Part:BBa_J61212

Tn5F|rbsGenR|FRT|R6K|CmR|FRT|P_T7|Tn5R||SpecR|OriTr|TnP

This is NOT actually a composite part, but rather a new plasmid created by cutting the composite parts out of their pSB1A2 plasmids and pasting them together to form this transposon bearing suicide plasmid. As such, the parts are not actually colinear, but rather the coding strand changes at the || indicated in the name. See box below for details; this is transposon/plasmid 2. The transposon has chloramphenicol resistance on a suicide plasmid bearing spectinomycin resistance.

This part is used in a project to assemble two different transposons on suicide plasmids.

Transposon/Plasmid 1
The first transposon/plasmid combination has a smaller transposable element which includes a spectinomicin resistance marker, a promoterless gentimicin marker (for selection of transposons that land inside a transcribed region), and a T7 promoter to help determine the region that the transposon inserted into. The rest of the plasmid contains a chloramphenicol resistance marker, a tranposase generating casette, an origin of transfer, and a conditional origin of replication (see box below about construction of transposon plasmids).
Preliminary experiments suggest that the suicide plasmid delivers the transposon with moderate efficiency, but that the ribosome binding site on the gentimicin gene is too weak to confer resistance from genomic transcription.


Transposon/Plasmid 2
The second transposon/plasmid combination has a larger transposable element which includes a chloramphenicol resistance marker, a promoterless gentimicin marker (for selection of transposons that land inside a transcribed region), and conditional origin of replication, and a T7 promoter. The FRT sites are an artifact of construction, and don't have any function in this case. Moving the origin of replication inside the transposon can greatly ease the process of decoding where the transposon inserted. The rest of the plasmid contains a spectinomycin resistance marker, a tranposase generating casette, and an origin of transfer.
Preliminary experiments suggest that the transposon is delivered with very poor efficiency, perhaps as a consequence of its significant size.

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 4213
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 4548
  Illegal PstI site found at 4213
  Illegal NotI site found at 4126
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 361
  Illegal BglII site found at 5048
  Illegal BamHI site found at 27
  Illegal XhoI site found at 4018
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 4213
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 4213
  Illegal NgoMIV site found at 2238
  Illegal NgoMIV site found at 3066
  Illegal NgoMIV site found at 3076
  Illegal NgoMIV site found at 3401
  Illegal AgeI site found at 4536
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4680
  Illegal BsaI.rc site found at 4501
  Illegal SapI site found at 2968
  Illegal SapI site found at 3637
  Illegal SapI.rc site found at 3949