Part:pSB2K0:Design

pSB2K0 (pSCANS-BNL; Replaced by pSB2K3)

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 1024
  Illegal XbaI site found at 2574
  Illegal SpeI site found at 1052
  Illegal PstI site found at 1745
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 1024
  Illegal NheI site found at 4319
  Illegal SpeI site found at 1052
  Illegal PstI site found at 1745
  Illegal NotI site found at 1068
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 1024
  Illegal BglII site found at 2620
  Illegal BglII site found at 3667
  Illegal BamHI site found at 1046
  Illegal XhoI site found at 30
  Illegal XhoI site found at 1013
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 1024
  Illegal XbaI site found at 2574
  Illegal SpeI site found at 1052
  Illegal PstI site found at 1745
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 1024
  Illegal XbaI site found at 2574
  Illegal SpeI site found at 1052
  Illegal PstI site found at 1745
  Illegal NgoMIV site found at 2063
  Illegal NgoMIV site found at 4305
  Illegal AgeI site found at 1939
  Illegal AgeI site found at 3598
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  

Design Notes

"A new plasmid vector, pSCANS, has been constructed which allows rapid

          generation of an ordered set of nested deletions from either strand of a cloned 

          DNA fragment. pSCANS is based on the low-copy F replicon. The size of the 

          vector DNA has been reduced to the 4.4-kbp range by removing the 2.5-kbp 

          sop (stability of plasmid genes) region. The resulting plasmid has the low copy 

          number typical of F plasmids and it remains stable enough to be easily maintained 

          by growth in the presence of kanamycin, the selective antibiotic. DNA in amounts 

          convenient for sequencing is readily obtained by amplification from an 

          IPTG-inducible P1 lytic replicon. The vector's multiple cloning region (MCR), which 

          has several unique sites for both shotgun and directional cloning, is flanked on one 

          side by recognition sequences for the extremely rare cutting intron encoded 

          nucleases I-CeuI and I-SceI, and on the other side by a recognition sequence 

          for another intron encoded enzyme, PI-PspI and a nicking site for the phage f1 

          protein, gpII, that initiates f1 rolling circle DNA replication. Cleavage with the intron 

          encoded enzymes leaves four-base 3' overhangs that are resistant to digestion with E. coli ExoIII. Between these sites and the MCR are recognition sites for several rare 8-base cutters that leave ExoIII sensitive termini. Double cutting with one intron encoded enzyme and an adjacent rare cutting restriction endonuclease allows for unidirectional 3' to 5' digestion across the insert with ExoIII. Alternatively, plasmid linearized with I-SceI can be blunt ended to produce an ExoIII sensitive end an

          ent generates a good distribution of deletion clones following electroporation. 

          Deletion clones are sized and sequenced using vector specific forward and 

          reverse primers. 

"