Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: David Caballero, Fernando Govantes
Date created: 2011-10-21 11:00:00
Date modified: 2015-05-08 01:12:30
pUC18R6KT-miniTn7BB-Gm-attTn7
| Types | DnaRegion |
| Roles | Composite engineered_region |
| Sequences | BBa_K510048_sequence (Version 1) |
Description
This plasmid allows the integration of BioBricks into microbial chromosomes, using prefix or suffix cloning sites, without removing the attachment site of the Tn7 transposon (attTn7). This fact makes posible to integrate another BioBrick into the chromosome of a strain in which miniTn7BB-Gm-attTn7 transposition has occurred previously.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.
| Access | Instance | Definition |
| public public | BBa_K510012 BBa_K510022 | BBa_K510012 BBa_K510022 |
| Sequence Annotation | Location | Component / Role(s) |
| BBa_K510012 BBa_K510022 | 1,4549 4558,4722 | BBa_K510012 BBa_K510022 |