Part:BBa_K5194000

pSB1K0 Yeast Shuttle Vector (sfGFP dropout) Type IIS

The plasmid backbone allows amplification in E. coli, and plasmid replication and expression in Pichia pastoris.

The plasmid backbone contains a high-copy origin and kanamycin resistance for growth and selection in E. coli, along with a GFP marker to help visualize successful assembly. It also includes PARS and Zeocin resistance for use in Pichia pastoris for replication and expression. To use this backbone, first select the necessary parts from the open yeast collection. For level 1 assembly, use Golden Gate cloning with BsaI. Success can be identified by checking kanamycin plates—successful colonies won’t show GFP fluorescence. For level 2 assembly, modify the backbone by adding a new E. coli selection marker using NotI and XhoI. Then, digest the level 1 assemblies with BbsI, and insert them into the modified backbone. Successful level 2 assemblies can be confirmed by growing colonies on plates with the new resistance marker (and no GFP fluorescence).

Components: High copy origin (BBa_J428318), Kanamycin resistance (sourced from BBa_K5194055), Origin of transfer (BBa_J435286), Pichia PARS element (BBa_J435251), Zeocin resistance (BBa_J435280) and sfGFP (BBa_K2541401).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 41
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 41
  Illegal NheI site found at 1371
  Illegal NheI site found at 2498
  Illegal NheI site found at 2521
  Illegal NotI site found at 3502
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 41
  Illegal BglII site found at 154
  Illegal BamHI site found at 69
  Illegal XhoI site found at 4555
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 41
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 41
  Illegal NgoMIV site found at 4968
  Illegal NgoMIV site found at 5094
  Illegal NgoMIV site found at 5104
  Illegal AgeI site found at 231
  Illegal AgeI site found at 5350
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 3436
  Illegal BsaI.rc site found at 2466
  Illegal SapI site found at 4625
  Illegal SapI site found at 4996
  Illegal SapI.rc site found at 2577