2012 Jan 1;3(1):38-43. In the Annotations and Tracks tab ensure the Concatenated Sequences annotation type is checked. [5] In this process, one needs to make sure that the introduced mutation will not affect the genetic function encoded by the sequence of interest. Existing primer_bind annotations with extension, 5. At this point we should also confirm that none of the sequences contain unwanted internal BsaI sites. The overhang can comprise any nucleotides, which allows BsaI to create 256 unique overhangs. Submit this PCR product sequence to you custom synthesis provider of choice. The inserts and cloning vectors are designed to place the Type IIS recognition site distal to the cleavage site, such that the Type IIS REase can remove the recognition sequence from the assembly. Unlike standard Type II restriction enzymes like EcoRI and BamHI, these enzymes cut DNA outside of their recognition sites and, therefore, can create non-palindromic overhangs. To confirm that our six fragments have recombined precisely to reform the GFP CDS We will define a CDS annotation for the region and examine the gene product. Golden Gate cloning or Golden Gate assembly[1] 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. We demonstrated proof … All of the Parts in this exercise, with the exception of the Backbone and GFP2, will require primer design and PCR in order to generate DNA suitable for the Golden Gate reaction. The Geneious Golden gate tool can only use a single type IIS enzyme. [10] This can be done using either level 2, or M and P. A variant version of level M and P is also provided by GoldenBraid. The alignment will show you that the reassembled sequence is identical to the original GFP sequence. Share. However, you might find that designing the right overhang sequences can be tedious, and Golden Gate Assembly is much less sequence independent than other cloning … The Golden Gate method uses Type IIs restriction enzymes in combination with DNA ligase. The GFP1 sequence provided for this tutorial contains a primer_bind annotation called “A primer“. Golden Gate cloning provides a precision module-based cloning technique for facile assembly of multiple genes in one construct. It make seamless (scarless) assembly of DNA fragments reaction is essentially irreversible 21. Alternately, you can use the Backbone: dropdown menu to specify that pGoldengate-SE7 should be the backbone  You can also use the Choose… button to navigate to this tutorial folder and select the pGoldengate-SE7 vector sequence as the backbone. The settings used are shown below. If you wish to design your Golden Gate primers with your own specific primer bind parameters, then you should design and add your own flanking primer_bind annotations to your sequences prior to performing assembly. Geneious will then choose one, or a combination of these, in order of precedence (see rules 1 to 6 below) to define the insert boundaries to be used for Golden gate recombination. Select the GFP1 ORF annotation (in orange), then hold down shift and select the GFP6 ORF annotation (in orange). [8], Level 2 vectors have two inverted BpiI sites from the insertion of level 1 modules. October 1, 2013. 3. By Karmella Haynes, 2013. A. For more information on the various options available in the Golden Gate window, you can click on the Help button in the bottom left corner of the window. -With Gibson, you can directly link the gene of interest with the tag with no extra amino acids added. Can assemble multiple components in a single reaction. A. This menu can be used to check, and if required, change whether existing primer_bind annotations are used as boundaries. In the past few years, a number of methods have been developed to facilitate and speed up this process. Does the Geneious Golden Gate tool exclude using overhang combinations where the same three or more consecutive nucleotides are present in another overhang used in the assembly? Video 1. [5] If starting from level -1 fragments, the level 0 modules do not need to be sequenced again, whereas if starting from level 0 modules, the modules must be sequenced. [7], MoClo utilizes a parallel approach, where all constructs from tier-one(level 0 modules) have restriction sites for BpiI on both sides of the inserts. Updated by Cassandra Barrett, 2016. Golden Gate Background: Golden Gate cloning is a strategy that allows ‘single-tube’ ordered assembly of a vector (Backbone) and one or more DNA fragments (Parts) into a single, usually circular, construct which is suitable for direct transformation of a bacterial host. The cloning steps consist of defining the part type, design primers containing BsaI restriction sites at the ends of the fragments, removing sites from internal sequences, and cloning the amplified fragments in a vector. Hierzu wird das TypIIS Restriktionsenzym (REase), das