Part:BBa_K4365023

pSB1KY-TRP1

backbone for shuttle vector

The pSB1KY-HIS3 is a shuttle plasmid backbone suitable for the iGEM RFC1000 assembly in Saccharomyces cerevisiae with HIS3 auxotrophy. It was adapted from pSB1K plasmid and optimized to be expressed in Escherichia coli cells and S. cerevisiae with his3 mutations.

Collection of shuttle vector backbones optimized for Saccharomyces cerevisiae

Backbone ID	Selection marker	Marker sequence source
BBa_K4365022	URA3	[http://freegenes.github.io/genes/BBF10K_000474.html Open Yeast Collection ScURA3-marker]
BBa_K4365023	TRP1	[http://freegenes.github.io/genes/BBF10K_000473.html Open Yeast Collection ScTRP1-marker]
BBa_K4365024	HIS3	[http://freegenes.github.io/genes/BBF10K_000468.html Open Yeast Collection ScHIS3-marker]

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal XbaI site found at 465
  Illegal SpeI site found at 1473
  Illegal PstI site found at 4
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal NheI site found at 2909
  Illegal SpeI site found at 1473
  Illegal PstI site found at 4
  Illegal NotI site found at 3112
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BamHI site found at 100
  Illegal XhoI site found at 4040
  Illegal XhoI site found at 5066
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal XbaI site found at 465
  Illegal SpeI site found at 1473
  Illegal PstI site found at 4
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal XbaI site found at 465
  Illegal SpeI site found at 1473
  Illegal PstI site found at 4
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  

Usage and Biology

HIS3 codes for the Imidazoleglycerol-phosphate dehydratase that catalyzes the sixth step in histidine biosynthesis. It complements his3 mutations allowing yeast to grow on media with no histidine supplements.

The pSB1KY-HIS3 was assembled similarly to pSB1KY-URA3

The pSB1KY-HIS3 was used by WWU-Münster 2022to clone and express in S. cerevisiae the afraGFP gene and the peroxisomal targeting signal 1 (PTS1) as a fusion protein K4188006. All peroxisomal proteins are translated in the cytoplasm and possess a peroxisomal targeting signal that is recognized by a receptor and can direct the protein to the peroxisome (Figure 1).

Figure 1: fluorescence microscopy of S. cerevisiae cells with strong afraGFP signal in (A) cytosol and (B) peroxisomes. Image provided by WWU-Münster 2022