Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Fernando Govantes
Date created: 2011-09-15 11:00:00
Date modified: 2015-05-08 01:12:30
pUC18SfiI-miniTn7BB-Km
| Types | DnaRegion |
| Roles | Plasmid plasmid |
| Sequences | BBa_K510002_sequence (Version 1) |
Description
The miniTn7BB-Km minitransposon 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 maintain the mini-Tn7-Km 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-Km 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 kanamycin resistance cassette (Km) 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.Notes
NcoI and SphI sites were added at the ends of the kanamycin resistance cassette to facilitate cloningSource
The pUC18Sfi-miniTn7BB-Km was created by replacing the gentamycin resistance cassette of pUC18Sfi-miniTn7-Gm (K510000) by a kanamycin resistance cassette amplified from pSB4K5 with the following primers: tatGCATGCCCATGGattggggctcactcaaagg and tatGCATGCCCATGGtcgacaatgtaactactag| Sequence Annotation | Location | Component / Role(s) |
| Suffix Prefix Tn7-L end Tn7-R end SfiI restriction site Ampicillin resistance (bla) ColEI (pUC18) Replication origin SfiI restriction site Flipase recombination sequence (FRT) Flipase recombination sequence (FRT) Kanamycin resistance | 2,21 4638,4658 82,347 3014,3213 348,360 966,1826 1965,2644 3002,3014 3329,3376 4467,4514 3467,4285 | engineered_region feature/BioBrick engineered_region feature/BioBrick feature/misc sequence_feature feature/misc sequence_feature feature/misc sequence_feature feature/cds CDS feature/dna sequence_feature feature/misc sequence_feature sequence_feature feature/misc sequence_feature feature/misc feature/cds CDS |