacement with a synonymous codon, wherein the synonymous codon is selected on the basis that it exhibits a higher translational efficiency in an epithelial cell of a mammal than the first codon in a comparison of translational efficiencies of codons in test cells of the same type as the epithelial cell; and
replacing the first codon with the synonymous codon to construct the synthetic polynucleotide.
2. The method of claim 1 , wherein the translational efficiencies of codons in the test cells are compared by:
separately introducing into the test cells individual synthetic constructs, each comprising a regulatory polynucleotide operably linked to a tandem repeat of a codon fused in frame with a reporter polynucleotide that encodes a reporter protein;
comparing expression of the reporter protein in the test cells to determine the relative translational efficiencies of the codons.
3. The method of claim 2 , wherein the synthetic constructs are introduced into the test cells by:
introducing the individual synthetic constructs into progenitors of the test cells; and
culturing the progenitors such that they differentiate to become the test cells.
4. The method of claim 2 , wherein the reporter protein is expressed from the synthetic construct comprising a tandem repeat of the synonymous codon at a level that is at least 110% of the level at which it is expressed from the synthetic construct comprising a tandem repeat of the first codon.
5. The method of claim 2 , wherein the tandem repeat of each of the synthetic constructs comprises at least three copies of the corresponding codon.
6. The method of claim 1 , wherein the translational efficiencies are compared by measuring the abundance of iso-tRNAs corresponding to the codons in the test cells.
7. The method of claim 6 , wherein the synonymous codon corresponds to an iso-tRNA which is in higher abundance in the test cells than the iso-tRNA corresponding to the first codon.
8. The method of claim 6 , wherein the synonymous codon corresponds to an iso-tRNA that is present in the test cells at a level which is at least 110% of the level of the iso-tRNA that corresponds to the first codon.
9. The method of claim 1 , wherein the first codon and the synonymous codon are selected such that the polypeptide is expressed from the synthetic polynucleotide in the test cells at a level which is at least 110% of the level at which the polypeptide is expressed from the parent polynucleotide.
10. The method of claim 1 , wherein the epithelial cell is an undifferentiated epithelial cell and wherein the comparison of translational efficiencies of the codons is represented by the following table:
Translational
Codon
Efficiency
Ala (GCA)
50.05
Ala (GCC)
46.85
Ala (GCG)
35.45
Ala (GCT)
29.95
Arg (AGA)
31.00
Arg (CGT)
26.95
Arg (CGG)
21.55
Arg (CGA)
16.25
Arg (CGC)
15.30
Arg (AGG)
5.12
Asp (GAC)
21.35
Asp (GAT)
16.70
Cys (TGC)
19.00
Cys (TGT)
5.92
Gln (CAG)
46.65
Gln (CAA)
35.00
Glu (GAA)
16.50
Glu (GAG)
3.86
Gly (GGA)
32.55
Gly (GGC)
22.95
Gly (GGG)
5.70
Gly (GGT)
5.57
His (CAT)
40.30
His (CAC)
34.80
Ile (ATC)
3.48
Ile (ATA)
3.37
Ile (ATT)
3.18
Leu (CTA)
6.43
Leu (CTG)
4.31
Leu (TTA)
3.47
Leu (TTG)
3.45
Leu (CTT)
3.14
Leu (CTC)
2.73
Lys (AAA)
17.60
Lys (AAG)
10.07
Phe (TTC)
4.35
Phe (TTT)
2.67
Pro (CGA)
31.00
Pro (CCG)
21.40
Pro (CCC)
10.48
Pro (CCT)
10.25
Ser (AGC)
62.40
Ser (TCG)
60.00
Ser (TCA)
33.95
Ser (TCT)
33.75
Ser (TCC)
32.90
Ser (AGT)
23.10
Thr (ACT)
40.70
Thr (ACG)
40.10
Thr (ACA)
38.85
Thr (ACC)
3.12
Val (GTA)
7.16
Val (GTC)
7.12
Val (GTG)
5.71
Val (GTT)
5.73.
