Part:BBa_K5103000:Design
Cry8Da with PCG004 unique BsaI cut sites
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 993
Illegal EcoRI site found at 1072
Illegal EcoRI site found at 2484
Illegal EcoRI site found at 3147
Illegal PstI site found at 124
Illegal PstI site found at 1565 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 993
Illegal EcoRI site found at 1072
Illegal EcoRI site found at 2484
Illegal EcoRI site found at 3147
Illegal PstI site found at 124
Illegal PstI site found at 1565 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 993
Illegal EcoRI site found at 1072
Illegal EcoRI site found at 2484
Illegal EcoRI site found at 3147
Illegal XhoI site found at 3455 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 993
Illegal EcoRI site found at 1072
Illegal EcoRI site found at 2484
Illegal EcoRI site found at 3147
Illegal PstI site found at 124
Illegal PstI site found at 1565 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 993
Illegal EcoRI site found at 1072
Illegal EcoRI site found at 2484
Illegal EcoRI site found at 3147
Illegal PstI site found at 124
Illegal PstI site found at 1565
Illegal AgeI site found at 1339
Illegal AgeI site found at 2221 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 3569
Illegal BsaI.rc site found at 7
Design Notes
BsaI is a type IIS digestion enzyme, which creates overhangs[1], so we must ensure that the sequences where the enzyme cuts on Cry8Da and PCG004 (refer to Part:BBa_K5103005) are complementary to each other. After designing and incorporating the cut sites with the Cry8Da[2] sequence, this fragment was ordered through IDT.
Source
The sequence of Cry8Da can be found in the NCBI GenBank under accession number KF148683.[3] The Cry8Da gene originates from the bacterium Bacillus thuringiensis (Bt), which is well known for producing Cry proteins.[4]
References
[1] New England Biolabs (2020). NEB.com. https://www.neb.com/en/products/r0535-bsai
[2] Gilbert, C., Howarth, M., Harwood, C. R., & Ellis, T. (2017). Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis. ACS synthetic biology, 6(6), 957–967. https://doi.org/10.1021/acssynbio.6b00292
[3] Xie, B., & Liu, Y. (2013). Cry8Da [Bacillus thuringiensis]. NCBI. https://www.ncbi.nlm.nih.gov/protein/543174970
[4] Shu, C., Yan, G., & Soberón, M. (2020). Characterization of two novel Bacillus thuringiensis Cry8 toxins reveals differential specificity of protoxins or activated toxins against Chrysomeloidea coleopteran superfamily. Toxins, 12(10), 642. https://doi.org/10.3390/toxins12100642