Difference between revisions of "Part:BBa K1431302"

Latest revision as of 03:44, 18 October 2014

SV40 promoter+SV40 PolyA

The SV40 (Simian Virus 40) promoter contains the SV40 enhancer promoter region and origin of replication for high-level expression and replication in cell lines expressing the large T antigen. It contains two 72bp SV40 enhancer repeats.

Mammalian vectors that contain the SV40 polyoma ori (usually part of the SV40 early promoter in our vectors) replicate episomally, but only in cells that express the SV40 large T antigen (e.g. COS-7 and 293T cells). They will not replicate episomally in the absence of the SV40 large T antigen.

Generating stable lines in cells expressing the SV40 large T antigen is very difficult, if not impossible, with any plasmid that contains the SV40 origin/promoter. This is because the SV40 large T antigen induces DNA replication at the SV40 ori in the vector sequence. If this occurs after the vector has integrated into the genome, it will result in isolated regions of the chromosome trying to replicate illegitimately, leading to chromosomal duplications and generally messing up the cell's genome. The best recommendation would be to use a cell line that does not express the SV40 large T antigen. Deleting the origin could be an option, however, this may affect promoter activity. Transient transfection of SV40 ori-containing plasmids into cells expressing the SV40 large T antigen results in replication of the plasmid (without hurting the host cell's chromosomes) and therefore an amplification of the gene of interest and subsequently very high levels of expression. Hence, we recommend the SV40 ori-containing vectors for transient use only in SV40 large T antigen-containing cells, but they can be used both transiently and stably in cells that do not have the SV40 large T antigen.(Source:[http://tools.lifetechnologies.com/content/sfs/productnotes/F_071215_MammalianExpressionVectors.pdf lifetechnoligies])

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 __NOTOC__
 <partinfo>BBa_K1431302 short</partinfo>
-SV40 promoter is a kind of eukaryocyte cell promoters, of course PolyA also needful. There are two BbsI sites, which recognize six bases[GAAGAC] but cut downstream sites between two bases and four bases, between promoter and PolyA which can make the coding sequence seamless gather with them. So that we can also test how many or what kind of nucleotides interval between promoter and coding sequence will make the highest expression efficiency. For example, if we want to test the efficiency of two nucleotides interval, we should design the the primer sequence like GAAGACNN'NN***, the first NN will be any bases, the second NN is the interval bases,and *** is the begining of coding sequence. If you still do not understand, you can search how BbsI work on the internet or connect with us.
+The SV40 (Simian Virus 40) promoter contains the SV40 enhancer promoter region and origin of replication for high-level expression and replication in cell lines expressing the large T antigen. It contains two 72bp SV40 enhancer repeats.
+Mammalian vectors that contain the SV40 polyoma ori (usually part of the SV40 early promoter in our vectors) replicate episomally, but only in cells that express the SV40 large T antigen (e.g. COS-7 and 293T cells). They will not replicate episomally in the absence of the SV40 large T antigen.
+Generating stable lines in cells expressing the SV40 large T antigen is very difficult, if not impossible, with any plasmid that contains the SV40 origin/promoter. This is because the SV40 large T antigen induces DNA replication at the SV40 ori in the vector sequence. If this occurs after the vector has integrated into the genome, it will result in isolated regions of the chromosome trying to replicate
+illegitimately, leading to chromosomal duplications and generally messing up the cell's genome. The best recommendation would be to use a cell line that does not express the SV40 large T antigen. Deleting the origin could be an option, however, this may affect promoter activity. Transient transfection of SV40 ori-containing plasmids into cells expressing the SV40 large T antigen results in replication of the plasmid (without hurting the host cell's chromosomes) and therefore an amplification of the gene of interest and subsequently very high levels of expression. Hence, we recommend the SV40 ori-containing vectors for transient use only in SV40 large T antigen-containing cells, but they can be used both transiently and stably in cells that do not have the SV40 large T antigen.(Source:[http://tools.lifetechnologies.com/content/sfs/productnotes/F_071215_MammalianExpressionVectors.pdf lifetechnoligies])
 <!-- Add more about the biology of this part here