Part:BBa_K3726015:Design
CDS_Lv0_BOH_2_A
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 461
Illegal PstI site found at 366
Illegal PstI site found at 614
Illegal PstI site found at 1915 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 461
Illegal BamHI site found at 1998
Illegal BamHI site found at 2337
Illegal XhoI site found at 3270 - 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 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 - 1000COMPATIBLE 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.
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 thay had few complementary nucleotides as overhangs.
References
X. Liu, R. Miao, P. Lindberg and P. Lindblad, "Modular engineering for efficient photosynthetic biosynthesis of 1-butanol from CO2in cyanobacteria", 2021.