Source:
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 |