Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: David Caballero & Fernando Govantes
Date created: 2011-09-17 11:00:00
Date modified: 2015-05-08 01:12:30
pUC18R6KT-miniTn7BB-Gm
| Types | DnaRegion |
| Roles | plasmid Plasmid |
| Sequences | BBa_K510012_sequence (Version 1) |
Description
pUC18R6KT-miniTn7BB-Gm is a vehicle vector for the minitransposon miniTn7BB-Gm. 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 bacteria. The structure of the mini-Tn7BB-Gm 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 gentamycin resistance cassette (Gm) 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 miniTn7-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-Gm is fully compatible with Assembly Standard 10. The complete transposon is flanked by SfiI restriction sites to facilitate subcloning in other vectors.Notes
Kyoung-Hee Choi, Jared B Gaynor, Kimberly G White, Carolina Lopez, Catharine M Bosio, RoxAnn R Karkhoff-Schweizer & Herbert P Schweizer (2005). A Tn7-based broad-range bacterial cloning and expression system. Nature Methods, vol.2 NO.6, June 2005, 443.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.
| Sequence Annotation | Location | Component / Role(s) |
| Suffix Tn7-L end VR (BBa_G00101) primer binding site Ampicillin resistance (bla) R6K replication origin oriT SfiI restriction site Tn7-R end double terminator (BBa_B0014) Flipase recombination sequence (FRT) SfiI restriction site NcoI restriction site Gentamicin resistance SphI restriction site NcoI restriction site SphI restriction site Prefix VF2 (G00100) primer binding site Flipase recombination sequence (FRT) E. coli his operon terminator Terminator | 2,21 182,347 157,176 1714,2574 929,1310 646,905 348,360 3176,3374 3385,3479 3490,3537 3163,3175 3548,3553 3554,4087 3542,3547 4345,4350 4351,4355 4528,4549 4410,4429 4357,4404 22,93 4487,4520 | engineered_region feature/BioBrick sequence_feature feature/misc primer_binding_site feature/primer_binding CDS feature/cds sequence_feature feature/dna sequence_feature feature/dna sequence_feature feature/dna feature/misc sequence_feature feature/BioBrick engineered_region sequence_feature feature/dna sequence_feature feature/dna sequence_feature feature/dna feature/cds CDS sequence_feature feature/dna sequence_feature feature/dna feature/dna sequence_feature feature/BioBrick engineered_region feature/primer_binding primer_binding_site sequence_feature feature/dna feature/stem_loop stem_loop feature/stem_loop stem_loop |