Composite

Part:BBa_K3726011:Design

Designed by: Santiago Barrag�n Ariza   Group: iGEM21_MADRID_UCM   (2021-10-18)

CDS_Lv0_BOH_3


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 461
    Illegal PstI site found at 366
    Illegal PstI site found at 614
    Illegal PstI site found at 1915
    Illegal PstI site found at 4930
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 461
    Illegal NheI site found at 3936
    Illegal NheI site found at 4164
    Illegal PstI site found at 366
    Illegal PstI site found at 614
    Illegal PstI site found at 1915
    Illegal PstI site found at 4930
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 461
    Illegal BglII site found at 5275
    Illegal BglII site found at 5392
    Illegal BamHI site found at 1998
    Illegal BamHI site found at 2337
    Illegal XhoI site found at 3270
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 461
    Illegal PstI site found at 366
    Illegal PstI site found at 614
    Illegal PstI site found at 1915
    Illegal PstI site found at 4930
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 461
    Illegal PstI site found at 366
    Illegal PstI site found at 614
    Illegal PstI site found at 1915
    Illegal PstI site found at 4930
    Illegal NgoMIV site found at 105
    Illegal NgoMIV site found at 205
    Illegal NgoMIV site found at 511
    Illegal NgoMIV site found at 2593
    Illegal AgeI site found at 883
    Illegal AgeI site found at 2121
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

In the upstream region the bases taatc corresponds with 5bp a spacer sequence to improve ribosomal translation, while AATG overhang creates an start codon that forces translation initiation of the downstream coding sequence.

In the downstream region, the extra “aa” bases allow the creation of an additional stop codon (TAA) at the end of the upstream coding sequence, ending the translation of the desired CDS in accordance with the Marburg Collection design guidelines.

The transcription initiation rate for each RBS element has been specifically adjusted to achieve optimal expression of the desired enzyme within the operon. The relative translation initiation rates for the operon can be observed in the picture below. Where this operon corresponds with the enzymes: CDS_NphT7, CDS_PhaB^173S , CDS_Ccr, CDS_PhaJ , CDS_PduP , CDS_Slr1192.

800px-T--MADRID_UCM--RBSBOH3.png


This polycistronic CDS sequence has been assembled following a modified procedure for golden gate domestication. To know more about the design process of this polycistronic lv.0 parts, visit the iGEM MADRID_UCM 2021 wiki page: https://2021.igem.org/Team:MADRID_UCM/Design .

Source

Gibson assembly composed by this synthesized basic parts: "BBa_K3726000" "BBa_K3726001" "BBa_K3726002" "BBa_K3726003" "BBa_K3726004" "BBa_K3726005" "BBa_K3726006" "BBa_K3726007" "BBa_K3726008" "BBa_K3726009" "BBa_K3726010"

References

X. Liu, R. Miao, P. Lindberg and P. Lindblad, "Modular engineering for efficient photosynthetic biosynthesis of 1-butanol from CO2in cyanobacteria", 2021.