![](https://parts.igem.org/images/partbypart/icon_plasmid_backbone.png)
Part:BBa_K4201015:Design
Kan BsaI
- 10INCOMPATIBLE WITH RFC[10]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 354
Illegal XbaI site found at 327
Illegal PstI site found at 315 - 12INCOMPATIBLE WITH RFC[12]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 354
Illegal NheI site found at 804
Illegal PstI site found at 315
Illegal NotI site found at 849
Illegal NotI site found at 2139 - 21INCOMPATIBLE WITH RFC[21]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 354
Illegal BamHI site found at 333 - 23INCOMPATIBLE WITH RFC[23]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 354
Illegal XbaI site found at 327
Illegal PstI site found at 315 - 25INCOMPATIBLE WITH RFC[25]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 354
Illegal XbaI site found at 327
Illegal PstI site found at 315
Illegal NgoMIV site found at 1747
Illegal NgoMIV site found at 1892
Illegal NgoMIV site found at 2103
Illegal NgoMIV site found at 2325
Illegal NgoMIV site found at 2571
Illegal NgoMIV site found at 2660
Illegal NgoMIV site found at 2800
Illegal AgeI site found at 1054 - 1000INCOMPATIBLE WITH RFC[1000]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal BsaI site found at 750
Illegal BsaI.rc site found at 68
Illegal SapI site found at 922
Design Notes
Notably, the backbone contains a left border overdrive sequence (nt 1-62) which greatly enhances cleavage during transformation4. It contains an E. coli origin of replication (nt 3584-4162) which enables plasmid replication in our model organism5. The Tet resistance gene was replaced with a NPTIII kanamycin resistance gene to make transformation easier, via PCR amplification and ligation. The blue/white selection marker (nt 67-756) is flanked by BsaI cut sites on either side, allowing for blue white selection of transformed colonies.
This backbone was designed for transformation in NEB 10-beta E. coli and GoldBio’s EHA105 Agrobacterium.
Source
This plasmid was made with a pLSUK vector. B/W selection is from pUC19 and kanamycin resistance is conveyed through a NPTIII gene.
References
1. Lee, S. New Binary Ti Vectors with Co-directional Replicons for Agrobacterium Tumefaciens-mediated Transformation of Higher Plants. 149.
2. Heeb, S. et al. Small, Stable Shuttle Vectors Based on the Minimal pVS1 Replicon for Use in Gram-Negative, Plant-Associated Bacteria. Mol. Plant-Microbe Interactions® 13, 232–237 (2000).
3. Yanisch-Perron, C., Vieira, J. & Messing, J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33, 103–119 (1985).
4. Toro, N., Datta, A., Yanofsky, M. & Nester, E. Role of the overdrive sequence in T-DNA border cleavage in Agrobacterium. Proc. Natl. Acad. Sci. U. S. A. 85, 8558–8562 (1988).
5. Hershfield, V., Boyer, H. W., Yanofsky, C., Lovett, M. A. & Helinski, D. R. Plasmid ColE1 as a Molecular Vehicle for Cloning and Amplification of DNA. Proc. Natl. Acad. Sci. U. S. A. 71, 3455–3459 (1974).