Part:BBa_K3579017

EpiFlex_pPBEF_bb1AB

This plasmid is the backbone of the level 1 plasmid of the EpiFlex MoClo toolkit for S. epidermidis. It has level 2 fusion sites A and B so it aims to be in position 1 of genetic circuits assembled in level 2 plasmid.

It is a E. coli -> S. epidermidis shuttle vectors. This means that they contain origins of replication for both E. coli and S. epidermidis as well antibiotic resistance selection for each microbe (Kanamycin for E. coli and erythromycin for S. epidermidis)

The level 1 plasmid aims to contain TU from individual parts assembly. The insertion of the parts in this backbone is done by golden gate assembly : This backbone has level 1 fusion sites A and H and BsaI restriction sites allowing the first and the last TU's parts insertion. A GFP dropout cassette is present in between the the pairs of level 1 fusion sites and BsaI restriction sites allowing to verify the insertion of the part. The removal of the GFP cassette is a visual indicator of successful transformants, as the lack of a green suggests successful interruption of the cassette.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2379
  Illegal SpeI site found at 552
  Illegal PstI site found at 682
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2379
  Illegal NheI site found at 6610
  Illegal SpeI site found at 552
  Illegal PstI site found at 682
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2379
  Illegal BglII site found at 409
  Illegal BglII site found at 491
  Illegal BamHI site found at 537
  Illegal XhoI site found at 530
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2379
  Illegal SpeI site found at 552
  Illegal PstI site found at 682
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2379
  Illegal SpeI site found at 552
  Illegal PstI site found at 682
  Illegal NgoMIV site found at 6758
  Illegal AgeI site found at 6227
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 6877
  Illegal BsaI.rc site found at 6024
  Illegal SapI site found at 3135
  Illegal SapI site found at 3911