Composite

Part:BBa_K3726038:Design

Designed by: Santiago Barrag�n Ariza   Group: iGEM21_MADRID_UCM   (2021-10-19)

CDS_Lv0_MCGII


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1099
    Illegal EcoRI site found at 1540
    Illegal EcoRI site found at 3299
    Illegal EcoRI site found at 4415
    Illegal EcoRI site found at 4499
    Illegal EcoRI site found at 7554
    Illegal PstI site found at 1684
    Illegal PstI site found at 1783
    Illegal PstI site found at 2368
    Illegal PstI site found at 3326
    Illegal PstI site found at 3554
    Illegal PstI site found at 5064
    Illegal PstI site found at 6078
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1099
    Illegal EcoRI site found at 1540
    Illegal EcoRI site found at 3299
    Illegal EcoRI site found at 4415
    Illegal EcoRI site found at 4499
    Illegal EcoRI site found at 7554
    Illegal NheI site found at 4638
    Illegal PstI site found at 1684
    Illegal PstI site found at 1783
    Illegal PstI site found at 2368
    Illegal PstI site found at 3326
    Illegal PstI site found at 3554
    Illegal PstI site found at 5064
    Illegal PstI site found at 6078
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1099
    Illegal EcoRI site found at 1540
    Illegal EcoRI site found at 3299
    Illegal EcoRI site found at 4415
    Illegal EcoRI site found at 4499
    Illegal EcoRI site found at 7554
    Illegal BglII site found at 41
    Illegal BglII site found at 2081
    Illegal BglII site found at 3445
    Illegal BamHI site found at 2179
    Illegal BamHI site found at 2194
    Illegal BamHI site found at 5893
    Illegal BamHI site found at 6351
    Illegal BamHI site found at 7324
    Illegal XhoI site found at 871
    Illegal XhoI site found at 1516
    Illegal XhoI site found at 1990
    Illegal XhoI site found at 4520
    Illegal XhoI site found at 5187
    Illegal XhoI site found at 5320
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1099
    Illegal EcoRI site found at 1540
    Illegal EcoRI site found at 3299
    Illegal EcoRI site found at 4415
    Illegal EcoRI site found at 4499
    Illegal EcoRI site found at 7554
    Illegal PstI site found at 1684
    Illegal PstI site found at 1783
    Illegal PstI site found at 2368
    Illegal PstI site found at 3326
    Illegal PstI site found at 3554
    Illegal PstI site found at 5064
    Illegal PstI site found at 6078
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1099
    Illegal EcoRI site found at 1540
    Illegal EcoRI site found at 3299
    Illegal EcoRI site found at 4415
    Illegal EcoRI site found at 4499
    Illegal EcoRI site found at 7554
    Illegal PstI site found at 1684
    Illegal PstI site found at 1783
    Illegal PstI site found at 2368
    Illegal PstI site found at 3326
    Illegal PstI site found at 3554
    Illegal PstI site found at 5064
    Illegal PstI site found at 6078
    Illegal NgoMIV site found at 1627
    Illegal NgoMIV site found at 3016
    Illegal NgoMIV site found at 3190
    Illegal NgoMIV site found at 3340
    Illegal NgoMIV site found at 6514
    Illegal AgeI site found at 3641
  • 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: ppc_CDS, gcl_CDS , CDS_mtkA , CDS_mtkB , CDS_mcl.

800px-T--MADRID_UCM--RBSMCGII.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 using this synthesized basic parts: "BBa_K3726029""BBa_K3726030" "BBa_K3726031""BBa_K3726032""BBa_K3726033"" BBa_K3726034""BBa_K3726035""BBa_K3726036""BBa_K3726037"

References

H. Yu, X. Li, F. Duchoud, D. Chuang and J. Liao, "Augmenting the Calvin–Benson–Bassham cycle by a synthetic malyl-CoA-glycerate carbon fixation pathway", 2021.