In this article we will learn about some of the cloning vectors commonly used in academic and industrial laboratories.

In the previous article, we learned about the various essential features of any generic cloning vector. In the light of that information, we will now discuss the specifics of those features for particular vector categories frequently used in regular cloning procedures: plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes and yeast artificial chromosomes. Bacteriophages are also very commonly used vectors in molecular cloning, as described in detail in this article.

Article Contents:




What are cos sites?

Utility in molecular cloning


Bacterial Artificial Chromosome (BAC)

Utility in molecular cloning

Yeast Artificial Chromosome (YAC)



For cloning and expressing DNA fragments of interest, plasmids are the more widely used vectors. Let us look at their specific distinguishing features:

Ori (origin of replication):

Each plasmid has an ori, or origin of replication, which ensures its replication within the host cell independent of the host genome replication. Plasmid replicons are compatible with replication machinery of bacterial hosts.

Whether two plasmid vectors would be compatible within one host cell is largely determined by their respective replicons and their corresponding mechanisms of replication regulation. Details of this concept are given in our earlier cloning vectors article.

Thus, the plasmid’s ori ensures its stable replication in the host cell, thereby achieving requisite copies of the cloned transgene, and determines its compatibility with other plasmid or other vectors.

Selectable marker:

Plasmids generally possess antibiotic resistance cassettes as their selectable markers. These markers enable researchers to confirm whether a cell was properly transformed with the proper plasmid containing the gene of interest.

This confirmation is based on whether a cell survives selection pressure, meaning, if the cell contains a plasmid with the antibiotic resistance marker, it will survive on a plate with the corresponding antibiotic.

Common antibiotic classes for which resistance cassettes are used in plasmids are macrolides (erythromycin), aminoglycosides (kanamycin, gentamicin), beta lactams (ampicillin), quinolones (nalidixic acid), and tetracyclines (tetracycline).

Another type of selection on some plasmids is enabled by auxotrophy complementation markers.

An auxotroph is mutant in its ability to synthesize at least one compound necessary for its growth. Commonly such mutations exist in amino acid biosynthesis pathways. Thus, an auxotrophic mutant strain fails to grow unless the culture media is supplemented with the specific compound for which it is deficient in biosynthetic capability.

A host cell, such as a bacterium or yeast that can synthesize on its own all of the compounds necessary for survival is called a prototroph.

The concept of auxotrophic mutation and how that is exploited using selectable markers on plasmids is illustrated in figure 1.

Auxotrophic mutants and their utility in selection of transformants
Figure 1: Auxotrophic mutants and their utility in selection of transformants

Figure 1A illustrates auxotrophic mutants. Auxotrophic mutants are deficient in the biosynthesis of a compound necessary for the cell's survival. Commonly this is an amino acid. When plated on minimal media, an auxotrophic mutant fails to survive, while a prototroph (wild type strain that can synthesize all necessary compounds for growth) produces colonies. When the auxotroph is grown on minimal media supplemented with that specific compound (for example, an amino acid) that it is deficient in synthesizing, it produces colonies. This is illustrated in figure 1B.

This scenario can be exploited during selection of transformants, for example, cells that have the vector. The auxotrophic strain does not grow when plated on minimal media. However, when it is transformed with a plasmid that complements for this mutation (that is, the plasmid has the biosynthetic gene that the host auxotrophic strain is mutant in), it produces colonies on minimal media.

Expression sequence elements:

Plasmids are usually used in experiments where bacteria and yeast strains serve as hosts. Therefore, the promoters and ribosome binding sequences are appropriate for bacterial/ yeast RNA polymerases and bacterial/ yeast ribosomes respectively.

Promoters on plasmids may be constitutively active, ensuring expression of the cloned transgene throughout the entire culture period.

Alternatively, and more often, they can be conditional. For example, plasmids can have:

  • Lac promoter (pLac) can be used for transgene transcription by adding lactose or its analog IPTG. Our article about IPTG induction has a lot more information about how this works.
  • Tac promoter (pTac) can be used similarly as pLac, but with tighter expression control.
  • araBAD promoter (pAra) can be induced for expression of cloned DNA fragment by adding arabinose to the culture media.

