![PBR322 color](https://upload.wikimedia.org/wikipedia/commons/0/01/PBR322_color.svg)
A cloning vector is a small piece of
DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for
cloning
Cloning is the process of producing individual organisms with identical or virtually identical DNA, either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction. In the field of biotechnology, cl ...
purposes. The cloning vector may be DNA taken from a
virus
A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea.
Since Dmitri Ivanovsky's 1 ...
, the
cell
Cell most often refers to:
* Cell (biology), the functional basic unit of life
Cell may also refer to:
Locations
* Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery ...
of a higher organism, or it may be the
plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
of a bacterium. The
vector
Vector most often refers to:
*Euclidean vector, a quantity with a magnitude and a direction
*Vector (epidemiology), an agent that carries and transmits an infectious pathogen into another living organism
Vector may also refer to:
Mathematic ...
contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of
restriction site Restriction sites, or restriction recognition sites, are located on a DNA molecule containing specific (4-8 base pairs in length) sequences of nucleotides, which are recognized by restriction enzymes. These are generally palindromic sequences (beca ...
s. The vector and the foreign DNA may be treated with a
restriction enzyme
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined together by
molecular ligation. After a DNA fragment has been cloned into a cloning vector, it may be further
subcloned into another vector designed for more specific use.
There are many types of cloning vectors, but the most commonly used ones are genetically engineered
plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
s. Cloning is generally first performed using ''
Escherichia coli
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
'', and cloning vectors in ''E. coli'' include plasmids,
bacteriophage
A bacteriophage (), also known informally as a ''phage'' (), is a duplodnaviria virus that infects and replicates within bacteria and archaea. The term was derived from "bacteria" and the Greek φαγεῖν ('), meaning "to devour". Bacteri ...
s (such as
phage λ),
cosmids, and
bacterial artificial chromosome A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually '' E. coli''. F-plasmids play a crucial role because they contain partition ...
s (BACs). Some DNA, however, cannot be stably maintained in ''E. coli'', for example very large DNA fragments, and other organisms such as yeast may be used. Cloning vectors in yeast include
yeast artificial chromosome
Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, ''Saccharomyces cerevisiae'', which is then ligated into a bacterial plasmid. By inserting large fragments of DNA, from 100–1000 ...
s (YACs).
Features of a cloning vector
All commonly used cloning vectors in
molecular biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
have key features necessary for their function, such as a suitable cloning site and selectable marker. Others may have additional features specific to their use. For reason of ease and convenience, cloning is often performed using ''
E. coli''. Thus, the cloning vectors used often have elements necessary for their propagation and maintenance in ''E. coli'', such as a functional
origin of replication
The origin of replication (also called the replication origin) is a particular sequence in a genome at which replication is initiated. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by semi ...
(ori). The
ColE1
ColE1 is a plasmid found in bacteria. Its name derives from the fact that it carries a gene for colicin E1 (the ''cea'' gene). It also codes for immunity from this product with the ''imm'' gene. In addition, the plasmid has a series of mobility ...
origin of replication is found in many plasmids. Some vectors also include elements that allow them to be maintained in another organism in addition to ''E. coli'', and these vectors are called
shuttle vector A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species. Therefore, DNA inserted into a shuttle vector can be tested or manipulated in two different cell types. The main advantage of these ...
.
Cloning site
All cloning vectors have features that allow a gene to be conveniently inserted into the vector or removed from it. This may be a
multiple cloning site A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only ...
(MCS) or polylinker, which contains many unique
restriction sites Restriction sites, or restriction recognition sites, are located on a DNA molecule containing specific (4-8 base pairs in length) sequences of nucleotides, which are recognized by restriction enzymes. These are generally palindromic sequences (beca ...
. The restriction sites in the MCS are first cleaved by restriction enzymes, then a
PCR-amplified target gene also digested with the same enzymes is ligated into the vectors using
DNA ligase
DNA ligase is a specific type of enzyme, a ligase, () that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. It plays a role in repairing single-strand breaks in duplex DNA in living organ ...
. The target DNA sequence can be inserted into the vector in a specific direction if so desired. The restriction sites may be further used for
sub-cloning
In molecular biology, subcloning is a technique used to move a particular DNA sequence from a ''parent vector'' to a ''destination vector''.
Subcloning is not to be confused with molecular cloning, a related technique.
Procedure
Restriction e ...
into another vector if necessary.
