atggcgggcctgctgaaagaaagcatgcgcattaaaatgtatatggaaggcaccgtgaac
ggccattattttaaatgcgaaggcgaaggcgatggcaacccgtttgcgggcacccagagc
atgcgcattcatgtgaccgaaggcgcgccgctgccgtttgcgtttgatattctggcgccg
tgctgccattatggcagcaaaacctttgtgcatcataccgcggaaattccggatttttgg
aaacagagctttccggaaggctttacctgggaacgcaccaccacctatgaagatggcggc
attctgaccgcgcatcaggataccagcctggaaggcaactgcctgatttataaagtgaaa
gtgcatggcaccaactttccggcggatggcccggtgatgaaaaacaaaagcggcggctgg
gaaccgattaccgaagtggtgtatccggaaaacggcgtgctgtgcggccgcgcggtgatg
gcgctgaaagtgggcgatcgccatctgatttgccatgcgtataccagctatcgcagcaaa
aaagcggtgcgcgcgctgaccatgccgggctttcattttaccgattatcgcctgcagatg
ctgcgcaaaaaaaaagatgaatattttgaactgtatgaagcgagcgtggcgcgctatagc
gatctgccggaaaaagcgaactaa

Results:

Note: This sequence corresponds to the most likely codons according to the default table (E. coli).

3.3. Codon Optimization

Prompt

Explain why you need to optimize codon usage. For which organism did you choose to optimize the sequence and why?

Answer

Why Optimize Codons?

Although the genetic code is universal, codon usage frequencies differ between organisms. Codon optimization improves translation efficiency and protein expression in the chosen host. It also helps control:

• Codon frequency (adaptation to the host)
• GC content (affects stability and transcription)
• Undesired mRNA secondary structures
• Internal restriction sites
• Repetitive sequences that complicate DNA synthesis

Organism Chosen for Optimization

For this exercise I used the default orientation toward E. coli (a practical choice for bacterial expression). Tools such as IDT provide organism-specific codon optimization options (e.g., E. coli, S. cerevisiae, Drosophila melanogaster, Mus musculus).

Example Optimized Sequence (Formatted in Codons)

Avoiding Specific Restriction Sites (IDT Optimization Settings)

Using the IDT Codon Optimization tool, it is also possible to exclude specific restriction enzyme recognition sites from the DNA sequence. This is particularly useful when designing constructs for cloning, since it prevents unwanted internal cutting within the gene of interest.

For this design, the following restriction sites were explicitly avoided:

• EcoRI
• BamHI
• SacII
• SalI
• HindIII
• EcoRV

By removing these internal restriction sites during optimization, the sequence becomes compatible with vectors that use these enzymes in their multiple cloning site (MCS), facilitating downstream cloning and assembly steps.

ATG GCA GGA TTG TTA AAA GAG TCA ATG CGT ATA AAA ATG TAT ATG GAA GGG
ACA GTC AAT GGT CAT TAT TTC AAA TGC GAA GGC GAA GGC GAT GGA AAC CCG
TTT GCG GGC ACC CAG TCC ATG CGT ATT CAT GTG ACC GAG GGC GCT CCC CTG
CCA TTT GCG TTT GAC ATC CTT GCG CCG TGT TGT CAT TAC GGT TCA AAG ACA
TTC GTC CAC CAT ACT GCA GAA ATT CCG GAT TTT TGG AAG CAG TCA TTT CCA
GAA GGT TTC ACG TGG GAA CGG ACA ACT ACT TAT GAA GAT GGC GGC ATT CTG
ACA GCC CAT CAA GAT ACA TCA TTA GAA GGC AAC TGT CTT ATA TAT AAG GTT
AAG GTC CAC GGG ACC AAT TTT CCT GCT GAC GGA CCA GTV ATG AAG AAT AAG
TCC GGC GGT TGG GAA CCT ATC ACC GAA GTC GTG TAC CCT GAA AAT GGA GTG
CTG TGT GGC CGC GCC GTT ATG GCT TTA AAA GTC GGG GAT CGT CAC CTT ATT
TGC CAT GCC TAC ACC AGC TAC CGC AGT AAA AAA GCG GTG CGT GCA TTA ACT
ATG CCT GGC TTT CAT TTC ACG GAC TAC CGT CTG CAA ATG CTG AGA AAA AAG
AAA GAT GAA TAC TTT GAA CTT TAC GAA GCC AGC GTA GCC AGA TAT TCA GAT
CTG CCT GAA AAG GCC AAC 

Detected Restriction Sites (Examples to Avoid or Consider)

• BglII — AGATCT
• BspEI — TCCGGA
• DraI — TTTAAA
• MlyI — GAGTC
• PmlI — CACGTG
• PstI — CTGCAG

3.4. You Have a Sequence — Now What?

Prompt

Which technologies could be used to produce this protein from your DNA? Describe how the DNA sequence can be transcribed and translated into your protein (cell-dependent and/or cell-free methods).

Answer

Cell-Dependent Methods

Expression in Bacteria (e.g., E. coli)

1. Clone the optimized sequence into an expression plasmid (strong promoter, RBS, terminator).
2. Transform the plasmid into competent E. coli.
3. Induce expression (e.g., IPTG in lac-based systems).
4. The cell transcribes DNA → mRNA and translates mRNA → protein.
5. After expression, the protein can be purified (e.g., chromatography).

Other possible systems include yeast (S. cerevisiae) or mammalian cells if post-translational modifications are required.