Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: David Caballero, Fernando Govantes
Date created: 2011-09-15 11:00:00
Date modified: 2015-05-08 01:12:30
pUC18Sfi-miniTn7BB-Gm
| Types | DnaRegion |
| Roles | Plasmid plasmid |
| Sequences | BBa_K510000_sequence (Version 1) |
Description
The miniTn7BB-Gm synthetic minitransposon was digested at the flanking SfiI sites and cloned into SfiI-digested pUC18-Sfi. This results in the elimination of the complete pUC18-SfiI multi-cloning site, except for the duplicated SfiI sites that remain on both sides of the transposon, thus facilitating its transfer to other vectors. This plasmid can be used to keep the mini-Tn7-Gm in E. coli and other enterics, and may be used for transposition into the genomes of non-enteric bacteria, in which it is non-replicative.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. The complete construction is flanked by SfiI restriction sites to facilitate cloning.
Notes
In order to make this vector compatible with assembly standard 10, several restriction sites were removed from the synthetic sequence.Source
The pUC18-SfiI cloning vector was kindly provided by the Microbiology laboratory of Centro Andaluz de Biolog??a del Desarrollo (CABD) in Sevilla.The miniTn7BB-Gm synthetic transposon was designed by the iGEM 2011 team UPO-Sevilla based on the set of miniTn7 derivatives developed by Choi et al. (2005) and commercially synthesized by Mr. Gene (Regensburg, Germany).
K.-H. Choi, J. B. Gaynor, K. G. White, C. Lopez, C. M. Bosio, R. R. Karkhoff-Schweizer & H. P. Schweizer (2005). A Tn7-based broad-range bacterial cloning and expression system. Nature Methods, vol.2 NO.6, June 2005, 443.
| Sequence Annotation | Location | Component / Role(s) |
| Suffix E. coli his operon terminator VR (BBa_G00101) primer binding site Tn7-L end SfiI restriction site Ampicillin resistance (bla) ColEI (pUC18) Replication origin SfiI restriction site Tn7-R end Flipase recombination sequence (FRT) Gentamycin resistance Flipase recombination sequence (FRT) Prefix double terminator (BBa_B0014) SphI restriction site NcoI restrictino site NcoI restriction site SphI restriction site VF2 (G00100) primer binding site Terminator | 2,21 22,93 157,176 182,347 348,360 966,1826 1965,2644 3002,3014 3014,3213 3329,3376 3393,3926 4196,4243 4367,4387 3224,3318 3381,3386 3387,3392 4184,4189 4190,4195 4249,4268 4326,4359 | engineered_region feature/BioBrick stem_loop feature/stem_loop feature/primer_binding primer_binding_site feature/misc sequence_feature sequence_feature feature/misc CDS feature/cds sequence_feature feature/dna feature/misc sequence_feature feature/misc sequence_feature feature/misc sequence_feature feature/cds CDS sequence_feature feature/misc engineered_region feature/BioBrick engineered_region feature/BioBrick sequence_feature feature/dna feature/dna sequence_feature sequence_feature feature/dna sequence_feature feature/dna feature/primer_binding primer_binding_site stem_loop feature/stem_loop |