11. The method of claim 1 , wherein the epithelial cell is an undifferentiated epithelial cell and wherein the first codon and the synonymous codon are selected from the foliowing table:
Synonymous
First Codon
Codon
Ala (GCC)
Ala (GCA)
Ala (GCG)
Ala (GCA)
Ala (GCT)
Ala (GCA)
Ala (GCG)
Ala (GCC)
Ala (GCT)
Ala (GCC)
Ala (GCT)
Ala (GCG)
Arg (CGT)
Arg (AGA)
Arg (CGG)
Arg (AGA)
Arg (CGA)
Arg (AGA)
Arg (CGC)
Arg (AGA)
Arg (AGG)
Arg (AGA)
Arg (CGG)
Arg (CGT)
Arg (CGA)
Arg (CGT)
Arg (CGC)
Arg (CGT)
Arg (AGG)
Arg (CGT)
Arg (CGA)
Arg (CGG)
Arg (CGC)
Arg (CGG)
Arg (AGG)
Arg (CGG)
Arg (CGC)
Arg (CGA)
Arg (AGG)
Arg (CGA)
Arg (AGG)
Arg (CGC)
Asp (GAT)
Asp (GAC)
Cys (TGT)
Cys (TGC)
Gln (CAA)
Gln (CAG)
Glu (GAG)
Glu (GAA)
Gly (GGC)
Gly (GGA)
Gly (GGG)
Gly (GGA)
Gly (GGT)
Gly (GGA)
Gly (GGG)
Gly (GGC)
Gly (GGT)
Gly (GGC)
His (CAC)
His (CAT)
Leu (CTG)
Leu (CTA)
Leu (TTA)
Leu (CTA)
Leu (TTG)
Leu (CTA)
Leu (CTT)
Leu (CTA)
Leu (CTC)
Leu (CTA)
Leu (TTA)
Leu (CTG)
Leu (TTG)
Leu (CTG)
Leu (CTG)
Leu (CTG)
Leu (CTT)
Leu (CTG)
Leu (CTC)
Leu (CTG)
Lys (AAG)
Lys (AAA)
Phe (TTT)
Phe (TTC)
Pro (CCG)
Pro (CGA)
Pro (CCC)
Pro (CGA)
Pro (CCT)
Pro (CGA)
Pro (CCC)
Pro (CCG)
Pro (CCT)
Pro (CCG)
Ser (TCA)
Ser (AGC)
Ser (TCT)
Ser (AGC)
Ser (TCC)
Ser (AGC)
Ser (AGT)
Ser (AGC)
Ser (TCA)
Ser (TCG)
Ser (TCT)
Ser (TCG)
Ser (TCC)
Ser (TCG)
Ser (AGT)
Ser (TCG)
Ser (AGT)
Ser (TCA)
Ser (AGT)
Ser (TCT)
Ser (AGT)
Ser (TCC)
Thr (ACC)
Thr (ACT)
Thr (ACC)
Thr (ACG)
Thr (ACC)
Thr (ACA)
Val (GTG)
Val (GTA)
Val (GTT)
Val (GTA)
Val (GTG)
Val (GTC)
Val (GTT)
Val (GTC).
12. The method of claim 1 , wherein the epithelial cell is an undifferentiated epithelial cell and wherein the first codon and the synonymous codon are selected from the following table:
Synonymous
First Codon
Codon
Arg (AGG)
Arg (AGA)
Arg (AGG)
Arg (CGG)
Arg (AGG)
Arg (CGA)
Arg (AGG)
Arg (CGC)
Cys (TGT)
Cys (TGC)
Glu (GAG)
Glu (GAA)
Gly (GGG)
Gly (GGA)
Gly (GGG)
Gly (GGC)
Gly (GGT)
Gly (GGC)
Leu (CTT)
Leu (CTA)
Leu (CTC)
Leu (CTA)
Leu (CTT)
Leu (CTG)
Leu (CTC)
Leu (CTG)
Pro (CCC)
Pro (CGA)
Pro (CCC)
Pro (CCG)
Pro (CCT)
Pro (CCG)
Ser (AGT)
Ser (AGC)
Ser (AGT)
Ser (TCG)
Thr (ACC)
Thr (ACT)
Thr (ACC)
Thr (ACG)
Thr (ACC)
Thr (ACA).
