Part:BBa_K678070

p68

p68 is a plasmid that can be used to assay promoter activity in Aspergillus nidulans. It contains the lacZ gene, the TtrpC terminator, a USER cassette, an argB marker cassette for selection, an ori for propagation in E. coli, and an ampR resistance cassette. It also contains up- and down stream regions for targeting to a specific site in the chromosome called IS1 situated 202 bp downstream of AN6638 and 245 bp upstream of AN6639. For homologous recombination to occur gene-targeting substrates have to contain these large homologous sequences around 1500 bp to ensure the targeted integration (1).

To insert a promoter by USER cloning p68 should be digested with AsiSI 2-8 hours and afterwards nicked with Nb.BstI for 1-3 hours. Instructions on how to perform the USER cloning and transformation of A. nidulans can be found [http://2011.igem.org/Team:DTU-Denmark-2/Team/Protocols#USER%20cloning here].

DTU-Denmark-2 2011 Plasmid map of p68. After digestion of the USER cassette a promoter of interest can be inserted.

Usage and Biology

This plasmid was successfully used to characterize the A. nidulans promoters PalcA and DMKP-P6. Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 6337
  Illegal EcoRI site found at 11000
  Illegal XbaI site found at 1257
  Illegal XbaI site found at 7126
  Illegal XbaI site found at 11325
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 6337
  Illegal EcoRI site found at 11000
  Illegal NotI site found at 1296
  Illegal NotI site found at 11282
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 6337
  Illegal EcoRI site found at 11000
  Illegal BglII site found at 3225
  Illegal BglII site found at 8299
  Illegal BglII site found at 10490
  Illegal BamHI site found at 6407
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 6337
  Illegal EcoRI site found at 11000
  Illegal XbaI site found at 1257
  Illegal XbaI site found at 7126
  Illegal XbaI site found at 11325
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 6337
  Illegal EcoRI site found at 11000
  Illegal XbaI site found at 1257
  Illegal XbaI site found at 7126
  Illegal XbaI site found at 11325
  Illegal NgoMIV site found at 7043
  Illegal AgeI site found at 8847
  Illegal AgeI site found at 8877
  Illegal AgeI site found at 10254
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2122
  Illegal BsaI site found at 8680
  Illegal BsaI site found at 9852
  Illegal BsaI site found at 10446
  Illegal BsaI site found at 10642
  Illegal BsaI site found at 10813
  Illegal BsaI.rc site found at 3137
  Illegal BsaI.rc site found at 6757
  Illegal SapI.rc site found at 2793
  

References

(1) Hansen, Bjarne G.; Bo Salomonsen; Morten T. Nielsen; Jakob B. Nielsen; Niels B. Hansen; Kristian F. Nielsen; Torsten B. Regueira; Jens Nielsen; Kiran R. Patil; and Uffe H. Mortensen; “Versatile enzyme expression and Characterization system for Aspergillus, with the Penicillium brevicompactum Polyketide Synthase Gene from the Mycophenolic Acid Gene Cluster as a Test Case.” American Society for Microbiology, 2011, 3044-3051.