Editing Cloning vector (section)

===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>