Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: David Caballero
Date created: 2011-09-17 11:00:00
Date modified: 2015-05-08 01:12:30
pUC18R6KT-miniTn7BB-Cm
| Types | DnaRegion |
| Roles | Plasmid plasmid |
| Sequences | BBa_K510013_sequence (Version 1) |
Description
pUC18R6KT-miniTn7BB-Cm is a vehicle vector for the minitransposon miniTn7BB-Cm. This transposon is a part of the miniTn7 BioBrick toolkit, a set of plasmids harboring Tn7 derivatives that can be used for integration of BioBricks in single copy at a conserved target (the attTn7 site), in multiple bacterial genomes.The structure of the mini-Tn7BB-Cm transposon is shown in the figure below. Tn7R and Tn7L are the right and left ends of Tn7 transposon, respectively, required for recognition of the transposase machinery and transposition. FRT is the Flp recombinase target site for excision of the resistance once the transposon is inserted in the genome of a living organism. The chloramphenycol resistance cassette (Cm) is flanked by restriction sites in order to facilitate the generation of variants with different antibiotic resistance markers. The BioBrick cloning site (BCS) includes the prefix and suffix restrictions sites used in assembly standard 10, and it is the place where BioBricks are inserted for genome integration.
This plasmid is replicative only in E. coli strains expressing the pir gene thanks to its R6K replication origin. Also it has an origin for transference (oriT) for conjugation purposes. This plasmid can be used to maintain the miniTn7BB-Gm transposon in E. coli pir+ strains and may be used for transposition into the genomes of any bacteria without the pir gen, in which it is non-replicative. As a BioBrick cloning vector, pUC18R6KT-miniTn7BB-Cm is fully compatible with Assembly Standard 10. The complete transposon is flanked by SfiI restriction sites to facilitate subcloning in other vectors.
Notes
The pUC18R6KT-mini-Tn7-Cm is a derivative of pUC18R6KT-mini-Tn7-Gm (BBa_K510012)by replacing the gentamycin resistance cassette by a chloramphenicol resistance cassette amplified from pSB1C3 with these primers: tatGCATGCCCATGGaacttggtctgacagctcgag and tatGCATGCCCATGGtcgggcacgtaagaggttcc.
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. In order to change the resistance cassettes NcoI digestions were made.
| Sequence Annotation | Location | Component / Role(s) |
| double terminator (BBa_B0014) E. coli his operon terminator VR (BBa_G00101) primer binding site Tn7-L end SfiI restriction site oriT R6K replication origin Ampicillin resistance (bla) SfiI restriction site Tn7-R end Suffix SphI restriction site NcoI restriction site Chloramphenicol resistance NcoI restriction site SphI restriction site Flipase recombination sequence (FRT) Flipase recombination sequence (FRT) Terminator VF2 (G00100) primer binding site T0 Terminator Prefix | 3385,3479 22,93 157,176 182,347 348,369 646,905 929,1310 1714,2574 3163,3175 3176,3374 2,21 4463,4468 4457,4462 3696,4355 3548,3553 3542,3547 4469,4516 3490,3537 4600,4632 4522,4541 3577,3683 4640,4661 | feature/BioBrick engineered_region feature/stem_loop stem_loop feature/primer_binding primer_binding_site sequence_feature feature/misc feature/dna sequence_feature sequence_feature feature/dna feature/dna sequence_feature CDS feature/cds feature/dna sequence_feature sequence_feature feature/misc engineered_region feature/BioBrick sequence_feature feature/dna feature/dna sequence_feature CDS feature/cds feature/dna sequence_feature feature/dna sequence_feature feature/dna sequence_feature feature/dna sequence_feature feature/stem_loop stem_loop feature/primer_binding primer_binding_site stem_loop feature/stem_loop engineered_region feature/BioBrick |