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Blasticidin Selection Plasmids

OG410 - pSF-CMV-Ub-Blast-SV40 Ori Sbfl

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Full Plasmid Details (PDF)

Sequence (txt)

This plasmid is compatible with all of the other DNA sequences available on this site, which are all sold in the same backbone. This flexibility allows you to create any vector you require, with a variety of functions, simply by compiling the relevant sections from our product range.

Vector: pSF-CMV-Ub-Blast-SV40 Ori SbfI

Product Code: OG410

Quantity Provided: 5µg

Size (bp): 6171

Bacterial Antibiotic Selection: Kanamycin

Origin and Compatibility: pUC high copy, derived from pBR322   

Copy Number: 500-700 copies per cell

Promoter: Cytomegalovirus (CMV) immediate early promoter

Purpose: This vector contains a Blasticidin (Blast) resistance expression cassette under the Ub promoter. It also contains the Simian Virus 40 (SV40) origin of replication. This allows the plasmid to be amplified in cells containing the SV40 large T antigen. This can be used to increase transgene signal following transient transfection. It is also possible to make cell lines containing the plasmid as a multi-copy episome. We have validated this activity in 293T cells. The entire Ub Blast cassette is flanked by AscI sites. This vector can also be used to force recombination into the genome of mammalian cells not expressing SV40 large T antigen following transfection and Blasticidin selection.

Quality Validation: This vector has been demonstrated to express reporter genes to high levels under the CMV promoter. The start codon of these reporters was positioned within the NcoI site and the stop codon was positioned within the XbaI site. Expression was validated in the A549 lung carcinoma cell line. We have also created stable cells lines using this plasmid in both large T antigen expressing, and non-large T antigen expressing cells, using 293 and 293T cells respectively.  

Quality Sequencing Analysis: This vector has been fully sequenced using primers F1-F10. The details and sequences of these primers are available through our website.

Genetic Modifications to standard parts: 
•    CMV promoter – an NcoI site has been ablated in the CMV promoter. This change does not reduce expression. 
•    KanR cassette – This KanR resistance region is not a wild type sequence, it has been modified to remove all restriction sites that conflict with the SnapFast system and to retain a high level of antibiotic resistance. It has been validated in E.coli.
•    Blasticidin resistance gene – The blast gene has been modified to remove StuI, BsgI and SacI sites. These changes did alter the amino acid sequence of the protein and do not effect resistance.  
•    The Ubiquitin (Ub) promoter in this vector has been modified to remove BseRI, StuI and SpeI sites. These changes do not reduce expression.

Restriction site notes: 
•    Bgl2, KpnI, SwaI and PmeI each cut the vector at two sites positioned to flank the promoter, start codon, origin, and KanR, respectively. 
•    BsgI and BseRI recognise non-palindromic DNA sequences and cleave upstream of their recognition sites, within the stop codon that is found inside the XbaI restriction site.