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CLONING AND SELECTABLE VECTORS
OriGene has two types available to our customers. All OriGene TrueClones are provided in a pCMV6 Cloning Vector and all are available for purchase as negative controls.
pCMV6-XL4
PCMV6-XL5
PCMV6-XL6
In addition, OriGene is offering a Neomycin resistant version of its pCMV cloning vector as a kit to those customer who wish to create a stable cell line from our TrueClones
pCMV6-NEO
Primer and Promoter sequences can be downloaded separately
Vector Description:
All of the cDNAs within the TrueClone collection are cloned into a series of pCMV vectors, enabling uniform screening methods. The vector plasmid was originally obtained from the laboratory of Dr. David Russell. The full-length cDNA fragments are directionally inserted downstream from a eukaryotic transcriptional promoter capable of driving heterologous gene expression in a variety of mammalian cell lines and transgenic mice.
The CMV promoter is covered under U.S. Patents 5,168,062 and 5,385,839 and its use is permitted for research purposes only. Any other use of the CMV promoter requires a license from the University of Iowa Research Foundation, 214 Technology Innovation Center, Iowa City, IA 52242.
All three OriGene pCMV6 vectors were constructed with the same features and multiple cloning site (MCS); with the exception, that pCMV-XL6 has an SP6 transcriptional promoter instead of T7. The basic backbone of the vector series is presented diagrammatically below.
Vector Sequences: Genbank accession number AF067196
pCMV6-XL4 pCMV6- XL5 pCMV6-XL6 pCMV6-Neo
Key Functional Features of pCMV6-XL4, XL5 & XL6 vectors:
- Vector size: 4.7kb
- Selection marker in E. coli: Ampicillin-resistance
- Selection marker in mammalian cells: None. For transient transfection only
- Promoter for in vivo expression in mammalian cells: CMV promoter
- Promoter for in vitro cell free system: T7 (for pCMV6-XL4 and pCMV6-XL5) and SP6 for (pCMV6-XL6)
- Cloning sites: EcoRI and SalI. While EcoRI is still preserved, SalI is destroyed upon cloning.
- Restriction sites for removing insert: NotI. *Two NotI sites are flanking the cloning sites in the vector.
- Cell line suitable for transfection: COS, 293, Hela, CHO, NIH3T3, Mouse L cell, etc.
- Transcription termination and polyadenylation signals: from human growth hormone (hGH) gene.
Extensive work has been done to engineer the vector to achieve high level of transgene expression level. When compared with another popular expression plasmid, pCDNA3.1 (Invitrogen), pCMV-based plasmids provide comparable if not higher level transgene expression.
Fig 1. Comparison of transgene expression level in pCMV6- and pCDNA3.1-based plasmids. CAT gene was cloned downstream of the promoters in the pCMV6 and pCDNA3.1 vectors. In three independent experiments, a same quantity of plasmid DNA was transfected into COS1 cell and the CAT activity was scored.
References:
Cloning, structure and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J Biol Chem. 1989 May 15; 264(14): 8222-9. Andersson S, Davis DL, Dahlback H, Jornvall H, Russell DW.
Expression cloning and regulation of steroid 5 alpha-reductase, an enzyme essential for male sexual differentiation. J Biol Chem. 1989 Sep 25;264(27):16249-55. Andersson S, Bishop RW, Russell DW
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