Most conditional promoters used in common plasmids exploit inducible or repressible promoter-driven gene expression mechanisms seen in bacterial and viral operons.

Multiple Cloning Site (MCS):

Each plasmid has its characteristic multiple cloning site, or MCS, containing multiple specific restriction endonuclease cleavage sites.

A plasmid MCS can be used to clone inserts typically less than 5kb in size, but at the most 10kb using specialized high-capacity plasmids.

Transmission to daughter cells: Plasmids have a highly organized mechanism by which they are partitioned to the two progeny cells during cell division. Though variations have been reported for specific plasmids, the general process of plasmid transmission is as follows. On the plasmid, there exists a site which is similar to eukaryotic centromeres. A protein called centromere-like region (on the plasmid) binding protein (CBP) binds to that region on the plasmid and pairs sister plasmids (two copies of the same plasmid). Then another protein called NTPase segregates each sister to each daughter cell thereby ensuring that each progeny receives exactly half the copies of each plasmid type. A schematic representation is depicted in figure 2.

Plasmid compatibility/ incompatibility determined by plasmid partitioning mechanism to daughter cells

Figure 2: Plasmid compatibility/ incompatibility determined by plasmid partitioning mechanism to daughter cells.

The left panel in figure 2 shows how compatible plasmids get transmitted to daughter cells from the parent. Here two different types of plasmids (type 1 and 2) are transmitted to daughter cells via two different centromere-like region binding proteins (CBPs). Each CBP binds to its corresponding plasmid type. As a result, each daughter cell receives both types of plasmids.

The right panel depicts the incompatibility issue between the two plasmids (type 1 and 2), rising from their improper transmission to daughter cells. Here the same CBP binds to both plasmid types. Thus, the daughter cells might end up with either plasmid type 1 or plasmid type 2 (that is, uneven distribution).

In the host cell, when two different plasmid types have the same centromere-like regions, they compete for the same CBP and NTPase proteins. If the concentration of any of these proteins is limited, then proper partitioning of each plasmid type to the two daughter cells is hampered.

Conversely, when two different plasmids have the same centromere-like regions, then the partition proteins cannot correctly identify and pair sister plasmids. In this case it is possible that both copies of the same plasmid end up getting transmitted to the same daughter cell and the other progeny is deprived of any copy of that plasmid leading to plasmid loss in that cell.

Thus, partitioning of two plasmids is another major factor that determines their compatibility.

While plasmids are the most used vectors, other vectors are also often used in molecular cloning. They are very similar to plasmids and differ mostly in two ways: how they are replicated, and the insert size they can hold.

Keeping this in mind, we will describe some other vector types highlighting their distinctive characteristics.


Phagemid (Phage+Plasmid) is a plasmid that also has certain bacteriophage-like properties that might be advantageous to have in a cloning vector. Let us illustrate this by describing the distinguishing features of phagemids, which lie in their method of replication.

Ori (origin of replication):

Phagemids, unlike genuine plasmids, have two origins of replication. The first ori is like a plasmid. It is suitable for the vector’s replication within the bacterial host cell. Therefore, ori, drives a double-stranded (ds) mode of vector DNA replication, characteristic of bacterial genomes and plasmid DNA sequences.

There is a second sequence element (for the second ori) in the vector’s DNA backbone which facilitates two things:

  • single-stranded (ss) replication of the phagemid DNA
  • subsequently, the packaging of this ss-phagemid DNA into a bacteriophage particle. Here lies the similarity of phagemids with bacteriophages, and hence its name.

Within the bacterial host cell, a phagemid behaves like a plasmid. It replicates copies using its plasmid-type ori. It confers resistance to a particular antibiotic(s), depending on its selectable marker gene cassette. Transgenes can be cloned into its MCS and expressed. Further, phagemids, just like plasmids, can be introduced into bacterial hosts in the laboratory setting using horizontal transfer methods like transformation.

Filamentous phages are a specific category of bacteriophages; for instance, the M13 phage is a common example.

