Composite

Part:BBa_K5181015:Design

Designed by: Mayukh Mitra   Group: iGEM24_IISER-TVM   (2024-09-04)


tet_RBS_P450-CPR_fusion protein (BC_linker)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 636
    Illegal PstI site found at 972
    Illegal PstI site found at 1650
    Illegal PstI site found at 3441
    Illegal PstI site found at 3720
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1125
    Illegal PstI site found at 972
    Illegal PstI site found at 1650
    Illegal PstI site found at 3441
    Illegal PstI site found at 3720
    Illegal NotI site found at 7
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 730
    Illegal BglII site found at 3104
    Illegal XhoI site found at 2061
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 636
    Illegal PstI site found at 972
    Illegal PstI site found at 1650
    Illegal PstI site found at 3441
    Illegal PstI site found at 3720
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 636
    Illegal PstI site found at 972
    Illegal PstI site found at 1650
    Illegal PstI site found at 3441
    Illegal PstI site found at 3720
    Illegal NgoMIV site found at 1101
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This is a bicistronic part that is codon optimized for Pseudomonas putida. The final part is assembled by Gibson assembly. The insert was PCR amplified using primers.

Forward primer: 5’ CGC CTA GGC CGC GGC CGC GCG AAT TCT TAG TGA TG 3’

Reverse primer: 5’ TTT TCC CAG TCA CGA CGC GGC CGC AAG CTT TTA AGA CCC 3’

The part is designed so that the tet promoter with RBS can be replaced with an araBAD promoter with RBS by using suitable primers and further testing for results.

Primers for promoter replacement:

Forward primer: 5’ ATATGCATATATCTCCTTCTTAAAAGATC 3’

Reverse primer: 5’ CGCAAGCTTTTATGACAAC 3’

Source

later

References