außerhalb seiner nicht-palindromischen Erkennungssequenz schneidet, und die T4 DNA Ligase mit den vorbereiteten Inserts und Vektoren zusammengebracht. Uncheck the Save intermediate products option as we do not need to see intermediate products. [8] Each cloning step needs to alternate the restriction site and the marker. Q. These sequences can then be submitted to an oligonucleotide synthesis company. To enable Golden Gate cloning into a single TII-RE site in common expression vectors, the first and last TIIS-RE sites of the assembled fragment array are … Existing primer_bind annotation(s) with valid type IIS cut site(s) on the extension, 4. For example, if you switch the order of fragments GFP1 and GFP2, you will see that the Backbone and GFP2 fragments turn red and are labelled Mismatched. Golden Gate Cloning is typically performed as an all-in-one-pot reaction. If Geneious finds a blunt end, and no suitable type IIS sites or primer_bind annotations are present, then a primer with an appropriate type IIS site extension will be designed. You should see the CDS translates to the complete GFP gene product starting MRK…, ending …LYK*, with no internal stop codons. You have the option to ignore or choose alternate primer_bind annotations associated with each Part. We wish to assemble this entire sequence. [8] Furthermore, two restriction enzymes are needed, where BpiI is used for releasing level 1 modules from level 1 constructs and BsaI/BsmBI is for digesting and opening the recipient level 2-n plasmid. The following reagents are supplied with this product: Show all Collapse all. Type IIS Assembly (Golden Gate) Updated 4/8/2016 9:53pm. You will notice that the first sequence, GFP1, is a blunt fragment with a central primer_bind annotation named A Primer. [7] Each tier-one construct and the vector have different overhangs on them yet complementary to the overhang of the next segment, and this determines the layout of the final multigene construct. Level M vectors are similar to level 2 vectors, but have a BsaI site located upstream of the two inverted BpiI sites. Sometimes referred to as MoClo, this strategy uses the Type IIS restriction enzymes BsaI and BpiI/BbsI to efficiently assemble up to six DNA fragments at a time. Email. To do this, select the Ligation of GFP1 – GFP2 – GFP3 – GFP4 – GFP5 – GFP6 into pGoldenGate-SE7 file. Linkedin. Since type … [5], Level -1 fragments are used to help cloning large level 0 modules. [8], As all level 1 vectors are binary plasmids, they are used for Agrobacterium mediated temporary expression in plants. The example below shows the GFP5 PCR product Part that would be generated during the exercise provided in this tutorial. In the multisegment assembly method Gateway, segments are added into the donor with additional att sequences, which overlap in those added segments, and this results in the segments separated by the att sequences. The Parts drop-down menu also provides an option to Reverse Complement Parts that are in the wrong orientation. The alignment will show you that the reassembled sequence is identical to the original GFP sequence. The six insert fragments, labelled GFP1 to GFP6 are also provided with this tutorial. [8] On one hand, this can induce more restriction sites in the construct, where this open construct allows additional genes be added. 07/18 Current best practices for assemblies of more than 10 modules often rely on two-step hierarchical approaches using different Type IIS restriction enzyme specificities at each step. If you wish, you can extract the CDS to a file and perform a pairwise alignment between the GFP reassembled sequence, and the original GFP sequence , which has also been provided with this tutorial. 0 . Golden Gate Assembly is a molecular cloning technique that utilizes simultaneous digestion with type IIS restriction enzymes and ligation by a DNA ligase to enable the scarless, ordered assembly of multiple fragments (1). A primer will be designed which incorporates the site. Prior to using the tool you should decide which type IIS restriction enzyme you are going to use. The Golden Gate tool will only map the primer_bind annotations to the output, if the primer_bind annotation matches the sequence 100%. Oracle Goldengate Step by Step Replication -1 Oracle Goldengate Architecture Create Replicat process on target DB Add Replicat Process GGSCI (Deveci) 1> add replicat RXFULL, exttrail /u01/goldengate/dirdat/x1, checkpointtable GOLDENGATE.CHKPTBL Start Replicat Process Login … With your backbone vector and six sequences selected, on the Tool bar click Cloning → Golden Gate… to start the Golden Gate tool. optimization. We use two Golden Gate cloning steps to generate new CRISPR-TSKO destination vectors. ... (known as Golden Gate cloning, PLoS ONE 3, e3647, 2008). [10] For counterselection, the