When a bacterial host that contains phagemids is infected by a filamentous phage, the virus provides the necessary machinery within the cell to initiate ss-replication from the specialized phagemid ori (please note that this is not the plasmid-type ori that drives ds-DNA replication).

This makes the phagemid produce ss-copies of itself, which then packages into the filamentous phage particle and emerges out of the bacterial host cell. A representation of phagemids as a cloning vector and how they can be transmitted using bacteriophages is depicted in Figure 3.

Representation of how a phagemid vector is used in molecular cloning.

Figure 3: Representation of how a phagemid vector is used in molecular cloning.

Figure 3 illustrates how DNA inserts can be cloned in phagemids just like how cloning is done with regular plasmids.

This typically involves digestion with restriction endonucleases and ligation of the insert into the digested vector. The recombinant phagemid produced can be introduced into the host cells via transformation techniques. However, when the transformant host cell is infected with a helper filamentous phage such as M13, the phage provides the necessary machinery to drive ss-DNA replication of the phagemid and its packaging into the viral particles that emerge from the host cell.

This ss-phagemid replication and packaging is driven by the sequence elements in the phage ori (shown in orange) on the vector backbone.

The following is detailed in figure 3: a) RE-digestion, and b) ligation of phagemid and insert fragment to produce the recombinant phagemid molecule c) transfer of the recombinant phagemid via transformation into the host bacterial cell. If this host is infected by a helper filamentous phage, the phagemid can undergo ss-DNA replication and get packaged into the virus d) host cells that contain the phagemid can be positively selected using the selectable marker gene present on the phagemid backbone.

For details about how a bacteriophage infects a bacterial cell and is instrumental in horizontal gene transfer, please refer to this article. In brief, the filamentous phage injects its genome within the host bacterial cytoplasm which now encodes the machinery necessary for the phagemid’s ss-DNA replication, packaging into new viral particles and secreting out of the host cell. Since the infecting filamentous phage ‘helps’ in phagemid transmission, it is known as a ‘helper’ phage.

For laboratory purposes, the helper phage is engineered so that the phage genome is less effective than the phagemid to replicate itself and package out as new phage particles. This ensures that following phage infection of the phagemid-containing bacterial culture, the resultant phage particles obtained primarily contain phagemid DNA instead of the phage genome.

Phagemid cloning capacity:

Another feature that sets phagemids apart from plasmids is in their capacity to hold larger DNA insert fragments - up to 20-25kb in size.


Cosmids are plasmid vectors containing specialized sequences that facilitate their packaging into lambda phage capsids. The specialized sequences are known as cos sites. Hence the name cosmid: cos site containing plasmid.

What are cos sites?

Cosmids are circular double-stranded DNA (ds-DNA) molecules like plasmids. However, they have a sequence where the ds-DNA backbone is nicked on each strand. The nicking sites on the two strands are situated 12 base pairs apart, making the circular molecule linear with 12-base single-stranded DNA overhangs.

Such a linear (rather than circular, like ordinary plasmids) structure is critical for the vector to be packaged into the lambda phage capsid.

The 12-base single-stranded overhangs are sticky ends that readily undergo cohesion; hence the name cos site. A representation of a cosmid as a cloning vector is depicted in Figure 4.

Representation of how cosmids are used as vectors in molecular cloning.

Figure 4: Representation of how cosmids are used as vectors in molecular cloning.

Cosmids, like phagemids and plasmids, need to be digested along with the insert (figure 4 a), followed by the ligation with the latter (figure 4 b) to create the recombinant cosmid vector.

However, transformation efficiency of recombinant cosmids is relatively low owing to their large size. To get around this issue, recombinant cosmids are packed into lambda phage particles by using their cos sites (figure 4 c) and introduced into the host cells by phage infection (figure 4 d). Recombinant host cell clones are positively selected using antibiotic resistance cassettes (selectable marker gene) encoded by the cosmids (figure 4 e).

