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Part:BBa_K3962355:Design

Designed by: Siheng Li   Group: iGEM21_Leiden   (2021-10-13)
Revision as of 11:36, 13 October 2021 by Registry (Talk | contribs)

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Constant expression of antitoxin CcdA by constitutive promoter J23117


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

BBa_J23117 has relatively low expression avoiding metabolic burden. We optimized the codon for the expression of the gene in E. coli. We used a PCR-based cloning method to clone the construct. This was done by fusing the sequence for the promoter to the forward primer and having an overlap with the coding sequence of CcdAin the primer. The total length of the forward primer was 85 bp. For the reverse primer, a simple 22 bp primer was used that has homology to the right flank of the biobrick. For the PCR a touch-down programme was used to get a higher degree of annealing specificity due to the long length of the primers.



Source

All sequences of subparts are from iGEM registry.


References