two levels of plasmids differ in their antibiotic resistance markers. CS1 maint: multiple names: authors list (, https://msbi.ipb-halle.de/GoldenMutagenesisWeb/, http://www.addgene.org/browse/article/28196591/, "A One Pot, One Step, Precision Cloning Method with High Throughput Capability", "A Modular Cloning System for Standardized Assembly of Multigene Constructs", "Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes", "Overview of post Cohen-Boyer methods for single segment cloning and for multisegment DNA assembly", "Bricks and blueprints: methods and standards for DNA assembly", "GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules", https://en.wikipedia.org/w/index.php?title=Golden_Gate_Cloning&oldid=984820598, Creative Commons Attribution-ShareAlike License, This page was last edited on 22 October 2020, at 08:54. Overview of modular cloning system (MoClo) that uses Golden Gate cloning for all assembly steps. Golden Gate Assembly (GGA) was first described in Engler C, Kandzia R, Marillonnet S (2008) and Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) as an efficient way to quickly assembly multiple DNA sequences, or parts, into a single plasmid. Do not unzip the tutorial. Hit the “Zoom to Selection” button to zoom in on this region. This will open the Golden Gate Window. If your Parts are in a different order to that shown in the figure above, drag and drop the Tags to rearrange them to the correct order. Twitter. Exercise: Golden Gate Exercise FAQ: Frequently (and not so frequently) Asked Questions. [6], Although Golden Gate Cloning speeds up multisegment cloning, careful design of donor and recipient plasmids is required. With the six insert sequences selected and visible in the sequence viewer, select the Annotations and Tracks tab and ensure ORF, primer_bind and Restriction Site annotations are displayed. [5], If the level 0 modules contains any unwanted restriction site, they can be mutated in silico by removing one nucleotide from the type IIS restriction site. If one or more of your sequences contain type IIS restriction sites that you do not want be involved in the assembly then you will need to engineer each site out of your fragment, taking care to avoid altering any gene product sequences. Q. Geneious will analyse your backbone (if defined), and each sequence passed to it, and will detect existing type IIS restriction sites, overhang annotations, primer_bind annotations and blunt ends. One of these methods, Golden Gate cloning, allows assembling up to nine fragments at a time in a recipient … Glyco-engineering. [7], Golden Gate assembly's cloning standards have two tiers. Successful constructs can easily be identified through blue-white screening. Type IIS restriction enzymes cut DNA at a location adjacent to their non-palindromic recognition site. The amplified product is cloned in a recipient vector using Golden Gate cloning. Werner S, Engler C, Weber E, Gruetzner R, Marillonnet S. annotation and hover over it to bring up a yellow tool tip showing the details of the Annotation. Theoretically, as many as 36 genes can be assembled in one construct using 6 parallel level M reactions (each required for assembly of 6 genes per level M construct) followed by one final level P reaction. Each part has a dropdown menu accessible via an inverted triangle. Based on this property, a cloning strategy called ‘Golden Gate’ cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. [10] There are two levels of destination plasmids, level α and level Ω. [3], A typical thermal cycler protocol oscillates between 37 °C (optimal for restriction enzymes) and 16 °C (optimal for ligases) many times. Golden Gate Assembly and its derivative methods exploit the ability of Type IIS restriction endonucleases (REases) to cleave DNA outside of the recognition sequence. At each step, a single DNA fragment is transferred from a donor plasmid or PCR product to a recipient vector. Removal of unwanted internal Type IIS sites. A. This allows a fragment containing all assembled genes to be excised from the vector and subcloned in a next level of cloning (Level P). However, Geneious will interpret the primer_bind annotation as a fusion boundary (as per Rules 3-6 described in the Tutorial Introduction). Once you have your Parts “built”, the in vitro assembly method involves combining the parts in equimolar concentrations, along with a suitable type IIS enzyme and DNA ligase, then cycling between a temperature that favours restriction enzyme activity, and a temperature that favours ligation. Geneious will also assume that you have this “sticky ended” DNA available and so will not design primers for PCR. Unless told otherwise, Geneious will assume this annotation defines a Part boundary. Geneious will also assume that you have DNA available to use, and so will not design