Other cloning vectors may use
topoisomerase
DNA topoisomerases (or topoisomerases) are enzymes that catalyze changes in the topological state of DNA, interconverting relaxed and supercoiled forms, linked (catenated) and unlinked species, and knotted and unknotted DNA. Topological issues i ...
instead of ligase and cloning may be done more rapidly without the need for restriction digest of the vector or insert. In this
TOPO cloning TOPO cloning is a molecular biology technique in which DNA fragments are cloned into specific vectors without the requirement for DNA ligases. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosi ...
method a linearized vector is activated by attaching topoisomerase I to its ends, and this "TOPO-activated" vector may then accept a PCR product by ligating both the 5' ends of the PCR product, releasing the topoisomerase and forming a circular vector in the process. Another method of cloning without the use of DNA digest and ligase is by
DNA recombination, for example as used in the
Gateway cloning system. The gene, once cloned into the cloning vector (called entry clone in this method), may be conveniently introduced into a variety of expression vectors by recombination.
Selectable marker
A
selectable marker A selectable marker is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used in laboratory microbiology, molecular biology, a ...
is carried by the vector to allow the selection of positively
transformed cells.
Antibiotic
An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting bacterial infections, and antibiotic medications are widely used in the treatment and prevention of ...
resistance is often used as marker, an example being the
beta-lactamase
Beta-lactamases, (β-lactamases) are enzymes () produced by bacteria that provide multi-resistance to beta-lactam antibiotics such as penicillins, cephalosporins, cephamycins, monobactams and carbapenems (ertapenem), although carbapenems ...
gene, which confers resistance to the
penicillin
Penicillins (P, PCN or PEN) are a group of β-lactam antibiotics originally obtained from ''Penicillium'' moulds, principally '' P. chrysogenum'' and '' P. rubens''. Most penicillins in clinical use are synthesised by P. chrysogenum using ...
group of
beta-lactam antibiotics
β-lactam antibiotics (beta-lactam antibiotics) are antibiotics that contain a beta-lactam ring in their chemical
structure. This includes penicillin derivatives (penams), cephalosporins and cephamycins (cephems), monobactams, carbapenems and ...
like
ampicillin
Ampicillin is an antibiotic used to prevent and treat a number of bacterial infections, such as respiratory tract infections, urinary tract infections, meningitis, salmonellosis, and endocarditis. It may also be used to prevent group B strepto ...
. Some vectors contain two selectable markers, for example the plasmid pACYC177 has both ampicillin and
kanamycin
Kanamycin A, often referred to simply as kanamycin, is an antibiotic used to treat severe bacterial infections and tuberculosis. It is not a first line treatment. It is used by mouth, injection into a vein, or injection into a muscle. Kanamycin ...
resistance gene. Shuttle vector which is designed to be maintained in two different organisms may also require two selectable markers, although some selectable markers such as resistance to
zeocin
Zeocin is a trade name for a formulation of phleomycin D1, a glycopeptide antibiotic and one of the phleomycins from ''Streptomyces verticillus'' belonging to the bleomycin family of antibiotics. It is a broad-spectrum antibiotic that is eff ...
and
hygromycin B
Hygromycin B is an antibiotic produced by the bacterium '' Streptomyces hygroscopicus''. It is an aminoglycoside that kills bacteria, fungi and higher eukaryotic cells by inhibiting protein synthesis.
History
Hygromycin B was originally develo ...
are effective in different cell types.
Auxotrophic
Auxotrophy ( grc, αὐξάνω "to increase"; ''τροφή'' "nourishment") is the inability of an organism to synthesize a particular organic compound required for its growth (as defined by IUPAC). An auxotroph is an organism that displays this ...
selection markers that allow an auxotrophic organism to grow in
minimal growth medium may also be used; examples of these are ''
LEU2'' and ''
URA3
URA3 is a gene on chromosome V in ''Saccharomyces cerevisiae'' (yeast). Its systematic name is YEL021W. URA3 is often used in yeast research as a "marker gene", that is, a gene to label chromosomes or plasmids. URA3 encodes Orotidine 5'-phosphate ...
'' which are used with their corresponding auxotrophic strains of yeast.
Another kind of selectable marker allows for the positive selection of plasmid with cloned gene. This may involve the use of a gene lethal to the host cells, such as
barnase
Barnase (a portmanteau of "BActerial" "RiboNucleASE") is a bacterial protein that consists of 110 amino acids and has ribonuclease activity. It is synthesized and secreted by the bacterium '' Bacillus amyloliquefaciens'', but is lethal to the cel ...
