Part:BBa_K510047:Design
pUC18Sfi-miniTn7BB-Gm-attTn7
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 4367
Illegal suffix found in sequence at 1 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 4367
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 4373 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 4367
Illegal BglII site found at 3051
Illegal BglII site found at 3322
Illegal BglII site found at 3608 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 4367
Illegal suffix found in sequence at 2 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 4367
Illegal XbaI site found at 4382
Illegal SpeI site found at 2
Illegal PstI site found at 16 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1681
Illegal SapI.rc site found at 2763
Design Notes
In order to construct the pUC18Sfi-miniTn7BB-Gm delivery plasmid, the miniTn7BB-Gm transposon was cleaved from the commercial plasmid pMA (Mr. Gene) with SfiI and ligated to SfiI-digested pUC18Sfi. This strategy removes all multi-cloning restriction sites from pUC18Sfi, except for the flanking SfiI, thus guaranteeing that the unique sites in the transposon are not duplicated. The remove of the multi-cloning restriction sites was verified by analytic digestions. The direction of miniTn7BB-Gm insertion was determined by digestion.
Source
The miniTn7BB-Gm minitransposon was synthesized commercially and pUC18SfiI is a commercial vector. The attTn7 was obtained from the purified genome of E. coli MC4100 by PCR (using a high accuracy Pfu polymerase) with the follow primers: gaattcgcggccgcttctagagTGCAGCTGCTGGCTTACCATG and ctgcagcggccgctactagtaACATGGAGTTGGCAGGATGTTTG.