Difference between revisions of "Part:BBa K4201013:Design"
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===Design Notes===
 
===Design Notes===
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Our Amp backbone uses the pUC191<sup>1</sup> vector, a commonly used <i>E. Coli</i> cloning vector, as a base. It contains an E. coli origin of replication (nt 1954-2542) which enables plasmid replication in <i>E. coli</i>. However, specific modifications have been made to maximize efficiency for Golden Gate synthesis and transformation. The pUC19 vector was shortened and a blue-white selection marker from pUC19 (nt 24-699) was inserted, along with flanking BsaI cut sites (nt 12-22, 701-711). Insertions were completed via PCR and ligation.  
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Our Amp backbone uses the pUC191<sup>1</sup> vector, a commonly used <i>E. Coli</i> cloning vector, as a base. It contains an E. coli origin of replication (nt 1954-2542) which enables plasmid replication in <i>E. coli</i><sup>2</sup>. However, specific modifications have been made to maximize efficiency for Golden Gate synthesis and transformation. The pUC19 vector was shortened and a blue-white selection marker from pUC19 (nt 24-699) was inserted, along with flanking BsaI cut sites (nt 12-22, 701-711). Insertions were completed via PCR and ligation.  
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Plasmids were transformed via electroporation or chemical transformation into NEB 10-beta competent cells. Transformation in cells was confirmed following sequencing of extracted DNA by Plasmidasaurus.
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Plasmids were transformed via electroporation or chemical transformation into NEB 10-beta competent cells. Transformation in cells was confirmed following sequencing of extracted DNA by Plasmidasaurus.
  
 
===Source===
 
===Source===
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===References===
 
===References===
  
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1. 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).
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1. 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>
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2. 2. 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).

Latest revision as of 05:44, 12 October 2022


Amp BsaI


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 304
    Illegal XbaI site found at 277
    Illegal PstI site found at 265
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 304
    Illegal PstI site found at 265
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 304
    Illegal BamHI site found at 283
    Illegal XhoI site found at 1641
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 304
    Illegal XbaI site found at 277
    Illegal PstI site found at 265
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 304
    Illegal XbaI site found at 277
    Illegal PstI site found at 265
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 701
    Illegal BsaI.rc site found at 17


Design Notes

Our Amp backbone uses the pUC1911 vector, a commonly used E. Coli cloning vector, as a base. It contains an E. coli origin of replication (nt 1954-2542) which enables plasmid replication in E. coli2. However, specific modifications have been made to maximize efficiency for Golden Gate synthesis and transformation. The pUC19 vector was shortened and a blue-white selection marker from pUC19 (nt 24-699) was inserted, along with flanking BsaI cut sites (nt 12-22, 701-711). Insertions were completed via PCR and ligation.

Plasmids were transformed via electroporation or chemical transformation into NEB 10-beta competent cells. Transformation in cells was confirmed following sequencing of extracted DNA by Plasmidasaurus.

Source

This part was ordered from Twist Bioscience, and is based on the pUC19 cloning vector.


References

1. 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).
2. 2. 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).