Part:BBa_K4201014:Design

Chlor AarI

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 320
  Illegal XbaI site found at 293
  Illegal PstI site found at 281
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 320
  Illegal PstI site found at 281
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 320
  Illegal BamHI site found at 299
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 320
  Illegal XbaI site found at 293
  Illegal PstI site found at 281
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 320
  Illegal XbaI site found at 293
  Illegal PstI site found at 281
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 14
  Illegal BsaI.rc site found at 736
  

Design Notes

Our Chlor backbone uses a pPTK-PARS vector as a base, which enables chloramphenicol resistance in bacteria¹. It contains an E. coli origin of replication (nt 1684-2272) which enables plasmid replication in our model organism². However, specific modifications have been made to maximize efficiency for Golden Gate synthesis and transformation. The pPTK-PARS vector was shortened and a blue-white selection marker from pUC19³ (nt 36-711) was inserted, along with flanking PaqCI cut sites (nt 25-39, 716-730). BsaI cut sites (nt 14-24, 731-741) flank the PaqCI cut sites, allowing for sequential insertion of genes using Golden Gate Synthesis. 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

Sources

This part was ordered for de novo gene synthesis from Twist Bioscience, and is based on the pPTK021-7-PARS cloning vector, using a pUC19 blue/white selection sequence.

References

1. Obst, U., Lu, T. K. & Sieber, V. A Modular Toolkit for Generating Pichia pastoris Secretion Libraries. ACS Synth. Biol. 6, 1016–1025 (2017).
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).
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).