BBa_K510012

BBa_K510012 Version 1

Component

Source:
http://parts.igem.org/Part:BBa_K510012
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
igem#experience
Works
 
igem#sampleStatus
It's complicated
igem#status
Planning
 
synbiohub#ownedBy
user/james
 
synbiohub#ownedBy
user/myers
 
synbiohub#topLevel
BBa_K510012/1