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Constitutive

OG412 - pSF-RecA-Fluc

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Vector: pSF-RecA-FLuc

Product Code: OG412

Quantity Provided: 5µg

Size (bp): 5475 bp

Bacterial Antibiotic Selection:
 Kanamycin

Origin and Compatibility:
 pUC high copy, derived from pBR322   

Copy Number:
 500-700 copies per cell

Promoter:
 Constitutive RecA bacterial promoter with the LexA repressor binding site deleted.

Purpose: The expression of the Firefly luciferase (FLuc, Photinus pyralis) reporter gene under the control of the RecA constitutive bacterial promoter. This plasmid can be used for monitoring reporter gene activity in bacterial cells or colonies using the luciferase substrate luciferin. 

Quality Validation: This vector has been demonstrated to express the luciferase reporter gene to high levels under the RecA promoter. The start codon of the luciferase reporter was positioned within the NcoI site and the stop codon was positioned within the XbaI site. Expression was validated in the Escherichia coli.  

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

Genetic Modifications to standard parts: 
•    RecA promoter – The LexA binding site has been ablated in this promoter to provide constitutive 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.

Restriction site notes: 
•    Bgl2, SwaI and PmeI each cut the vector at two sites positioned to flank the promoter, 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.tic Modifications to standard parts:

· RecA promoter – The LexA binding site has been ablated in this promoter to provide constitutive 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.

 

 

Restriction site notes:

· Bgl2, SwaI and PmeI each cut the vector at two sites positioned to flank the promoter, 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.