pVITRO1-MCS

Expression plasmids pVITRO1-MCS are developed mainly for in vitro studies. pVITRO1-MCS allow the ubiquitous and constitutive co-expression of two genes of interest.

pVITRO1-MCS plasmids can be stably transfected in mammalian cells and the genes of interest are expressed at high levels.

pVITRO1-MCS plasmids carry two elongation factor 1 alpha (EF-1α) promoters, from rat and mouse origins combined to the CMV and SV40 enhancers respectively.

pVITRO1-MCS plasmids are available with different selectable markers that are active both in E. coli and mammalian cells.

pVITRO1-mcs plasmids contain two multiple cloning sites (MCS) for the convenient cloning of your cDNAs of interest.

Specifications

Promoters: CMV enhancer / rat EF-1α & SV40 enhancer / mouse EF-1α

Selection markers active both in E. coli and mammalian cells:

•   bsr (blasticidin resistance)
•   hph (hygromycin resistance)
•   neo (kanamycin/G418 resistance).

Cloning sites:

•   MCS1: 5’- Bsp EI, Bst 1107I, Bam HI, Bsi WI, Avr II -3’
•   MCS2: 5’- Age I, Eco RV, Bgl II, Bsr GI, Nhe I -3’

Contents

pVITRO1-neo-mcs

• 20 µg of lyophilized DNA

pVITRO1-hygro-mcs

• 20 µg of lyophilized DNA
• 1 ml of Hygromycin B Gold (hygromycin) at 100 mg/ml

pVITRO1-blasti-mcs

• 20 µg of lyophilized DNA
• 2 x 1 ml blasticidin at 10 mg/ml

Product is shipped at room temperature

Description

Gene Combinations in a Single Plasmid

Co-expression of two or more genes from a single vector is more  efficient and convenient than using two separate vectors. pVITRO1-MCS  are multigenic plasmids that contain two distinct transcription units  (TU).

Strong and Constitutive Expression of Two Transgenes

Each pVITRO plasmid features two constitutive promoters that drive  high levels of expression from two separate TU in a large number of  mammalian cell lines. Transcriptional interference is minimized by using  promoters of different origins, and strong polyadenylation signals  (polyA).
pVITRO1-MCS plasmids carry two elongation factor 1 alpha  (EF-1α) promoters, from rat and mouse origins combined to the CMV and  SV40 enhancers respectively.

Single Selection Marker for E. coli and Mammalian Cells

pVITRO-MCS plasmids are available with a selectable marker that is active both in E. coli and mammalian cells: bsr (blasticidin resistance), hph (hygromycin resistance), neo (kanamycin/G418 resistance).
In bacteria, the resistance gene is expressed from the E. coli EM7 promoter. In mammalian cells, it is transcribed from the promoter  located 3’ of the Ori, as a polycistronic mRNA and translated through  the IRES of the Foot and Mouth Disease Virus.

Available with Two MCS

• pVITRO1-MCS plasmid contains two multiple cloning sites (MCS) for the convenient cloning of cDNAs. Open reading frames could be chosen from InvivoGen’s Gene A-List.

pVITRO1 is also available with  two reporter genes:
- GFP and LacZ
- GFP and SEAP
- Lucia luciferase and SEAP

pVITRO1-rep plasmids can be used as control plasmids or cloning  vectors.

Details

PLASMID FEATURES

• rEF1 and mEF1 prom: pVITRO1-MCS plasmids carry two elongation factor 1 alpha (EF-1α) promoters, from rat and mouse origins. Similarly to their human counterpart, both promoters display a strong activity that yield similar levels of expression. EF-1α promoters are expressed at high levels in all cell cycles and lower levels during G0 phase. EF-1α promoters are also non-tissue specific; they are highly expressed in all cell types.

• SV40 enhancer allows the enhancement of gene expression in a large host range. The enhancement varies from 2-fold in non-permissive cells to 20-fold in permissive cells.

• CMV enhancer: The major immediate early enhancer of the human
cytomegalovirus (HCMV) is composed of unique and repeated sequence motifs. The HCMV enhancer can substitute for the 72-bp repeats of SV40 and is severalfold more active than the SV40 enhancer.

• SV40 pAn: the Simian Virus 40 late polyadenylation signal enables efficient cleavage and polyadenylation reactions resulting in high levels of steady-state mRNA.

• pMB1 ori: a minimal E. coli origin of replication to limit vector size, but with the same activity as the longer Ori.

• FMDV IRES: The internal ribosome entry site of the Foot and Mouth Disease Virus enables the translation of two open reading frames from one mRNA with high levels of expression.

• EM7 is a bacterial promoter that enables the constitutive expression of the antibiotic resistance gene in E. coli.

•  Antibiotic resistance gene :
- Resistance to Blasticidin S is conferred by the bsr gene from Bacillus cereus.
- hph gene confers resistance to Hygromycin B both in E. coli and mammalin cells.
- The neo gene from Tn5 confers resistance to Kanamycin in E.coli and G418 in mammalian cells.
In bacteria, the resistance gene is expressed from the constitutive E. coli EM7 promoter. In mammalian cells, the resistance gene is transcribed from the rat EF-1α promoter as a polycistronic mRNA and translated via the FMDV IRES.

• EF1 pAn is a strong polyadenylation signal.

• MCS1 and MCS2: Each multiple cloning site contains several restriction sites that are compatible with many other enzymes, thus facilitating cloning.
MCS1 contains the following restriction sites: Bsp EI, Bst 1107I, Bam HI, Bsi WI and Avr II
- Bsp EI is compatible with Age I and Sgr AI.
- Bst 1107I (blunt-end restriction enzyme)
- Bam HI is compatible with Bgl II, Bst YI and Bcl I.
- Bsi WI is compatible with Acc 65I, Ban I and Bsr GI.
- Avr II is compatible with Xba I, Spe I and Nhe I.
MCS2 contains the following restriction sites: Age I, Eco RV, Bgl II, Bsr GI, and Nhe I
- Age I is compatible with Bsp EI and Sgr AI.
- Eco RV (blunt-end restriction enzyme)
- Bgl II is compatible with Bam HI, Bst YI and Bcl I.
- Bsr GI is compatible with Acc 65I, Ban I and Bsi WI.
- Nhe I is compatible with Xba I, Spe I and Avr II.