Part:BBa_J61023

Tn5 Transposase [Tnp1-PN] (with PstI and NotI)

High-activity variant of Tn5 transposase-expressing cassette from pRL27. Cassette includes a complete promoter/rbs/orf cassette. The promoter is Ptet, but not derived from r0040. There are PstI and NotI sites in this part, so care must be taken when doing assembly not to use those sites. Part is used in conjunction with transposons derived from J61021 and J61022.

pSB1A2-Bca1014

This part belongs to a family useful for the construction of transposons and knockin cassettes.

FRT The FRT sequence is the recombination substrate for Flp recombinase. Like the Cre/Lox system, sequences flanked by FRT sites oriented in the same direction get excised by expression of Flp. Cassettes flanked by FRT allow the introduction of selectable markers into the genome followed by subsequent removal by the recombinase.

Tn5 The two Tn5 elements are the substrates for the Tnp transposase in both orientations. Sequences flanked by the two Tn5 elements (in opposing orientation) are mobilizable elements. In the presence of Tnp, they are excised from their plasmid and introduced randomly into the genome or plasmids present in the cell.

Tnp The gene for a high-activity mutant of Tn5 transposase.

OriTr Special strains of E. coli with remnants of the RP4 plasmid, including Rlambda (part ) and BW20767, can transfer plasmids with oriTr (part J01003) to diverse species of bacteria.

OriTf Special strains of E. coli with remanants of the F plasmid, such as J23055, can transfer plasmids with oriTf (part J01002) to other E. coli.

R6K A conditional origin of transfer requiring the pir gene expressed in trans.

Construction of Transposon Plasmids
Transposition mutagenesis is usually carried out by mating a donor strain containing a transposon on a plasmid into a recipient strain. The plasmid with the transposon and transposase transfers to the recipient strain, the transposon is excised from the plasmid, and inserts randomly into the genome. A selectable marker in the transposon and in the recipient strain allows selection of cells receiving the transposon over both the donor and recipient strain.

Construction of the transposon donor strain requires a conjugative donor strain harboring a plasmid with the following elements:

• A conjugative origin of transfer
• A conditional origin of replication, usually R6K, so that the plasmids cannot replicate within the recipient strain (so no second origin of replication)
• A transposon containing a selectable marker flanked by Tn5 ends
• The transposase gene

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 885
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1220
  Illegal PstI site found at 885
  Illegal NotI site found at 798
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1720
  Illegal XhoI site found at 690
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 885
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 885
  Illegal NgoMIV site found at 73
  Illegal AgeI site found at 1208
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1352
  Illegal BsaI.rc site found at 1173
  Illegal SapI site found at 309
  Illegal SapI.rc site found at 621