Part:pSB6A1

pBR322 converted to BioBrick vector (formally BBa_I739201)

Plasmid pBR322BB1 (Fig. 1) is based on pBR322 (Fig. 2), a low-copy cloning vector (15-20 copies per cell) that confers ampicillin (ApR) and tetracylin (TcR) resistance and harbours the pMB1 origin of replication.

Fig. 1: Map of the pBR322BB1 plasmid

Fig. 2: Map of the original pBR322 plasmid

In order to make this plasmid compatible with the BioBrick Standard assembly process, the original pBR322 was digested at its EcoRI and BamHI restriction sites and an appropriate multiple cloning site (MCS) was introduced in this position. However, this leads to a loss of the original tetracyclin resistance and only ampicillin resistance is left. Since the original plasmid also contains a PstI restriction site in the bla gene, the GCA codon at this position was changed to GTA by site directed mutagenesis. The term "BB1" in pBR322BB1 refers to "BioBrick version 1".

Purpose

This plasmid was designed for the [http://2007.igem.org/ETHZ ETHZ iGEM 2007 project] and is used for the following BioBricks: Part 1, Part 2, Part 3 and the composites BBa_I739012 and BBa_I739013. More information on the plasmid and the used assembly strategy can be found [http://2007.igem.org?title=ETHZ/Biology/Lab here].

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 4001
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 4007
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4001
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 4001
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 4001
  Plasmid lacks a suffix.
  Illegal XbaI site found at 4016
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 43
  Illegal NgoMIV site found at 411
  Illegal NgoMIV site found at 571
  Illegal NgoMIV site found at 925
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 3075
  Illegal SapI site found at 1992