Utility in molecular cloning

Cosmids provide all the features a plasmid offers in cloning. They replicate within bacterial host cells. Using antibiotic resistance cassettes, presence of cosmids in host cells can be positively selected for. Further, their MCS has a much larger insert loading capacity than plasmids of around 40-45kb.

Additionally, they can be transmitted via lambda phage mediated transduction due to their cos site, which is not possible with regular plasmids.


Fosmid is a variation of a cosmid with a very low copy number of one per host cell.

This copy number of one copy per cell is because the ori driving replication of this vector in the bacterial host is derived from an F plasmid. The ori for intra-cellular replication of this plasmid is such that it drives only one to two copies per cell.

F plasmid gets its name because it confers fertility to the bacterial cell that possesses it. It was the first plasmid to be reported. A bacterial cell that has this plasmid is capable of conjugation, a method of horizontal gene transfer in bacteria (relevant link to previous article). This plasmid has two oris. One drives the vector’s low copy number replication within the host cell; this ori is used in fosmids. The second ori drives inter cellular conjugative transfer.

Fosmids have very high cloning capacity of 100kb insert size. They are used in constructing genomic DNA libraries from big and complicated genomes. Fosmids mostly have been optimized to be used with E. coli as hosts. The selectable markers mostly reported in fosmids are kanamycin and ampicillin.

Bacterial Artificial Chromosome (BAC)

Another type of vector based on F plasmid is BAC. Like a chromosome, this vector can hold very large insert fragments of around 150-300kb size. This explains the phrase artificial chromosome. Additionally, they are stably maintained in E. coli. Taken together, this justifies the name of this vector bacterial artificial chromosome.

Utility in molecular cloning

Because of their ability to hold very large cloned sequences, complex eukaryotic genes with all their regulatory elements can be cloned in their native orientation using BACs.

Human disease-causing genetic loci can be big with highly complicated gene-specific regulatory elements such as promoters. To study these genes properly it is often necessary to clone them along with all their original regulatory sequences in proper orientation to obtain a real holistic picture of the gene’s regulation and dysregulation. BACs with their huge cloning capacity facilitate such studies. Cancer-related genetic loci within the human genome and those associated neurogenerative illnesses such as Alzheimer’s and Parkinson’s disease have extensively been studied using BACs.

Yeast Artificial Chromosome (YAC)

A vector similar to BAC, based on yeast’s genomic DNA is the yeast artificial chromosome (YAC). In a YAC, yeast DNA is joined to a bacterial plasmid. The host in this case is yeast.

A YAC can accommodate up to 1000kb of cloned DNA. Because of this, they were used during the initial days of analysis of whole genomes of higher organisms such as mouse and the plant Arabidopsis.

Further, YACs were initially used in the Human Genome Project, but were replaced with BACs due to some of these technical reasons:

  • Yield of YAC DNA from a yeast recombinant clone is very low.
  • Cloning efficiency is low.
  • YACs are significantly less stable than BACs. In YAC clones there frequently (as high as 50%) occur deletions, insertions, recombination, and other rearrangements within the cloned fragment. This can lead to erroneous interpretation of results when YACs are used in chromosome mapping.
  • BACs can be created faster than YACs.


Lai. C. BAC transgenics: Cell type specific expression in the nervous system. Encyclopedia of neuroscience. 2009.

Nierman W.C. and Feldblyum T.V. Genomic library. Encyclopedia of genetics. 2001. Pages 865-872.

Saraswathy. N and Ramalingam. P. High capacity vectors. Concepts and techniques in genomics and proteomics. 2011. Pages 49-56.

Qi. H et al, 2012. Phagemid vectors for phage display, properties, characteristics and construction. J Mol Biol. 2012. 417(3):129-43. Doi: 10.1016/j.jmb.2012.01.038

Rosenberg S.M and Hastings P.J. F-factor. Encyclopedia of genetics. 2001. Pages 877-880.

Thomas C.M. Plasmid incompatibility. Molecular life sciences. 2014.

Yang X.W and Lu X.H. The BAC transgenic approach to study Parkinson’s disease in mice. Parkinson’s disease: Molecular and therapeutic insights from model systems. 2008. Chapter 19: 247-268.