Part:BBa_K812200

Biobricked pCS2+ plasmid with Elastase promoter for frogs and chicken

This plasmid is a biobricked version of the plasmid pSC2+(<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K812000">BBa_K812000</a>)with Elastase promoter. The CMV promoter was replaced by elastase promoter with SalI and HindIII restriction sites. This plasmid is made of a bacterial ampicilin resistant backbone. The Biobrick site is surrounded by a 5'UTR expression enhancer, and a poly-A tail with a SV40 3' UTR that enhance the stabilisation of the mRNA and the expression of the protein.

This plasmid is meant to be micro-injected in frog eggs or chicken eggs. This plasmid does not replicate, so it gets diluted and degraded as the animal grow, leaving no chance for DNA propagation to the breeding.

To use this plasmid, you simply have to insert your protein between the EcoRI and PstI site. It is better to add a kozac sequence in front of your gene to enhance its expression. This plasmid contains the standard registry insert J04450 as a negative cloning control (Bacteria with the BioBrick

Elastase is a tissue specific promoter for the pancreas. This backbone is characterized using sfGFP (<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K812031">BBa_K812031</a>) as reporter.

Tadpole at stage 47 produced sfGFP in pancreas, you can compare the auto-fluorescence with the control and the picture with RFP filter compare to GFP. <img src="" alt="perdu" width="880px"/>

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3291
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 248
  Illegal NotI site found at 3297
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3291
  Illegal BamHI site found at 3276
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 3291
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 3291
  Plasmid lacks a suffix.
  Illegal XbaI site found at 3306
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 2611
  Illegal NgoMIV site found at 3032
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 1634
  Illegal SapI site found at 551