Difference between revisions of "Part:BBa K4201015:Design"

 
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===Design Notes===
 
===Design Notes===
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Notably, the backbone contains a left border overdrive sequence (nt 1-62) which greatly enhances cleavage during transformation. Moreover, the psV1 replicon 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.
 
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Notably, the backbone contains a left border overdrive sequence (nt 1-62) which greatly enhances cleavage during transformation<sup>4</sup>. 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.
  
  
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===References===
 
===References===
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1. Lee, S. New Binary Ti Vectors with Co-directional Replicons for Agrobacterium Tumefaciens-mediated Transformation of Higher Plants. 149. <br> 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).<br>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). <br> 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).

Revision as of 05:02, 12 October 2022


Kan BsaI


Assembly Compatibility:
  • 10
    INCOMPATIBLE 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
  • 12
    INCOMPATIBLE 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
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 354
    Illegal BamHI site found at 333
  • 23
    INCOMPATIBLE 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
  • 25
    INCOMPATIBLE 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
  • 1000
    INCOMPATIBLE 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. 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.


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).