Part:pSB3K3

Low to medium copy BioBrick standard vector

pSB3K3-1 is a medium copy plasmid with kanamycin resistance. It has a p15A pMR101-derived replication origin, copy number 20-30. (Lutz and Bujard, 1997) pSB3K3-1 has a terminator upstream of its MCS, which is oriented to prevent transcription from *inside* the MCS from reading out into the vectorl. A second terminator (E.coli His operon-derived) is downstream of the MCS, again insulating the vector from transcription reading out of the MCS. Ideally, future versions of standard biobrick vectors would have terminators bracketing the MCS that were 100% efficient in terminating transcription both into and out of the MCS region.

2017 CCA_San_Diego Part Improvment

Replacement of p15A origin:

K2491030 (https://parts.igem.org/Part:BBa_K2491030)

In 2017, Philippe Hansen-Estruch from the CCA_San_Diego team created a new plasmid (K2491030) based off the pSB3K3 plasmid. The p15A origin of replication was replaced with a RK2 origin of replication. The goal of this improvement was to replace p15a origin of replication (ori) with the RK2 ori to generate a shuttle vector as a broad host range vector (i.e. can replicate in E.coli and other microorganisms).

The RK2 origin of replication is a broad-host-range plasmid belonging to the incP incompatibility group that can be maintained in a large number of bacteria. The minimal region for replication and maintenance consists of an origin of replication, oriV, and the plasmid replication initiator protein TrfA protein, that activates oriV. The copy number of RK2 is about 4-7 per cell in E. coli, 3 in Pseudomonas aeruginosa, and 4-7 in Agrobacterium.

Improvement by Evry_Paris-Saclay 2017

The pSB3K3 backbone vector contains a BsaI cloning site between the p15A origin of replication and the VF2 primer binding site. Its presence prevents from using the Golden Gate assembly technique with this backbone. To circumvent this issue, we performed a site-directed mutagenesis (A2499T) and created the pSB3K3 BsaI free backbone (BBa_K2448037).

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 2729
  Illegal NheI site found at 1384
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 2735
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2729
  Illegal XhoI site found at 177
  Illegal XhoI site found at 1020
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 2729
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 2729
  Plasmid lacks a suffix.
  Illegal XbaI site found at 2744
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal AgeI site found at 1470
  Illegal AgeI site found at 1793
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 2498