Translational_Unit

Part:BBa_K4249003:Design

Designed by: Wenxin Wang   Group: iGEM22_HBUT-China   (2022-10-11)


The expression cassette carrying an TEF1 promoter, CpEgt2 coding sequence and an CYC1 terminator


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1497
    Illegal XbaI site found at 425
    Illegal XbaI site found at 843
    Illegal XbaI site found at 1188
    Illegal XbaI site found at 1476
    Illegal SpeI site found at 419
    Illegal PstI site found at 1219
    Illegal PstI site found at 2033
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1497
    Illegal SpeI site found at 419
    Illegal PstI site found at 1219
    Illegal PstI site found at 2033
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1497
    Illegal BglII site found at 840
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1497
    Illegal XbaI site found at 425
    Illegal XbaI site found at 843
    Illegal XbaI site found at 1188
    Illegal XbaI site found at 1476
    Illegal SpeI site found at 419
    Illegal PstI site found at 1219
    Illegal PstI site found at 2033
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1497
    Illegal XbaI site found at 425
    Illegal XbaI site found at 843
    Illegal XbaI site found at 1188
    Illegal XbaI site found at 1476
    Illegal SpeI site found at 419
    Illegal PstI site found at 1219
    Illegal PstI site found at 2033
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 168


Design Notes

During the experiment, we found that some regions of the promoter sequence templates are rich in GC residues, which tend to fold into complex secondary structures and might not melt during the annealing phase of the PCR cycle. Also, the primers used to amplify GC-rich regions often have a high capacity to form self- and cross-dimers and a strong tendency to fold into stem-loop structures that can impede the progress of the DNA polymerase along the template molecule. Therefore, instead of PCR method, we obtained the gene expression cassettes by enzyme digestion.


Source

The complete coding sequence was amplified by primer pairs, and the obtained fragment was inserted into the plasmid pTEF2, which was double digested by BamHI and EcoRI. Then, the complete gene expression cassette was gained by enzyme digestion.


References