BBa_K390000

BBa_K390000 Version 1

Component

Source:
http://parts.igem.org/Part:BBa_K390000
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Cody Tramp
Date created: 2010-07-31 11:00:00
Date modified: 2015-05-08 01:12:19

pSB1A3_Int (BamHI, XhoI, NdeI, NheI added)



Types
DnaRegion

Roles
Plasmid_Backbone

plasmid_vector

Sequences BBa_K390000_sequence (Version 1)

Description

This part is based on pSB1A3, with BamHI, XhoI, NdeI and NheI sites added through site-directed mutagenesis. This plasmid backbone retains compatibility with BioBricks (except with RFC 21 - BamBgl), its antibiotic resistance, and its high copy number in E. coli.

The additional restriction sites allow the addition of DNA sequences homologous to genomic sequences in Synechocystis sp. PCC 6803 and other species, enabling the plasmid to be used to integrate the BioBrick construct and an antibiotic resistance gene into the chromosome at a specific location through homologous recombination. Integration sequences can be designed so that the BioBrick construct is added in the middle of a gene knocking the gene out in the process, or between two genes without interfering with native expression.

The BioBrick construct can be assembled in E. coli cells without changes to the standard procedure, and when completely assemble, the plasmid can be introduced into the target species.

Synechocystis sp. PCC 6803 naturally takes up DNA from its environment, and if sequences are homologous to regions of the genome, will naturally undergo homologous recombination. Other species may lack one or both of these abilities, and the addition of another plasmid carrying a recombinase gene may be necessary for genomic integration.

Notes

BamHI, XhoI, NdeI, and NheI sites were mutated in using Stratagene's site-directed mutagenesis kit, the sites were mutated in by changing the nucleotide sequence, without the addition or removal of nucleotides.

Source

pSB1A3 with BamHI, XhoI, NdeI, and NheI sites mutated in outside of the terminator regions.

Sequence Annotation Location Component / Role(s)
suffix
E. coli his operon terminator
terminator stem loop
deletion of two bases
VR
NdeI site
g > a
NheI Site
c > t
rep(pMB1) (rev)
Ori
AmpR (rev)
BamHI Site
t > a
XhoI Site
c > t
a > g
VF2
end of terminator
terminator stem loop
prefix
1,21
22,93
30,64
34,35
157,176
216,221
219,219
230,235
232,232
261,875
276,276
1025,1895
1992,1997
1995,1995
2009,2014
2010,2010
2014,2014
2017,2037
2094,2097
2107,2126
2136,2157
engineered_region feature/BioBrick
sequence_feature feature/misc
feature/stem_loop stem_loop
feature/misc sequence_feature
feature/primer_binding primer_binding_site
sequence_feature feature/misc
feature/mutation sequence_alteration
sequence_feature feature/misc
sequence_alteration feature/mutation
feature/cds CDS
sequence_feature feature/misc
feature/cds CDS
sequence_feature feature/misc
sequence_alteration feature/mutation
feature/misc sequence_feature
sequence_alteration feature/mutation
sequence_alteration feature/mutation
feature/primer_binding primer_binding_site
feature/misc sequence_feature
feature/stem_loop stem_loop
engineered_region feature/BioBrick
igem#experience
Works
 
igem#sampleStatus
It's complicated
igem#status
Planning
 
synbiohub#ownedBy
user/james
 
synbiohub#ownedBy
user/myers
 
synbiohub#topLevel
BBa_K390000/1