13. The method of claim 1 , wherein the epithelial cell is an undifferentiated epithelial cell and wherein the first codon and the synonymous codon are selected from the following table:
Synonymous
First Codon
Codon
Arg (AGG)
Arg (AGA)
Arg (AGG)
Arg (CGG)
Cys (TGT)
Cys (TGC)
Gly (GGG)
Gly (GGA)
Gly (GGT)
Gly (GGA)
Gly (GGG)
Gly (GGC)
Gly (GGT)
Gly (GGC)
Pro (CCC)
Pro (CGA)
Pro (CCT)
Pro (CGA)
Pro (CCC)
Pro (CCG)
Pro (CCT)
Pro (CCG)
Thr (ACC)
Thr (ACT)
Thr (ACC)
Thr (ACG)
Thr (ACC)
Thr (ACA).
14. The method of claim 10 , wherein the undifferentiated epithelial cell is a undifferentiated keratinocyte.
15. The method of claim 10 , wherein the undifferentiated epithelial cell is a Cos-1 cell.
16. The method of claim 1 , wherein the polypeptide is a viral capsid protein.
17. The method of claim 16 , wherein the viral capsid protein is selected from the group consisting of papillomavirus L1 protein, papillomavirus L2 protein, polyomavirus VP1-3 protein, blue tongue virus VP1-6 protein, and adenovirus capsid proteins.
18. The method of claim 1 , wherein the polypeptide is a papillomavirus protein.
19. The method of claim 18 , wherein the papillomavirus protein is a capsid protein.
20. The method of claim 19 , wherein the capsid protein is selected from the group consisting of L1 and L2.
21. The method of claim 20 , wherein the capsid protein is L1 and wherein the synthetic polynucleotide comprises the sequence set forth in SEQ ID NO:3.
22. The method of claim 20 , wherein the capsid protein is L2 and wherein the synthetic polynucleotide comprises the sequence set forth in SEQ ID NO:7.
23. The method of claim 1 , wherein the mammal is a human.
24. A method of constructing a synthetic polynucleotide, the method comprising:
selecting a first codon of a parent polynucleotide that encodes a polypeptide for replacement with a synonymous codon, wherein the synonymous codon is selected on the basis that it exhibits a lower translational efficiency in an epithelial cell of a mammal than the first codon in a comparison of translational efficiencies of codons in test cells of the same type as the epithelial cell; and
replacing the first codon with the synonymous codon to construct the synthetic polynucleotide.
25. The method of claim 24 , wherein the translational efficiencies of codons in the test cells are compared by:
separately introducing into the test cells individual synthetic constructs, each comprising a regulatory polynucleotide operably linked to a tandem repeat of a codon fused in frame with a reporter polynucleotide that encodes a reporter protein;
comparing expression of the reporter protein in the test cells to determine the relative translational efficiencies of the codons.
26. The method of claim 25 , wherein the synthetic constructs are introduced into the test cells by:
introducing the individual synthetic constructs into progenitors of the test cells; and
culturing the progenitors such that they differentiate to become the test cells.
27. The method of claim 25 , wherein the reporter protein is expressed from the synthetic construct comprising a tandem repeat of the first codon at a level that is at least 110% of the level at which it is expressed from the synthetic construct comprising a tandem repeat of the synonymous codon.
28. The method of claim 25 , wherein the tandem repeat of each of the synthetic constructs comprises at least three copies of the corresponding codon.
29. The method of claim 24 , wherein the translational efficiencies are compared by measuring the abundance of iso-tRNAs corresponding to the codons in the test cells.
30. The method of claim 29 , wherein the synonymous codon corresponds to an iso-tRNA which is in lower abundance in the test cells than the iso-tRNA corresponding to the first codon.
31. The method of claim 29 , wherein the first codon corresponds to an iso-tRNA that is present in the test cells at a level which is at least 110% of the level of the iso-tRNA that corresponds to the synonymous codon.