primers for PCR. pGGA is a 2,714 bp cloning vector useful for Golden Gate Assembly. We constructed a simplified acceptor plasmid, called pDOC-GG, for the assembly of multiple DNA fragments precisely and simultaneously to form a donor plasmid using Golden Gate assembly. However, generation of donor plasmids typically requires multiple cloning and screening steps. If the pGoldenGate-SE7 sequence is not leftmost, then select and drag it to the leftmost position. One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. If Geneious detects a single primer_bind annotation with a suitable type IIS site Geneious will assume you wish to use it and a new primer will not be designed. Die Golden Gate Assembly Reaktion läuft als simultane Single-Tube Reaktion ab. This cloning system should be useful for generating the multiple construct variants that will be required for developing gene networks encoding novel functions, and fine- tuning the expression levels of the various genes involved. [10] Each level of plasmids can be used as entry plasmids for the other level of plasmids for multiple times because both levels of plasmids have different type IIS restriction sites that are in inverted orientation. Based on this property, a cloning strategy called 'Golden Gate' cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. [5] For the purpose of Golden Gate Cloning, the internal sequences of level 0 modules should not contain type IIS restriction enzymes sites for BsaI, BpiI, and Esp3I while surrounded by two BsaI restriction sites in inverted orientation. This means that all DNA parts, the type IIs restriction enzyme and a ligase are mixed in a PCR tube and put into a thermocycler. [7] First-tier Golden Gate assembly constructs the single-gene construct by adding in genetic elements such as promoter, open reading frames, and terminators. Repeated cloning in level M and P vectors forms a loop that can be repeated indefinitely to assemble progressively large constructs. We provide here a protocol for DNA assembly using Golden Gate cloning, taking as an example the level of assembly of gene fragments to complete coding sequences, a level of cloning that can be used to perform DNA shuffling. [10] To add more genes to the construct, restriction sites of a different type IIS restriction enzyme need to be added to the destination vector. Inserts und Vektoren werden so designed, dass die TypIIS Erkennungsstelle distal zur Schnittstelle … [10], The Golden Gate Cloning principle can also be applied to perform mutagenesis termed Golden Mutagenesis. Checking the correct primer_bind boundaries are used. [5], Restriction enzyme DNA assembly has cloning standards to minimize the change in cloning efficiency and the function of the plasmid, which can be caused by compatibility of the restriction sites on the insert and those on the vector. Geneious Prime tutorials are installed by either 'Dragging and dropping' the zip file into Geneious Prime or using File → Import → From File... in the Geneious Prime menu. This will create a PCR product sequence for each Part. Die Golden-Gate-Klonierung (engl. If Geneious detects a primer annotation with an extension which does not contain a valid type IIS site then the 5′ terminus of the extension will be considered the fusion point and the extension will be extended to introduce a valid type IIS recognition site, resulting in a new primer sequence. The Golden Gate tool currently does not exclude using palindromic overhangs. Q. [5] Meanwhile, the original restriction sites, which are not ligated, can be redigested so that they can add more fragments into the plasmid. An introduction to Type IIS restriction enzymes and how they differ from classical typeII restriction enzymes. doi: 10.1371/journal.pone.0016765. In some cases, for instance when a preexisting type IIS site is utilised, the primer bind region will lie external to the fragment that is assembled, and so will be removed by digestion, and not be present in the final assembly. Mit Hilfe von Typ IIs Restriktionsenzymen und T4 DNA-Ligase ist der gleichzeitige Zusammenbau mehrerer DNA-Fragmente in vitroin korrekter Orientierung möglich. