pTRIOZ-hIgG4 (S228P)

PLASMID FEATURES

Cassette 1: mAb Heavy chain

• AldA enh/ hFerH: This composite promoter combines the human aldehyde dehydrogenase (aldA) enhancer and the core promoter of the human ferritin heavy chain gene.
• MCS1: To facilitate cloning of the variable heavy (VH) chain, the multiple cloning site contains restriction sites that are compatible with many different enzymes,
• hIgG4: The constant region of the human immunoglobulin IgG4 heavy chain with the core-hinge stabilizing S228P mutation.
• βGlo pAn: The human beta-globin 3’UTR and polyadenylation sequence allow the efficient arrest of the transgene transcription.

Cassette 2: mAb Light chain

• hCMV enh / hFerL prom: This composite promoter combines the human cytomegalovirus (CMV) immediate-early gene 1 enhancer and the core promoter of the human ferritin light chain gene.
• MCS2: To facilitate cloning of the variable light (VL) chain, the multiple cloning site contains restriction sites that are compatible with many different enzymes,
• Human κ light chain: The constant region of the human kappa light chain
• SV40 pAn: The Simian Virus 40 late polyadenylation signal enables efficient cleavage and polyadenylation reactions resulting in high levels of steady-state mRNA

Cassette 3: Zeocin™ selection

• mCMV/hEF1-HTLV prom: This composite promoter combines mouse cytomegalovirus (mCMV) immediate-early gene 1 enhancer, the elongation Factor-1α (EF-1α) core promoter, as well as the R  segment and part of the U5 sequence (R-U5’) of the Human T-Cell Leukemia Virus (HTLV) type 1 long terminal repeat. The EF-1α promoter exhibits a strong activity and yields a long-lasting expression of a transgene in vivo. The R-U5’ has been coupled to the EF-1α core promoter to enhance the stability of RNA.
• EM7 prom: This is a bacterial promoter that enables the constitutive expression of the antibiotic resistance gene in E. coli. EM7 is located within an intron and is spliced out in mammalian cells.
• Sh Ble gene: Resistance to Zeocin™ is conferred by the Sh ble gene from Streptoalloteichus hindustanus. The same gene confers resistance in both mammalian cells and E. coli.
• hEF-1alpha pAn: This provides a strong polyadenylation signal. InvivoGen uses a sequence that starts after the stop codon of the EF1 cDNA and finishes after a bent structure rich in GT.

General features: pTRIOZ

• 5’ UTR: The 5’ UTR enhances mRNA stability and protein translation.
• Ori: A minimal E. coli origin of replication.