32. The method of claim 24 , wherein the epithelial cell is an undifferentiated epithelial cell and the comparison of translational efficiencies of the codons is represented by the following table:
Translational
Codon
Efficiency
Ala (GCA)
50.05
Ala (GCC)
46.85
Ala (GCG)
35.45
Ala (GCT)
29.95
Arg (AGA)
31.00
Arg (CGT)
26.95
Arg (CGG)
21.55
Arg (CGA)
16.25
Arg (CGC)
15.30
Arg (AGG)
5.12
Asp (GAC)
21.35
Asp (GAT)
16.70
Cys (TGC)
19.00
Cys (TGT)
5.92
Gln (CAG)
46.65
Gln (CAA)
35.00
Glu (GAA)
16.50
Glu (GAG)
3.86
Gly (GGA)
32.55
Gly (GGC)
22.95
Gly (GGG)
5.70
Gly (GGT)
5.57
His (CAT)
40.30
His (CAC)
34.80
Ile (ATC)
3.48
Ile (ATA)
3.37
Ile (ATT)
3.18
Leu (CTA)
6.43
Leu (CTG)
4.31
Leu (TTA)
3.47
Leu (TTG)
3.45
Leu (CTT)
3.14
Leu (CTC)
2.73
Lys (AAA)
17.60
Lys (AAG)
10.07
Phe (TTC)
4.35
Phe (TTT)
2.67
Pro (CGA)
31.00
Pro (CCG)
21.40
Pro (CCC)
10.48
Pro (CCT)
10.25
Ser (AGC)
62.40
Ser (TCG)
60.00
Ser (TCA)
33.95
Ser (TCT)
33.75
Ser (TCC)
32.90
Ser (AGT)
23.10
Thr (ACT)
40.70
Thr (ACG)
40.10
Thr (ACA)
38.85
Thr (ACC)
3.12
Val (GTA)
7.16
Val (GTC)
7.12
Val (GTG)
5.71
Val (GTT)
5.73.
33. The method of claim 24 , wherein the epithelial cell is an undifferentiated epithelial cell and the first and synonymous codons are selected from the following table:
Synonymous
First Codon
Codon
Ala (GCA)
Ala (GCC)
Ala (GCA)
Ala (GCG)
Ala (GCA)
Ala (GCT)
Ala (GCC)
Ala (GCG)
Ala (GCC)
Ala (GCT)
Ala (GCG)
Ala (GCT)
Arg (AGA)
Arg (CGT)
Arg (AGA)
Arg (CGG)
Arg (AGA)
Arg (CGA)
Arg (AGA)
Arg (CGC)
Arg (AGA)
Arg (AGG)
Arg (CGT)
Arg (CGG)
Arg (CGT)
Arg (CGA)
Arg (CGT)
Arg (CGC)
Arg (CGT)
Arg (AGG)
Arg (CGG)
Arg (CGA)
Arg (CGG)
Arg (CGC)
Arg (CGG)
Arg (AGG)
Arg (CGA)
Arg (CGC)
Arg (CGA)
Arg (AGG)
Arg (CGC)
Arg (AGG)
Asp (GAC)
Asp (GAT)
Cys (TGC)
Cys (TGT)
Gln (CAG)
Gln (CAA)
Glu (GAA)
Glu (GAG)
Gly (GGA)
Gly (GGC)
Gly (GGA)
Gly (GGG)
Gly (GGA)
Gly (GGT)
Gly (GGC)
Gly (GGG)
Gly (GGC)
Gly (GGT)
His (CAT)
His (CAC)
Leu (CTA)
Leu (CTG)
Leu (CTA)
Leu (TTA)
Leu (CTA)
Leu (TTG)
Leu (CTA)
Leu (CTT)
Leu (CTA)
Leu (CTC)
Leu (CTG)
Leu (TTA)
Leu (CTG)
Leu (TTG)
Leu (CTG)
Leu (CTG)
Leu (CTG)
Leu (CTT)
Leu (CTG)
Leu (CTC)
Lys (AAA)
Lys (AAG)
Phe (TTC)
Phe (TTT)
Pro (CGA)
Pro (CCG)
Pro (CGA)
Pro (CCC)
Pro (CGA)
Pro (CCT)
Pro (CCG)
Pro (CCC)
Pro (CCG)
Pro (CCT)
Ser (AGC)
Ser (TCA)
Ser (AGC)
Ser (TCT)
Ser (AGC)
Ser (TCC)
Ser (AGC)
Ser (AGT)
Ser (TCG)
Ser (TCA)
Ser (TCG)
Ser (TCT)
Ser (TCG)
Ser (TCC)
Ser (TCG)
Ser (AGT)
Ser (TCA)
Ser (AGT)
Ser (TCT)
Ser (AGT)
Ser (TCC)
Ser (AUT)
Thr (ACT)
Thr (ACC)
Thr (ACG)
Thr (ACC)
Thr (ACA)
Thr (ACC)
Val (GTA)
Val (GTG)
Val (GTA)
Val (GTT)
Val (GTC)
Val (GTG)
Val (GTC)
Val (GTT).