,
Ccda, and the
parD/parE toxins. This typically works by disrupting or removing the lethal gene during the cloning process, and unsuccessful clones where the lethal gene still remains intact would kill the host cells, therefore only successful clones are selected.
Reporter gene
Reporter genes are used in some cloning vectors to facilitate the screening of successful clones by using features of these genes that allow successful clone to be easily identified. Such features present in cloning vectors may be the
''lacZ''α fragment for α complementation in
blue-white selection, and/or
marker gene In biology, a marker gene may have several meanings. In nuclear biology and molecular biology, a marker gene is a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA. In particular, there are tw ...
or
reporter gene
In molecular biology, a reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. Such genes are called reporters because the charac ...
s in frame with and flanking the
MCS to facilitate the production of
fusion protein
Fusion proteins or chimeric (kī-ˈmir-ik) proteins (literally, made of parts from different sources) are proteins created through the joining of two or more genes that originally coded for separate proteins. Translation of this ''fusion gene'' r ...
s. Examples of fusion partners that may be used for screening are the
green fluorescent protein
The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish ''Aequorea ...
(GFP) and
luciferase
Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. The name was first used by Raphaël Dubois who invented the words ''luciferin'' and ''luciferase'', ...
.
Elements for expression
A cloning vector need not contain suitable elements for the
expression
Expression may refer to:
Linguistics
* Expression (linguistics), a word, phrase, or sentence
* Fixed expression, a form of words with a specific meaning
* Idiom, a type of fixed expression
* Metaphorical expression, a particular word, phrase, o ...
of a cloned target gene, such as a
promoter and
ribosomal binding site A ribosome binding site, or ribosomal binding site (RBS), is a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome during the initiation of translation. Mostly, RBS refers ...
(RBS), many however do, and may then work as an
expression vector
An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for ...
. The target
DNA may be inserted into a site that is under the control of a particular promoter necessary for the expression of the target gene in the chosen host. Where the promoter is present, the expression of the gene is preferably tightly controlled and
inducible
Induction, Inducible or Inductive may refer to:
Biology and medicine
* Labor induction (birth/pregnancy)
* Induction chemotherapy, in medicine
* Induced stem cells, stem cells derived from somatic, reproductive, pluripotent or other cell typ ...
so that proteins are only produced when required. Some commonly used promoters are the
T7 and
''lac'' promoters. The presence of a promoter is necessary when screening techniques such as
blue-white selection are used.
Cloning vectors without promoter and RBS for the cloned DNA sequence are sometimes used, for example when cloning genes whose products are toxic to ''
E. coli'' cells. Promoter and RBS for the cloned DNA sequence are also unnecessary when first making a
genomic
Genomics is an interdisciplinary field of biology focusing on the structure, function, evolution, mapping, and editing of genomes. A genome is an organism's complete set of DNA, including all of its genes as well as its hierarchical, three-dim ...
or
cDNA library of clones since the cloned genes are normally subcloned into a more appropriate expression vector if their expression is required.
Some vectors are designed for transcription only with no heterologous protein expressed, for example for ''in vitro'' mRNA production. These vectors are called transcription vectors. They may lack the sequences necessary for polyadenylation and termination, therefore may not be used for protein production.
Types of cloning vectors
A large number of cloning vectors are available, and choosing the vector may depend upon a number of factors, such as the size of the insert, copy number and cloning method. Large insert may not be stably maintained in a general cloning vector, especially for those with a high copy number, therefore cloning large fragments may require more specialised cloning vector.
Plasmid
Plasmids are autonomously replicating circular extra-chromosomal DNA. They are the standard cloning vectors and the ones most commonly used. Most general plasmids may be used to clone DNA insert of up to 15 kb in size. One of the earliest commonly used cloning vectors is the
pBR322
pBR322 is a plasmid and was one of the first widely used '' E. coli'' cloning vectors. Created in 1977 in the laboratory of Herbert Boyer at the University of California, San Francisco, it was named after Francisco Bolivar Zapata, the postdoctora ...
plasmid. Other cloning vectors include the
pUC series of plasmids, and a large number of different cloning plasmid vectors are available. Many plasmids have high copy number, for example
pUC19
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers. The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the abbreviation for the University of California, where early ...
which has a copy number of 500-700 copies per cell, and high copy number is useful as it produces greater yield of recombinant plasmid for subsequent manipulation. However low-copy-number plasmids may be preferably used in certain circumstances, for example, when the protein from the cloned gene is toxic to the cells.
