Composite

Part:BBa_K3726012:Design

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

CDS_Lv0_BOH_1_A


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
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 461
    Illegal PstI site found at 366
    Illegal PstI site found at 614
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 461
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 461
    Illegal PstI site found at 366
    Illegal PstI site found at 614
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 461
    Illegal PstI site found at 366
    Illegal PstI site found at 614
    Illegal NgoMIV site found at 105
    Illegal NgoMIV site found at 205
    Illegal NgoMIV site found at 511
    Illegal AgeI site found at 883
  • 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.

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

Firstly we made two PCR domestication with specific primers and CDS_LV0_BOH_3 ("BBa_K3726011") as template obtaining as products two sequences that afterwards were linked with Golden Gate. That sequences were able to be attached by golden gate because they had few complementary nucleotide as overhangs. Between "BBa_K3726000"and "BBa_K3726001" we added this RBS: (RBS*) "BBa_K3726093" . Additionally, we used 4 nucleotides (AACG) as overhang to link both CDS ("BBa_K3726000" "BBa_K3726001")

References

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