34. The method of claim 24 , wherein the epithelial cell is an undifferentiated epithelial cell and the first and synonymous codons are selected from the following table:
Synonymous
First Codon
Codon
Arg (AGA)
Arg (AGG)
Arg (CGT)
Arg (AGG)
Arg (CGG)
Arg (AGG)
Arg (CGA)
Arg (AGG)
Arg (CGC)
Arg (AGG)
Cys (TGC)
Cys (TGT)
Glu (GAA)
Glu (GAG)
Gly (GGA)
Gly (GGG)
Gly (GGA)
Gly (GGT)
Gly (GGC)
Gly (GGG)
Gly (GGC)
Gly (GGT)
Leu (CTA)
Leu (CTT)
Leu (CTA)
Leu (CTC)
Leu (CTG)
Leu (CTT)
Leu (CTG)
Leu (CTC)
Pro (CGA)
Pro (CCC)
Pro (CGA)
Pro (CCT)
Pro (CCG)
Pro (CCC)
Pro (CCG)
Pro (CCT)
Ser (AGC)
Ser (AGT)
Ser (TCG)
Ser (AGT)
Thr (ACT)
Thr (ACC)
Thr (ACG)
Thr (ACC)
Thr (ACA)
Thr (ACC).
35. The method of claim 24 , wherein the epithelial cell is an undifferentiated epithelial cell and the first and synonymous codons are selected from the following table:
Synonymous
First Codon
Codon
Arg (AGA)
Arg (AGG)
Arg (CGG)
Arg (AGG)
Cys (TGC)
Cys (TGT)
Gly (GGA)
Gly (GGG)
Gly (GGA)
Gly (GGT)
Gly (GGC)
Gly (GGG)
Gly (GGC)
Gly (GGT)
Pro (CGA)
Pro (CCC)
Pro (CGA)
Pro (CCT)
Pro (CCG)
Pro (CCC)
Pro (CCG)
Pro (CCT)
Thr (ACT)
Thr (ACC)
Thr (ACG)
Thr (ACC)
Thr (ACA)
Thr (ACC).
36. The method of claim 32 , wherein the undifferentiated epithelial cell is an undifferentiated keratinocyte.
37. The method of claim 32 , wherein the undifferentiated epithelial cell is a Cos-1 cell.
38. The method of claim 24 , wherein the mammal is a human.
39. A method of constructing a synthetic polynucleotide, the method comprising:
selecting a first codon of a parent polynucleotide that encodes a polypeptide for replacement with a synonymous codon, wherein the synonymous codon is selected on the basis that it exhibits a higher translational efficiency in a first type of epithelial cell of a mammal than in a second type of epithelial cell of the mammal; and
replacing the first codon with the synonymous codon to construct the synthetic polynucleotide.
40. The method of claim 39 , wherein the first codon and the synonymous codon are selected by:
comparing translational efficiencies of individual codons in cells of the first type and in cells of the second type; and
selecting the synonymous codon such that it has a higher translational efficiency in cells of the first type than in cells of the second type.
41. The method of claim 40 , wherein the first and synonymous codons are selected by:
comparing translational efficiencies of individual codons in cells of the first type and cells of the second type by:
(a) separately introducing into cells of the first type and into cells of the second type individual synthetic constructs, each of which comprises a regulatory polynucleotide operably linked to a tandem repeat of a codon fused in frame with a reporter polynucleotide that encodes a reporter protein; and
(b) comparing expression of the reporter protein in cells of the first and second types to determine the relative translational efficiencies of the codons; and
selecting the synonymous codon such that it has a higher translational efficiency in cells of the first type than in cells of the second type.
