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ClonePro™ SLIC Enzyme (CPS1001)

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Quick Overview

CloneProTM SLIC enzyme (CPS1001) in ATCGbio Life Technology Inc. is developed based on the technique of Sequence and Ligation Independent Cloning (SLIC).  In 20 min reaction, PCR product can be inserted in immediately.
SLIC (Sequence and Ligation Independent Cloning) technique employs in vitro directional homologous recombination at cohesive ends created by 5’→3’ or 3’→5’ exonuclease activity reaction.  It has been developed for years since the first report using T4 DNA polymerase in 1993(1), and this protocol was restated by Mamie Z Li and Stephen J Elledge(2) in 2007. Besides using T4 DNA polymerase, the protocol using exonuclease III(3), T7 exonuclease (4), T5 exonuclease(5) and vaccinia virus DNA polymerase(6) were also reported.  Commercial products are also available using these enzymes and others in proprietary combination.  In-Fusion® (Takara), GENEART® Seamless cloning and assembly enzyme mix (Invitrogen), Gibson Assembly® Master Mix (NEB) are such enzyme kits and are made to optimize exonuclease activity and filled-in polymerase activity. However, those enzymes are more suitable for long homologous (more than 20-30 bp) and long fragment (more than 100 bp).
CloneProTM SLIC enzyme has similar or more effective function as In-Fusion® (Takara), GENEART® Seamless cloning, assembly enzyme mix (Invitrogen), Gibson Assembly® Master Mix (NEB) and others.
                                  5’-exonuclease activity    ------------->      
5’-NNNNNNNNNNNNNNN                                                             NNN-3’     
3’-NN                                                         NNNNNNNNNNNNNNNNNN-5’
                  <---------- 5’-exonuclease activity

 NNNNNNNNNNNNN and NNNNNNNNNNNNNNN are cohesive ends that can anneal spontaneously.

Features of CloneProTM SLIC enzyme
CloneProTM SLIC enzyme in ATCGbio Life Technology Inc. is developed to be suitable for the follows:
1. Short (~15 bp) homologous recombination
2. Fragments of length from less than 100 bp to 6kb
3. Assembling multiple fragments. 
It is based on 5’3’ exonuclease activity and is stable at -20 °C for more than 3 months. The cloning reaction requires only 20 minutes at 50 °C with 10 µl in volume. 
Kit component
20µl CloneProTM SLIC enzyme (10 x solution) is enough for 20 reactions of SLIC reaction. Keep at -20°C (will be stable at least 3 months). Centrifuge briefly before use.

ClonePro™ SLIC Enzyme (CPS1001)


Design and creation of homologous region by PCR

1. Insert one fragment to blunt ends vector

Following is an example showing CloneProTM SLIC enzyme (CPS1001) mediated cloning of FOXO3 gene into pENTR1A® vector (and other pENTR® vectors).

Gateway®cloning system is useful to express a gene of interest in multiple systems (bacteria, mammalian) and with different tags (GST, HIS tag, etc). However, cloning a target gene into Entry® vector is the first challenge in this system. With CloneProTM SLIC enzyme, this can be done easily with near 100% efficiency.

pENTR1A® has two multiple cloning sites flanking ccdB gene and gene of interest should be inserted between these cloning sites.  For SLIC reaction, XmnI and EcoRV (both produce blunt end) are used to cut and produce the following sequence.

                             XmnI           EcoRV      

5’-----GGCTTTAAAGGAACC            ATCTAGACCCAGCTT------------------3’

Since AACC is Kozak consensus sequence, start codon ATG can be immediately placed.

Interest of gene in this case is human FOXO3 which has 2022bp and sequence is


To make PCR primer for CloneProTM SLIC reaction, first design cloning forward primer from ATG which should yield Tm=~60 °C.  (This task can be done easily using SerialCloner®.)

Following 18bp sequence produces Tm=60.7°C.


To make homologous region to pENTR1A vector (XmnI site), add 15bp GGCTTTAAAGGAACC at the front of cloning sequence.


Second, in similar way choose from stop codon TGA to 5’ direction to give Tm= ~60 °C.


Add 15bp pENTR1A (EcoRV site) homologous region at the end.


Reverse primer should be anti-parallel (reverse complement) of above sequence showing as: 


With these forward and reverse primers, PCR products have homologous regions to linearized pENTR1A vector at both ends.


G     F     K    G     T     M    A             G     *                             

This sequence is in-frame at 5’ site. So, it can be used for native and 5’ tagged protein expression.

To create C-terminus tagged protein, reverse primer needs to change. First, remove stop codon TGA. Second, add one nucleotide before AT CTA GAC CCA GCT T to make C-terminus in-frame. So, reverse primer will be anti-parallel of 


N can be chosen by the user. (TAG) should not be included.

