Part:BBa_J61031

Amplifiable BAC plasmid

This plasmid is derived originally from pBeloBAC11 with multiple modifications to make it compatible with biobricks and more useable. Without modification, J61031 is a high-copy plasmid in normal laboratory strains of E. coli due to the colE1 origin of replication located within the biobrick part region. It also confers ampicillin resistance, kanamycin resistance, and constitutively-expressed RFP. Upon replacement of the SpeI/XbaI region, the colE1, ampR, and RFP features are removed, leaving behind the F plasmid replicon, the KanR gene, and an R6K origin of replication. At this point, the plasmid will replicate as a medium or high copy plasmid in pir+ or pir116 strains such as Ec100D::pir116. In non-pir strains (most lab strains), the plasmid will replicate at near-single copy levels.

The restriction sites EcoRI, SpeI, NotI, and XbaI are all unique in this plasmid. There are several PstI sites, however.

pBACr-NESd

• RS 17:40, 4 September 2008 (UTC): This plasmid does not seem to have an XbaI site.

• RS 5:34, 20 October 2009 (UTC): The constitutively-expressed RFP can be visualized even without fluorescent microscopy. However, if the RFP coding region is recombined to a T7 promoter, the red fluorescence do not seem to be visible without fluorescent microscopy. We obtain this part from the iGEM07 team, which seems to show no Kan resistance.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 6527
  Illegal SpeI site found at 2
  Illegal PstI site found at 2001
  Illegal PstI site found at 3542
  Illegal PstI site found at 5068
  Illegal PstI site found at 8975
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 6527
  Illegal NheI site found at 8816
  Illegal SpeI site found at 2
  Illegal PstI site found at 2001
  Illegal PstI site found at 3542
  Illegal PstI site found at 5068
  Illegal PstI site found at 8975
  Illegal NotI site found at 9
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 6527
  Illegal BglII site found at 2350
  Illegal BglII site found at 4421
  Illegal BglII site found at 4855
  Illegal XhoI site found at 4554
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 6527
  Illegal SpeI site found at 2
  Illegal PstI site found at 2001
  Illegal PstI site found at 3542
  Illegal PstI site found at 5068
  Illegal PstI site found at 8975
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 6527
  Illegal SpeI site found at 2
  Illegal PstI site found at 2001
  Illegal PstI site found at 3542
  Illegal PstI site found at 5068
  Illegal PstI site found at 8975
  Illegal NgoMIV site found at 3860
  Illegal NgoMIV site found at 5519
  Illegal AgeI site found at 1613
  Illegal AgeI site found at 2506
  Illegal AgeI site found at 3736
  Illegal AgeI site found at 10369
  Illegal AgeI site found at 10481
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 7384
  Illegal BsaI site found at 9050
  Illegal BsaI.rc site found at 759
  Illegal BsaI.rc site found at 5690
  Illegal BsaI.rc site found at 6256
  Illegal BsaI.rc site found at 6434
  Illegal SapI.rc site found at 1756
  Illegal SapI.rc site found at 2966
  Illegal SapI.rc site found at 5368
  Illegal SapI.rc site found at 5578
  Illegal SapI.rc site found at 8466