Part:BBa_K812300

Biobricked pCS2+ plasmid with HSP70 promoter for frogs and chicken

This plasmid is a biobricked version of the plasmid pCS2+(BBa_K812000)with HSP70 promoter. The CMV promoter was replaced by Hsp70 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 ment to be micro-injected in frog eggs or chicken eggs for a transient expression of the protein at the early ages of development of the embryo. 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.

The Heat Shock Promoter 70 induce the expression of the protein which is after HSP70. The induction can occur heating tadpole from 21°C to 34°C.

The results of its sequencage presented some divergences with the reference sequence. That's why, all the results have to be consireded with caution.

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 3570
  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 3576
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3570
  Illegal BglII site found at 3407
  Illegal BamHI site found at 3555
  Illegal XhoI site found at 3126
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 3570
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 3570
  Plasmid lacks a suffix.
  Illegal XbaI site found at 3585
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 2611
  
• 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
  Illegal SapI.rc site found at 3419