DNA

Part:BBa_K4943005:Design

Designed by: Neha Suresh   Group: iGEM23_NCSU   (2023-10-11)


GEM-1:Plasmid for codon optimized Cas9


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 26
    Illegal XbaI site found at 657
    Illegal XbaI site found at 2331
    Illegal XbaI site found at 3860
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 3645
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3513
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 26
    Illegal XbaI site found at 657
    Illegal XbaI site found at 2331
    Illegal XbaI site found at 3860
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 26
    Illegal XbaI site found at 657
    Illegal XbaI site found at 2331
    Illegal XbaI site found at 3860
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Design Considerations: a. Codon usage - The codon usage of the native cas9 gene was analyzed and altered to match the preferred codons of b. Clostridium. This helps ensure efficient translation. b. GC content - The GC content was adjusted to fit the typical range of Clostridium genes. Extreme GC content can negatively impact transcription and translation. c. Restriction sites - Common restriction enzyme sites were avoided in the optimized sequence to facilitate future cloning efforts. d. Repeats - Long repeats and inverted repeats were minimized to avoid unwanted secondary structures in the mRNA. e. RBS - An optimal ribosome binding site was included upstream of the start codon to promote translation initiation. f. mRNA structure - Potential mRNA secondary structures that could inhibit translation were analyzed and disrupted. g. Toxic sequences - Known toxic motifs like endonuclease recognition sites were removed. Sequence continuity - The optimized sequence was designed to keep overall continuity relative to the native gene. h. Synthesis - The optimized sequence parameters were set to enable successful gene synthesis by GenScript.


Source

This codon optimized cas9 gene was synthesized based on the native cas9 sequence from Streptococcus pyogenes. The DNA sequence was optimized to match Clostridium codon after extensive literature review and using a Genscript algorithm. Codon optimization helps improve expression in the target host by replacing rare codons and removing detrimental sequences like internal promoters. We hope that this will enable The synthesized gene was cloned into a vector backbone provided by GenScript


References