Some plasmids contain an
M13 bacteriophage
M13 is one of the Ff phages (fd and f1 are others), a member of the family filamentous bacteriophage ( inovirus). Ff phages are composed of circular single-stranded DNA ( ssDNA), which in the case of the m13 phage is 6407 nucleotides long and i ...
origin of replication and may be used to generate single-stranded DNA. These are called
phagemid
A phagemid or phasmid is a DNA-based cloning vector, which has both bacteriophage and plasmid properties. These vectors carry, in addition to the origin of plasmid replication, an origin of replication derived from bacteriophage. Unlike commonly u ...
, and examples are the
pBluescript series of cloning vectors.
Bacteriophage
The bacteriophages used for cloning are the
λ phage
''Enterobacteria phage λ'' (lambda phage, coliphage λ, officially ''Escherichia virus Lambda'') is a bacterial virus, or bacteriophage, that infects the bacterial species ''Escherichia coli'' (''E. coli''). It was discovered by Esther Leder ...
and
M13 phage
M13 is one of the Ff phages (fd and f1 are others), a member of the family filamentous bacteriophage ( inovirus). Ff phages are composed of circular single-stranded DNA ( ssDNA), which in the case of the m13 phage is 6407 nucleotides long and ...
. There is an upper limit on the amount of DNA that can be packed into a phage (a maximum of 53 kb), therefore to allow foreign DNA to be inserted into phage DNA, phage cloning vectors may need to have some non-essential genes deleted, for example the genes for
lysogeny
Lysogeny, or the lysogenic cycle, is one of two cycles of viral reproduction (the lytic cycle being the other). Lysogeny is characterized by integration of the bacteriophage nucleic acid into the host bacterium's genome or formation of a circu ...
since using phage λ as a cloning vector involves only the lytic cycle. There are two kinds of λ phage vectors - insertion vector and replacement vector. Insertion vectors contain a unique cleavage site whereby foreign DNA with size of 5–11 kb may be inserted. In replacement vectors, the cleavage sites flank a region containing genes not essential for the lytic cycle, and this region may be deleted and replaced by the DNA insert in the cloning process, and a larger sized DNA of 8–24 kb may be inserted.
There is also a lower size limit for DNA that can be packed into a phage, and vector DNA that is too small cannot be properly packaged into the phage. This property can be used for selection - vector without insert may be too small, therefore only vectors with insert may be selected for propagation.
Cosmid
Cosmids are plasmids that incorporate a segment of bacteriophage λ DNA that has the cohesive end site (''cos'') which contains elements required for packaging DNA into λ particles. It is normally used to clone large DNA fragments between 28 and 45 Kb.
Bacterial artificial chromosome
Insert size of up to 350 kb can be cloned in
bacterial artificial chromosome A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually '' E. coli''. F-plasmids play a crucial role because they contain partition ...
(BAC). BACs are maintained in ''E. coli'' with a copy number of only 1 per cell.
BACs are based on
F plasmid
F, or f, is the sixth letter in the Latin alphabet, used in the modern English alphabet, the alphabets of other western European languages and others worldwide. Its name in English is ''ef'' (pronounced ), and the plural is ''efs''.
Hist ...
, another artificial chromosome called the
PAC is based on the
P1 phage
P1 is a temperate bacteriophage that infects ''Escherichia coli'' and some other bacteria. When undergoing a lysogenic cycle the phage genome exists as a plasmid in the bacterium unlike other phages (e.g. the lambda phage) that integrate into t ...
.
Yeast artificial chromosome
Yeast artificial chromosome are used as vectors to clone DNA fragments of more than 1 mega base (1Mb=1000kb) in size. They are useful in cloning larger DNA fragments as required in mapping genomes such as in the
Human Genome Project
The Human Genome Project (HGP) was an international scientific research project with the goal of determining the base pairs that make up human DNA, and of identifying, mapping and sequencing all of the genes of the human genome from both a ...
. It contains a telomeric sequence, an autonomously replicating sequence (features required to replicate linear chromosomes in yeast cells). These vectors also contain suitable restriction sites to clone foreign DNA as well as genes to be used as selectable markers.