42. The method of claim 41 , wherein the synthetic constructs are introduced into cells of the first type and into cells of the second type by:
introducing the individual synthetic constructs into progenitor cells selected from the group consisting of progenitors of cells of the first type and progenitors of cells of the second type; and
culturing the progenitor cells such that they differentiate to become cells of the corresponding type.
43. The method of claim 41 , wherein the reporter protein is expressed from the synthetic construct comprising a tandem repeat of the synonymous codon in cells of the first type at a level that is at least 110% of the level at which it is expressed from the same synthetic construct in cells of the second type.
44. The method of claim 41 , wherein the tandem repeat of each of the synthetic constructs comprises at least three copies of the corresponding codon.
45. The method of claim 40 , wherein the translational efficiencies of individual codons are compared by measuring the abundance of the iso-tRNAs corresponding to the individual codons in cells of the first and second types.
46. The method of claim 45 , wherein the synonymous codon corresponds to an iso-tRNA which is more abundant cells of the first type than in cells of the second type.
47. The method of claim 45 , wherein the synonymous codon corresponds to an iso-tRNA that is present in cells of the first type at a level which is at least 110% of the level of the iso-tRNA that is present in cells of the second type.
48. The method of claim 39 , wherein the synonymous codon is selected from the group consisting of (1) a codon used at relatively high frequency by genes of cells of the first type, (2) a codon used at relatively high frequency by genes of the mammal, (3) a codon used at relatively low frequency by genes of cells of the second type, and (4) a codon used at relatively low frequency by genes of an organism other than the mammal.
49. The method of claim 39 , wherein the first codon is selected from the group consisting of (a) a codon used at relatively high frequency by genes of cells of the second type, (b) a codon used at relatively low frequency by genes of cells of the first type, (c) a codon used at relatively low frequency by genes of the mammal, and (d) a codon used at relatively high frequency by genes of an organism other than the mammal.
50. The method of claim 39 , wherein the first and synonymous codons are selected such that the polypeptide is expressed from the synthetic polynucleotide in cells of the first type at a level which is at least 110% of the level at which the polypeptide is expressed from the parent polynucleotide in cells of the second type.
51. The method of claim 39 , wherein the second type of epithelial cell is a precursor of the first type of epithelial cell.
52. The method of claim 39 , wherein the second type of epithelial cell is derived from the first type of epithelial cell.
53. The method of claim 39 , wherein the polypeptide is not substantially expressible in the second type of epithelial cell.
54. The method of claim 39 , wherein the first and second types of epithelial cells are at different stages of differentiation.
55. The method of claim 54 , wherein the first type of epithelial cell is undifferentiated and the second type of epithelial cell is differentiated.
56. The method of claim 55 , wherein the first type of epithelial cell is an undifferentiated keratinocyte and the second type of epithelial cell is a differentiated keratinocyte.
57. The method of claim 39 , wherein the polypeptide is a viral capsid protein.
58. The method of claim 57 , wherein the viral capsid protein is selected from the group consisting of papillomavirus L1 protein, papilloma virus L2 protein, polyomavirus VP1-3 protein, blue tongue virus VP1-6 protein, and an adenovirus capsid protein.
59. The method of claim 39 , wherein the polypeptide is a papillomavirus protein.
60. The method of claim 59 , wherein the papillomavirus protein is a capsid protein.
61. The method of claim 60 , wherein the capsid protein is selected from the group consisting of L1 and L2.
62. The method of claim 60 , wherein the capsid protein is L1 and wherein the synthetic polynucleotide comprises the sequence set forth in SEQ ID NO:3.
63. The method of claim 60 , wherein the capsid protein is L2 and wherein the synthetic polynucleotide comprises the sequence set forth in SEQ ID NO:7.
64. The method of claim 39 , wherein the first and second types of epithelial cells are at different stages of the cell cycle.
65. The method of claim 39 , wherein the mammal is a human.
66. A method of constructing a synthetic polynucleotide, the method comprising:
selecting a first codon of a parent polynucleotide that encodes a polypeptide for replacement with a synonymous codon, wherein the synonymous codon is selected on the basis that it exhibits either a higher translational efficiency or a lower translational efficiency in a single type of cells of a mammal than in a single type of cells of another organism; and
replacing the first codon with the synonymous codon to construct the synthetic polynucleotide.