pVITRO2-MCS

Dual expression plasmids - Ferritin promoters - two MCS

Expression plasmids pVITRO2-MCS are developed mainly for in vitro studies.

pVITRO2-MCS allows the ubiquitous and constitutive co-expression of two genes of interest.

pVITRO2-MCS plasmids carry two human ferritin composite promoters, FerH (heavy chain) and FerL (light chain). To eliminate the iron regulation, their 5’UTRs have been replaced by the 5’UTR of the mouse and chimpanzee EF-1α genes.

pVITRO2-MCS plasmids are available with different selectable markers that are active both in E. coli and mammalian cells.

pVITRO2-MCS plasmids contain two multiple cloning sites (MCS) for the convenient cloning of your cDNAs of interest.

Specifications

Promoters: SV40/hFerH/mEF1α & CMV/hFerL/chEF1α

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

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

Cloning sites:

•   MCS1: 5’- Age I, Eco RV, Bam HI, Mlu I, Cla I, Sal I, Avr II -3’
•   MCS2: 5’- Sgr AI, Fsp I, Bgl II, Eco RI, Bst BI, Xho I, Nhe I -3’

Contents

pVITRO2-neo-mcs

• 20 µg of lyophilized DNA

pVITRO2-hygro-mcs

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

pVITRO2-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. pVITRO2-MCS are multigenic plasmids that contain two distinct transcription units (TU).

Strong and Constitutive Expression of Two Transgenes

Each pVITRO2-MCS 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 that are coordinately activated, and strong polyadenylation signals (polyA). pVITRO2-MCS plasmids contain two human ferritin composite promoters, FerH (heavy chain) and FerL (light chain), combined with the SV40 and CMV enhancers, respectively. To eliminate the iron regulation, their 5’UTRs have been replaced by the  5’UTR of the mouse and chimpanzee EF-1α genes.

Single Selection Marker for E. coli and Mammalian Cells

pVITRO2-MCS plasmids are available with a selectable marker that is active both in E. coli and mammalian cells: bsr (blasticidin resistance), hph (hygromycin resistance) or 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

pVITRO2-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.

pVITRO2 is also available with  two reporter genes:
- GFP and LacZ
- GFP and SEAP
- Lucia luciferase and SEAP

pVITRO2 reporter plasmids can be used as control plasmids or cloning vectors.

Details

• hFerH and hFerL composite promoters: Ferritin is a 24 subunit protein composed of two subunit types, termed H (heavy) and L (light). Ferritin is ubiquitously expressed. Its synthesis is highly regulated by the iron status of the cell. To eliminate the iron regulation of the ferritin promoters, the 5’UTR of FerH and FerL have been replaced by the 5’UTR of the mouse and chimpanzee elongation factor 1 (EF1) genes, respectively.

• SV40 enhancer which is comprised of a 72-base-pair repeat 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 includes the following restriction sites:
Age I, Eco RV, Bam HI, Sal I and Avr II
- Age I is compatible with Bsp EI and Sgr AI.
- Eco RV (blunt-end restriction enzyme)
- Bam HI is compatible with Bgl II, Bst YI and Bcl I.
- Sal I is compatible with Ava I and Xho I.
- Avr II is compatible with Xba I, Spe I and Nhe I.
MCS2 includes the following restriction sites:
Sgr AI, Bgl II, Xho I and Nhe I
- Sgr AI is compatible with Bsp EI and Age I.
- Bgl II is compatible with Bam HI, Bst YI and Bcl I.
- Xho I is compatible with Ava I and Sal I.
- Nhe I is compatible with Xba I, Spe I and Avr II.