Part:BBa_K3888006

UppE Downstream Flanking Region

UppE Downstream Flanking Region
This basic part is the downstream flanking region of UppE for R. palustris. Together with UppE upstream flanking region(BBa_K3888005), it forms UppE Deletion plasmid(BBa_K3888012). Upp encodes part of the unipolar polysaccharide (UPP) and it has been showed that UPP is important for adhesion when light and organic substrates are used for growth for R. palustris. UppE(RPA2750) is located on 3120549..3122105 on the complete genome of R. palustris.

Figure 1: Illustration of the general form of plasmid used to create marked knockout strains of R. palustris.

The origin of replication (ori), origin of transfer (oriT) and sacB (blue) are part of the delivery plasmid. The green sequence is inserted into the multiple cloning site of the delivery plasmid. The left and right fragments (green) are sequences amplified from the R. palustris genome and used in overlap extension PCR to generate a recombinant product containing (a deletion and a restriction site）. The restriction site is used to introduce the resistance cassette (purple). This interrupts the gene even if no deletion is present, and allows selection of successful transformants.
According to this model, we constructed UppE knockout plasmid below.

Figure 2: Deletion plasmid of UppE constructed according to the method mentioned above. UppE Downstrem Flanking region is located from 1bp to 1020bp.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 513
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 513
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 513
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 513
  Illegal NgoMIV site found at 172
  Illegal NgoMIV site found at 318
  Illegal NgoMIV site found at 442
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1001