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by BsaI recognition sites in different orientations (B: ggtctcn, B: ngagacc).A linear and distinct assembly of those DNA fragments is ensured by the design of … [6], Golden Gate assembly uses type II restriction enzymes cutting outside their recognition sequences. For Geneious R9 and above. Q. Most commonly used Type IIS enzymes include BsaI, BsmBI, and BbsI. Gibson Assembly and Golden Gate Assembly (S2 Alvey) Flashcards Preview ... -Tradition cloning: Two extra amino acids due to the 6 base restriction site, which might interfere with the structure of the protein. All assembly steps are completed using Golden Gate cloning. The advantages of such an arrangement are … The principle of Golden Gate cloning is based on the special ability of type IIS restriction enzymes to cleave outside of their recognition site . You also have the option to Reset reaction if you have previously specified non-default boundary options for the Part. [8], Therefore, constructs of more than six genes need successive cloning steps, which requires end-linkers containing BsaI or BsmBI internal restriction sites and blue or purple markers. [8] Each cloning allows 2-6 genes to be inserted in the same vector. If not already selected, set the Enzyme: to BsaI. The plasmid contains two BsaI sites; digestion with BsaI releases a 41 bp fragment and a 2,133 bp vector backbone fragment to receive your insert or assembly. If Geneious detects a pair of appropriately orientated type IIS sites with unique overhangs, then it will assume you wish to use them. Will the Autoarrange button sort based on sequence names? Preexisting BsaI sites are also visible as well as a number of primer_bind annotations. [5] However, if the level 0 module is too large, cloning will start from level -1 fragments, which have to be sequenced, to help cloning the large construct. The Geneious Golden Gate tool will then complete the analysis and save a circular construct that represents your assembled sequences, plus the primer sequences required to create the Parts. To reduce the screen clutter turn off display of Primer Bind and Ligation annotations. By cycling back and forth 10 to 50 times between 37°C and 20°C, the DNA parts get digested and ligated over and over again. This will force Geneious to use the annotations as Part boundaries. It does not sort based on sequence name. If the BsaI sites in the Parts are preexisting, such as the two in the backbone, the Tag will be labelled with Cut by BsaI, or Enzyme. A. Check the option to Save Intermediate products. Usually the “receiving” type IIS sites already present in your backbone will define the site you will use. [3] Since 256 potential overhang sequences are possible, multiple fragments of DNA can be assembled by using combinations of overhang sequences. [6] If the overhangs are carefully designed, the segments are ligated without scar sequences between them, and the final construct can be quasi-scarless, where the restriction enzyme sites remain on both sides of the insert. Then, click Add Annotation, and define a new annotation for this range called GFP Reassembled of Type: CDS, and click ok. A new yellow CDS annotation should appear. The technology is easy to implement as a web tool is available for primer design (https://msbi.ipb-halle.de/GoldenMutagenesisWeb/) and the vectors are deposited at addgene (http://www.addgene.org/browse/article/28196591/).[11]. Q.  I want to have my part synthesized for Golden Gate rather than do PCR – what should I do? pot cloning steps, resulting in a 50 kb construct containing 17 eukaryotic trans-cription units. Additionally, because the final product does not have a Type II restriction enzyme recognition site, the correctly-ligated product cannot be cut again by the restriction enzyme, meaning the reaction is essentially irreversible. Facebook. The following link takes you to an exercise using the Golden Gate tool. The advantages of such an arrangement are … Introduction . Die Schnittstellen können vorab mittels PCR Primern integriert werden. [5] The vector used in cloning level -1 fragments cannot contain type IIS restriction site BpiI that is used for the following assembly step. If the assembly goes to plan, Golden Gate-mediated recombination of the six sequences will regenerate a complete CDS encoding the GFP and insert it into a vector “backbone”. We present here a versatile resource for plant biologists comprising a set of cloning vectors and 96 standardized parts to enable Golden Gate construction of multigene constructs for plant transformation. If only one type IIS cut site is detected, then Geneious will assume you wish to use it. A. Existing primer_bind annotation(s) without extension(s), Overriding the default use of primer_bind annotations as Part fusion points (Rules 4 & 5). Step 4: 25°C for 30 minutes (optimal ligation temperature for T7 ligase) Step 5: 65°C for 20 minutes (heat inactivation of T7 ligase) Once these predefined blocks are constructed, the first cloning step utilizes Golden Gate cloning to assemble the entry vectors sequentially into an appropriate Gateway … For example BsaI creates a 4 nucleotide 3′-recessed overhang adjacent to the recognition site (See Figure below). ReddIt. If Geneious detects a pair of valid overhangs compatible with the specified type IIS site, then it will assume you wish to use them. The following rules apply regardless of the type IIS enzyme selected for assembly. Golden Gate Assembly and its derivative