When G is chosen for N, reverse primer will be


After PCR, C-terminus side becomes 


               P     G     D     L    D     P     A

2. Design for cohesive end cut:

If PCR fragment is inserted to cohesive end cut region of the plasmid, homologous region should be design as follows.

In this example, the fragment below is inserted to HindIII cut site.


HindIII recognition site of the plasmid (circular) is



HindIII will cut this site like the follows:

5’-GAGTCGACCTGCAGA-3’                                  5’-AGCTTGCCTCGAGCAGCGC-3’

3’-CTCAGCTGGACGTCTTCGA-5’                                 3’ -ACGGAGCTCGTCGCG-5’

CloneProTM SLIC enzyme (CPS1001) produce cohesive end by 5’→3’ exonuclease activity. Thus, in left side, the bottom strand is chewed up 5’→ 3’ direction and the top strand remains intact. In right side, the bottom strand remains intact. 15bp homologous region should be design to recognize intact strands (Left side (forward) → top, Right side (reverse) → bottom). 

Thus, forward primer is:


Reverse primer is anti-parallel of


If you wish to keep HindIII site at both ends of the insert,

Forward primer is:


Reverse primer is anti-parallel of



  1. Plasmid preparation. Cloning site should be cut by appropriate restriction enzyme. Typically, this can be done by incubate 1 µg plasmid in 20 µl reaction solution for 30 min-1 hr, and followed by heat-inactivation of enzyme such as 80 °C for 10 min. Store at -20 °C if not used immediately.
  2. PCR insert preparation. PCR product should be produced by proofreading DNA polymerase. Typically, this can be done 20-40ng template with 0.3 µM primers 24 cycles in 50 µl reaction volume. Annealing temperature should be set 55 °C (Note we design Tm = ~60 °C). Then, PCR product should be should be run on agarose gel and stained with Ethidium bromide or SYBR®Safe (Invitrogen). Appropriate band is cut and purified with gel-purification kit. Typically, this can be done with many commercialized kits with 30 µl elution volume. Store at -20°C if not used immediately.
  3. CloneProTMSLIC reaction. We recommend to process at 0.2 ml PCR tube with a thermal cycler. Program should be 50 °C for 20 min followed by 4 °C. Heat lid can be on. Transfer 1 µl plasmid prepared above (0.5 µg) and 2-4 µl of PCR product into a tube and bring the volume to 9 µl with nuclease free water. Start thermal cycler program, add 1 µl CloneProTM SLIC enzyme into the tube, vortex briefly and put on the heating block. Prepare competent E.Coli on ice. After finishing reaction in 20 min, vortex and centrifuge briefly, then transform E.Coli with 3-4 µl of the solution by heat-shock or electroporation.

Expected Result

CloneProTM SLIC enzyme (CPS1001) reaction is very specific meaning that if the plasmid is completely cut and homologous insert is not present, very few (1-4) colonies can grow. Therefore, most of the colonies should have correct inserts. To be sure in-frame cloning, sequencing is recommended.  

Increasing number of false-negative (E.Coli colony do not have insert) colonies reflects incomplete cut of the plasmid. We recommend to use advanced restriction enzyme system such as FastDigest (Thermoscientific) to cut more completely. This problem is virtually non-existent when pENTR1A vector is used because of presence of ccdB gene. The other aberrant results could be happened by residual template plasmid. This sometimes cause multiple bands contaminated. This problem can be solved by digesting PCR products by DpnI which does not digest DNA created by PCR. Typically this can be done by add 1 µl of 10X digestion buffer and 0.5 µl DpnI (10 U) to PCR products (8.5 µl) and incubating it at 37 °C for 30 min; then proceed CloneProTM SLIC reaction.

If there is no colony, first check homologous site design. Second, check purified PCR product whether it exists without degradation. Third, check possibility of DNA damage caused by UV light during cutting. Commercialized UV lamp is ~260, ~300 and ~360 nm. Shorter the wavelength, more cause damage. Use ~360 nm wavelength UV light which is much less problem.


1. Construction of recombinant DNA by exonuclease recession. Yih-Sheng Yang, et.al. Nucleic Acids Research 21:1889, 1993

2. Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Mamie Z Li and Stephen J Elledge. Nature Methods 4:251, 2007

3. Exonuclease III induced ligase-free directional subcloning of PCR products. Ku-chuan Hsiao. Nucleic Acids Research 21:5528, 1993

4. An improved ligase-free method for directional subcloning of PCR amplified DNA. Meixia Zhou and Zafer Hatahet, Nucleic Acids Research 23:1089, 1995

5. Enzymatic assembly of DNA molecules up to several hundred kilobases. Daniel G Gibson, et al. Nature Methods 6:343, 2009.

6. Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase. Michael D Hamilton, et al. Nucleic Acids Research 35:143, 2007.

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