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OG51 - pSF-U6

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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.

Product Code: OG51

Size (bp): 3932

Bacterial Antibiotic Selection: Kanamycin

Origin and Compatibility: pUC high copy, derived from pBR322

Copy Number: 500-700 copies per cell

Promoter: U6 promoter

Purpose: The expression of short hairpin RNA in eukaryotic cells. The U6 is transcribed by RNA polymerase III (RNA pol III), which terminates transcription at a string of thymidine residues, typically a minimum of five residues in mammalian cells. Unlike most SnapFastTM vectors, this vector contains an RNA pol III terminator downstream of the XhoI site, although it is more common to insert a terminator sequence at the end of the sequence you insert into the MCS that encodes a shRNA.

Transcription Termination: This plasmid contains an RNA pol III termination sequence (TTTTTT) between the XhoI and XbaI restriction sites. However, it is common to insert an additional terminator at the end of the shRNA sequence that you ligate into the vector. This plasmid also contains three alternative transcription terminators for mammalian, bacterial and bacteriophage (T7) expression, however, these relate to other products that we sell in the same backbone. This also allows you to exchange the RNA pol III promoter in this vector for any of our RNA pol II promoters if you want a standard protein expression plasmid. The presence of each terminator does not reduce expression in the alternative systems.

Inserting an siRNA: We recommend inserting shRNAs into this plasmid using the NotI as the 5' restriction site and the XhoI as the 3' restriction site. This should minimise the presence of extra nucleotides at either end of the shRNA following transcription.

The following sequence is an example of a shRNA sequence that could be generated by creating two oligo nucleotides that are complementary to each other, and produce overhangs that are compatible with NotI and XhoI. Green letters highlight the overhangs and blue letters highlight the remaining sequences of the NotI and XhoI sites (these could be removed from the oligos if the sites are not required after insertion into the vector). Italic letters highlight sequences that are complementary to the pGL3 luciferase coding sequence. The first string of italic letters match the luciferase coding sequence 5' to 3', and the second series of italic letters are self-complementary to this same region). Bold letters highlight the loop at the head of the hairpin and the underlined sequences highlight an inserted RNA pol III terminator that minimises the length of the shRNA produced.    

sirna-structure-for-h1-and-u6-pages

This is just one possible structure of an shRNA. Many other strategies and sequences are possible and may enhance knockdown. We cannot guarantee that this sequence will produce a functional siRNA. This sequence was used to validate the vector, and when used with other shRNAs it may not be the optimal sequence. 

Intellectual Property Status: According to our IP-friendly policy, this plasmid is sold free of reach-through rights on any derivatives you may create.

Quality Validation: This plasmid has been demonstrated to induce knockdown of mRNA transcripts from reporter genes when transfected into eukaryotic cells following the insertion of reporter gene specific hairpin sequences.   

Quality Sequencing Analysis: This vector has been fully sequenced using primers F1-F10. The sequences and details of these primers are available through our website.. Primer F2 does not bind this plasmid.

Genetic Modifications to standard parts:

  • 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.
  • MCS – See the expanded multiple cloning site below (page 3).

Restriction site notes:

  • SwaI and PmeI each cut the plasmid 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.