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Features: Origin of Replication

Modern artificially-constructed vectors contain several essential components to function properly, including an origin of replication to maintain the vector in host cells, a promoter to drive transcription of the vector's genes, and a cloning site to insert foreign DNA. Vectors also contain additional optional features like genetic markers, antibiotic resistance genes, epitopes, and reporter genes to identify cells containing the vector. Expression vectors may further include targeting sequences or protein purification tags.

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58 views2 pages

Features: Origin of Replication

Modern artificially-constructed vectors contain several essential components to function properly, including an origin of replication to maintain the vector in host cells, a promoter to drive transcription of the vector's genes, and a cloning site to insert foreign DNA. Vectors also contain additional optional features like genetic markers, antibiotic resistance genes, epitopes, and reporter genes to identify cells containing the vector. Expression vectors may further include targeting sequences or protein purification tags.

Uploaded by

AviKumar
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Features

Modern artificially-constructed vectors contain essential components as well as


other additional features:

Origin of replication: Necessary for the replication and maintenance of the


vector in the host cell.

Promoter: Promoters are used to drive the transcription of the vector's


transgene as well as the other genes in the vector such as the antibiotic
resistance gene. Some cloning vectors need not have a promoter for the
cloned insert but it is an essential component of expression vectors so that
the cloned product may be expressed.

Cloning site: This may be a multiple cloning site or other features that allow
for the insertion of foreign DNA into the vector through ligation.

Genetic markers: Genetic markers for viral vectors allow for confirmation
that the vector has integrated with the host genomic DNA.

Antibiotic resistance:

Vectors

with

antibiotic-resistance open

reading

frames allow for survival of cells that have taken up the vector in growth
media containing antibiotics through antibiotic selection.

Epitope: Vector contains a sequence for a specific epitope that is


incorporated into the expressed protein. Allows for antibody identification of
cells expressing the target protein.

Reporter genes: Some vectors may contain a reporter gene that allow for
identification of plasmid that contains inserted DNA sequence. An example
is lacZ- which codes for the N-terminus fragment of -galactosidase, an

enzyme that digests galactose. A multiple cloning site is located within lacZ-,
and an insert successfully ligated into the vector will disrupt the gene
sequence, resulting in an inactive -galactosidase. Cells containing vector
with an insert may be identified using blue/white selection by growing cells in
media containing an analogue of galactose (X-gal). Cells expressing galactosidase (therefore doesn't contain an insert) appear as blue colonies.
White colonies would be selected as those that may contain an insert. Other
commonly used reporters include green fluorescent protein andluciferase.

Targeting sequence: Expression vectors may include encoding for a


targeting sequence in the finished protein that directs the expressed protein to
a specific organelle in the cell or specific location such as the periplasmic
space of bacteria.

Protein purification tags: Some expression vectors include proteins or


peptide sequences that allows for easier purification of the expressed protein.
Examples include polyhistidine-tag, glutathione-S-transferase, and maltose
binding protein. Some of these tags may also allow for increased solubility of
the target protein. The target protein is fused to the protein tag, but a protease
cleavage site positioned in the polypeptide linker region between the protein
and the tag allows the tag to be removed later.

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