Human artificial chromosome
Human artificial chromosome A human artificial chromosome (HAC) is a microchromosome that can act as a new chromosome in a population of human cells. That is, instead of 46 chromosomes, the cell could have 47 with the 47th being very small, roughly 6–10 megabases (Mb) in si ...
may be potentially useful as a gene transfer vectors for gene delivery into human cells, and a tool for expression studies and determining human chromosome function. It can carry very large DNA fragment (there is no upper limit on size for practical purposes), therefore it does not have the problem of limited cloning capacity of other vectors, and it also avoids possible insertional mutagenesis caused by integration into host chromosomes by viral vector.
Animal and plant viral vectors
Viruses that infect plant and animal cells have also been manipulated to introduce foreign genes into plant and animal cells. The natural ability of viruses to adsorb to cells, introduce their DNA and replicate have made them ideal vehicles to transfer foreign DNA into eukaryotic cells in culture. A vector based on
Simian virus 40 (SV40) was used in first cloning experiment involving mammalian cells. A number of vectors based on other type of viruses like
Adenoviruses
Adenoviruses (members of the family ''Adenoviridae'') are medium-sized (90–100 nm), nonenveloped (without an outer lipid bilayer) viruses with an icosahedral nucleocapsid containing a double-stranded DNA genome. Their name derives from the ...
and
Papilloma virus
''Papillomaviridae'' is a family of non- enveloped DNA viruses whose members are known as papillomaviruses. Several hundred species of papillomaviruses, traditionally referred to as "types", have been identified infecting all carefully inspected ...
have been used to clone genes in mammals. At present, retroviral vectors are popular for cloning genes in mammalian cells. In case of plants like
Cauliflower mosaic virus
Cauliflower mosaic virus (CaMV) is a member of the genus ''Caulimovirus'', one of the six genera in the family ''Caulimoviridae'', which are pararetroviruses that infect plants. Pararetroviruses replicate through reverse transcription just like ...
,
Tobacco mosaic virus
''Tobacco mosaic virus'' (TMV) is a positive-sense single-stranded RNA virus species in the genus ''Tobamovirus'' that infects a wide range of plants, especially tobacco and other members of the family Solanaceae. The infection causes characteri ...
and
Gemini viruses have been used with limited success.
Screening: example of the blue/white screen
Many general purpose vectors such as
pUC19
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers. The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the abbreviation for the University of California, where early ...
usually include a system for detecting the presence of a cloned DNA fragment, based on the loss of an easily scored phenotype. The most widely used is the gene coding for ''E. coli''
β-galactosidase
β-Galactosidase (EC 3.2.1.23, lactase, beta-gal or β-gal; systematic name β-D-galactoside galactohydrolase), is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides.
β ...
, whose activity can easily be detected by the ability of the enzyme it encodes to hydrolyze the soluble, colourless substrate
X-gal
X-gal (also abbreviated BCIG for 5-bromo-4-chloro-3-indolyl-β--galactopyranoside) is an organic compound consisting of galactose linked to a substituted indole. The compound was synthesized by Jerome Horwitz and collaborators in 1964. The formal c ...
(5-bromo-4-chloro-3-indolyl-beta-d-galactoside) into an insoluble, blue product (5,5'-dibromo-4,4'-dichloro indigo). Cloning a fragment of DNA within the vector-based ''lacZα'' sequence of the β-galactosidase prevents the production of an active enzyme. If X-gal is included in the selective agar plates, transformant colonies are generally blue in the case of a vector with no inserted DNA and white in the case of a vector containing a fragment of cloned DNA.
See also
*
Vector (molecular biology)
In molecular cloning, a vector is any particle (e.g., plasmids, cosmids, Lambda phages) used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vec ...
*
Plant transformation vector
Plant transformation vectors are plasmids that have been specifically designed to facilitate the generation of transgenic plants. The most commonly used plant transformation vectors are termed binary vectors because of their ability to replicat ...
*
IMAGE cDNA clones
IMAGE cDNA clones are a collection of DNA vectors containing cDNAs from various organisms including human, mouse, rat, non-human primates, zebrafish, pufferfish, Xenopus (frogs), and cow. Together they represent a more or less complete set of e ...
*
fosmid Fosmids are similar to cosmids but are based on the bacterial F-plasmid. The cloning vector is limited, as a host (usually '' E. coli'') can only contain one fosmid molecule. Fosmids can hold DNA inserts of up to 40 kb in size; often the source of t ...
*
Golden Gate Cloning
Golden Gate Cloning or Golden Gate assembly is a molecular cloning method that allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIS restriction enzymes and T4 DNA ligase. Th ...
References
{{Reflist, 2
Genetics techniques
Molecular biology
Cloning
Plasmids