Editing Cloning vector (section)

== Features of a cloning vector ==
All commonly used cloning vectors in [[molecular biology]] 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]] (ori).  The [[ColE1]] 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]].

===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]] (MCS) or polylinker, which contains many unique [[restriction enzyme|restriction sites]].  The restriction sites in the MCS are first cleaved by restriction enzymes, then a [[Polymerase chain reaction|PCR]]-amplified target gene also digested with the same enzymes is ligated into the vectors using [[DNA ligase]]. 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]] into another vector if necessary.{{citation needed|date=September 2022}}

Other cloning vectors may use [[topoisomerase]] 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]] 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.<ref>{{cite web |url=http://www.invitrogen.com/site/us/en/home/brands/Product-Brand/topo/The-Technology-Behind-TOPO-Cloning.html |title=The Technology Behind TOPO® Cloning |work=Invitrogen }}</ref>  Another method of cloning without the use of DNA digest and ligase is by [[Site-specific recombination|DNA recombination]], for example as used in the [[Gateway Technology|Gateway cloning system]].<ref>{{Cite book |chapter=Gateway cloning for protein expression |vauthors=Esposito D, Garvey LA, Chakiath CS |title=High Throughput Protein Expression and Purification |series=Methods in Molecular Biology |year=2009 |volume=498 |pages=[https://archive.org/details/highthroughputpr00shar/page/31 31–54] |doi=10.1007/978-1-59745-196-3_3 |pmid=18988017 |isbn=978-1-58829-879-9 |chapter-url-access=registration |chapter-url=https://archive.org/details/highthroughputpr00shar/page/31 }}</ref><ref>{{cite web |url=http://www.embl.de/pepcore/pepcore_services/cloning/cloning_methods/recombination/gateway/ |title=Cloning Methods - Recombination cloning systems |work=EMBL }}</ref> 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.<ref>{{cite web |url=http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning/Gateway-Technology.html |title=Gateway® Recombination Cloning Technology |work=Invitrogen}}</ref>

===Selectable marker===
A [[selectable marker]] is carried by the vector to allow the selection of positively [[Transformation (genetics)|transformed]] cells.  [[Antibiotic]] resistance is often used as marker, an example being the [[beta-lactamase]] gene, which confers resistance to  the [[penicillin]] group of [[beta-lactam antibiotics]] like [[ampicillin]].  Some vectors contain two selectable markers, for example the plasmid pACYC177 has both ampicillin and [[kanamycin]] resistance gene.<ref name = "Casali_2003">{{cite book | vauthors = Casali N, Preston A  |title= ''E. coli'' plasmid vectors |series=Methods in Molecular Biology |volume=235 |page=23 |url=https://books.google.com/books?id=r6QC0hTwsrwC&pg=PA23 |isbn=978-1-58829-151-6 |year=2003 }}</ref> 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]] and [[hygromycin B]] are effective in different cell types.  [[Auxotrophic]] selection markers that allow an auxotrophic organism to grow in [[minimal growth medium]] may also be used; examples of these are ''[[Leucine|LEU2]]'' and ''[[URA3]]'' which are used with their corresponding auxotrophic strains of yeast.<ref>{{cite journal | vauthors = Romanos MA, Scorer CA, Clare JJ | title = Foreign gene expression in yeast: a review | journal = Yeast | volume = 8 | issue = 6 | pages = 423–488 | date = June 1992 | pmid = 1502852 | doi = 10.1002/yea.320080602 | s2cid = 15674832 }}</ref>

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]],<ref>{{cite journal | vauthors = Yazynin SA, Deyev SM, Jucovic M, Hartley RW | title = A plasmid vector with positive selection and directional cloning based on a conditionally lethal gene | journal = Gene | volume = 169 | issue = 1 | pages = 131–132 | date = February 1996 | pmid = 8635737 | doi = 10.1016/0378-1119(95)00814-4 | url = https://zenodo.org/record/1258531 }}</ref> [[CcdA/CcdB Type II Toxin-antitoxin system|Ccda]],<ref>{{cite journal | vauthors = Bernard P | title = Positive selection of recombinant DNA by CcdB | journal = BioTechniques | volume = 21 | issue = 2 | pages = 320–323 | date = August 1996 | pmid = 8862819 | doi = 10.2144/96212pf01 | doi-access = free }}</ref> and the [[ParDE type II toxin-antitoxin system|parD/parE]] toxins.<ref>{{cite journal | vauthors = Gabant P, Van Reeth T, Drèze PL, Faelen M, Szpirer C, Szpirer J | title = New positive selection system based on the parD (kis/kid) system of the R1 plasmid | journal = BioTechniques | volume = 28 | issue = 4 | pages = 784–788 | date = April 2000 | pmid = 10769758 }}</ref><ref>{{cite journal | vauthors = Kim HG, Kim HS, Hwang HJ, Chung SK, Lee JM, Chung DK | title = Construction of a pTOC-T vector using GST-ParE toxin for direct cloning and selection of PCR products | journal = Biotechnology Letters | volume = 26 | issue = 21 | pages = 1659–1663 | date = November 2004 | pmid = 15604816 | doi = 10.1007/s10529-004-3518-z | s2cid = 10312859 }}</ref>  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 [[lac operon|''lacZ''α fragment]] for α complementation in [[blue white screen|blue-white selection]], and/or [[marker gene]] or [[reporter gene]]s in frame with and flanking the [[Multiple cloning site|MCS]] to facilitate the production of [[fusion protein]]s.  Examples of fusion partners that may be used for screening are the [[green fluorescent protein]] (GFP) and [[luciferase]].

===Elements for expression===
{{main |Expression vector}}
A cloning vector need not contain suitable elements for the [[Gene expression|expression]] of a cloned target gene, such as a [[Promoter (biology)|promoter]] and [[ribosomal binding site]] (RBS),  many however do, and may then work as an [[expression vector]].  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 [[Enzyme induction and inhibition|inducible]] so that proteins are only produced when required.  Some commonly used promoters are the [[T7 phage|T7]] and [[lac operon|''lac'' promoters]].  The presence of a promoter is necessary when screening techniques such as [[Blue white screen|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 ''[[Escherichia coli|E. coli]]'' cells.  Promoter and RBS for the cloned DNA sequence are also unnecessary when first making a [[Genomic library|genomic]] 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.