Difference between revisions of "Part:BBa K510048:Design"
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The pUC18R6KT vector was amplified by PCR using as template a pTNS2 plasmid (GenBank accession number: AY884833) provided by Herbert P. Schweizer. The primers were designed to flanked the pUC18R6KT with SfiI restriction sites and allow the insertion of the mini-Tn7-Gm. Primers: attcGGCCTAGGCGGCCgtcgttttacaacgtcgtgac and attcGGCCGCCTAGGCCggaagcataaagtgtaaagcctg. | The pUC18R6KT vector was amplified by PCR using as template a pTNS2 plasmid (GenBank accession number: AY884833) provided by Herbert P. Schweizer. The primers were designed to flanked the pUC18R6KT with SfiI restriction sites and allow the insertion of the mini-Tn7-Gm. Primers: attcGGCCTAGGCGGCCgtcgttttacaacgtcgtgac and attcGGCCGCCTAGGCCggaagcataaagtgtaaagcctg. | ||
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The miniTn7BB-Gm minitransposon was synthesized commercially, and then digested at the flanking SfiI sites and cloned into SfiI-digested pUC18R6KT PCR products. | The miniTn7BB-Gm minitransposon was synthesized commercially, and then digested at the flanking SfiI sites and cloned into SfiI-digested pUC18R6KT PCR products. | ||
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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. | 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. | ||
===References=== | ===References=== | ||
Revision as of 16:00, 22 October 2011
pUC18R6KT-miniTn7BB-Gm-attTn7
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 4528
Illegal suffix found in sequence at 1 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 4528
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 4534 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 4528
Illegal BglII site found at 3212
Illegal BglII site found at 3483
Illegal BglII site found at 3769 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 4528
Illegal suffix found in sequence at 2 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 4528
Illegal XbaI site found at 4543
Illegal SpeI site found at 2
Illegal PstI site found at 16 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1854
Illegal SapI site found at 518
Design Notes
The portable attTn7 (BBa_K510022) BioBrick was inserted within the BCS of pUC18R6KT-miniTn7BB-Gm (BBa_K510012) by EcoRI and PstI clonning.
Source
The pUC18R6KT vector was amplified by PCR using as template a pTNS2 plasmid (GenBank accession number: AY884833) provided by Herbert P. Schweizer. The primers were designed to flanked the pUC18R6KT with SfiI restriction sites and allow the insertion of the mini-Tn7-Gm. Primers: attcGGCCTAGGCGGCCgtcgttttacaacgtcgtgac and attcGGCCGCCTAGGCCggaagcataaagtgtaaagcctg.
The miniTn7BB-Gm minitransposon was synthesized commercially, and then digested at the flanking SfiI sites and cloned into SfiI-digested pUC18R6KT PCR products.
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.