methods exploit the ability of Type IIS restriction endonucleases (REases) to cleave DNA outside of the recognition sequence. Several level M constructs with compatible fusion sites can be subcloned into a level P vector in one step. When these recognition sites are placed to the far 5′ and 3′ end of any DNA fragment in inverse orientation, they are removed in the cleavage process, allowing two DNA fragments flanked by compatible sequence overhangs to be ligated seamlessly. Kit Components. Die bei dieser Methode eingesetzten Typ IIs Restriktionsenzyme, wie BsaI, BsmBI und BbsI, schneiden außerhalb ihrer Erkennungssequenz und könn… This vector contains two BsaI sites with unique overhangs, that will be used to “receive” our six-Part insert. FAQ: Frequently (and not so frequently) Asked Questions. [5] Level 0 modules without type IIS restriction sites flanking can add the BsaI sites during the process of Golden Gate cloning. Assembly of multigene constructs using Golden Gate Cloning. One-Step Cloning. Example of a DNA fragment with 2 BsaI sites. [4] While this technique can be used for a single insert, researchers have used Golden Gate cloning to assemble many pieces of DNA simultaneously. [5] Moreover, the vector should also have a different selection marker from the destination vector in next assembly step, for example, if spectinomycin resistance is used in level 0 modules, level -1 fragments should have another antibiotic resistance like ampicillin. Parts that require design of a primer pair will be labelled PCR product or Primer. You will see each fragment is annotated with an ORF annotation which defines a region corresponding to a portion of the GFP CDS. In standard Golden Gate Cloning, the restriction sites from the previous tier construct cannot be reused. Therefore, make sure the option to Save used Primers is checked. Step-by-step. [7] To achieve second-tier assembly, modular cloning(MoClo) system and GoldenBraid2.0 standard are used. I assembled by combining a promoter, Cas9, N- and C-tags, and a terminator in entry vectors. by Tara Lee. [8] There are fourteen available level 1 vectors, which differ only by the sequence of the flanking fusion sites while being identical in the internal fusion sites. [2] This assembly is performed in vitro. Geneious will assume that you already have the corresponding primers, and new primers will not be designed for the region. The protocol for this assembly method can be found here (Marillonnet S et al. If preexisting type IIS sites on your fragments are incompatible with sites on adjacent fragments then Geneious will highlight the Tag red and the offending overhangs will also be red. You should see the CDS translates to the complete GFP gene product starting MRK…, ending …LYK*, with no internal stop codons. Will they all have similar melting temperatures in the initial rounds of PCR? This exercise has been devised to demonstrate how the rules outlined in the introductory section of this tutorial are implemented, and also, to demonstrate the general procedure for use of the Golden Gate tool. If a primer_bind annotation without an extension is found, then an extension will be appended to introduce a valid type IIS recognition site, resulting in a new primer sequence. [6] As additional segments can be inserted into the vectors without scars within an open reading frame, Golden Gate is widely used in protein engineering. A. A. Golden Gate cloning is a strategy that allows ‘single-tube’ ordered assembly of a vector (Backbone) and one or more DNA fragments (Parts) into a single, usually circular, construct which is suitable for direct transformation of a bacterial host. The inserts and cloning vectors are designed to place the Type IIS recognition site distal to the cleavage site, such that the Type IIS REase can remove the recognition sequence from the assembly. Digestion with BsaI releases a central fragment with unique 4 nucleotide overhangs and no longer contains a BsaI motif. Can the Geneious Golden gate tool consider multiple type IIS restriction enzymes? [7] Then, second-tier Golden Gate assembly combine several constructs made in first-tier assembly to make a multigene construct. If required, Geneious will then design a primer pair for PCR amplification of each Part. Restriction enzyme DNA assembly has cloning standards to minimize the change in cloning efficiency and the function of the plasmid, which can be caused by compatibility of the restriction sites on the insert and those on the vector. The Autoarrange option will try to identify a unique sorting of the sequences based on the available overhangs generated via preexisting sites in your Parts. Such protocol requires the following steps: (1) selecting fusion sites within parental sequences (sites at which parental sequences will be recombined), (2) amplifying …