Part:BBa_K3726013:Design
CDS_Lv0_BOH_1_B
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 85
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 85
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 168
Illegal BamHI site found at 507
Illegal XhoI site found at 1416 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 85
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 85
Illegal NgoMIV site found at 763
Illegal AgeI site found at 291 - 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.
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_K3726002"and "BBa_K3726003" we added this RBS: (RBS*) "BBa_K3726093" . Additionally, we used 4 nucleotides (AACG) as overhang to link both CDS ("BBa_K3726002" "BBa_K3726003")
References
X. Liu, R. Miao, P. Lindberg and P. Lindblad, "Modular engineering for efficient photosynthetic biosynthesis of 1-butanol from CO2in cyanobacteria", 2021.