Subcloning Tool

The DNADynamo subcloning tool will place an 'Insert' sequence into a 'Vector' sequence and create a 'Construct' sequence. In some modes, the subcloning tool will also suggest PCR primers to generate the DNA fragments required for the subcloning process.

You don't have to use this tool, you may find it easier to just copy and paste your insert sequence into your vector sequence manually and/or manually design PCR primers via the 'Oligo' button -> oligo editor functions.

How To Use The SubCloning Tool



From the window containing the 'insert' sequence, open the Subcloning Tool by selecting 'Subclone into Vector' from the 'Subcloning' menu.

Next, select the subcloning mode required using the mode buttons.



The following modes are available...
the following modes also suggest PCR primers to generate fragments required in the subcloning process


'Restriction Sites' Mode



(note - to design PCR primers that add restriction sites to your insert, use Add Restriction Sites' mode)

Summary - Drag 'insert' restriction sites to 'vector' restriction sites with the mouse to specify joints as illustrated below If the ends to be joined do not represent two compatible cohesive ends or two blunt ends, DNADynamo fills in 5' overhangs to create a blunt end or chews back 3' overhangs to the blunt end and joins the two blunt ends together


1) Displaying graphic maps containing the required restriction sites for the insert and vector

From the main window that contains the 'Insert' DNA sequence, select 'Subclone into Vector' from the 'Vector' pull down menu. Select the vector you wish to subclone into from the list on the left hand side of the display.

In the right hand side display, two graphic restriction enzyme maps are displayed. The insert is on top, the vector on the bottom. By default only the Multiple Cloning Site of the selected vector (as defined by its MCS Annotation/Vector Element) is shown, unless you uncheck the 'MCS Only' control.

Enzymes from the 'Dynamo' box are used to generate the restriction maps. In the current version of DNADynamo, the 'Dynamo' box is a subset of the 'uniqueSites' box that produce blunt or non-ambigious sticky ends. If you've updated from a previous version of DNADynamo that doesn't contain a 'uniqueSites' box (where the 'Dynamo' box is a subset of prototypes) you can press the 'RESET' button in the 'Enzyme Box' editor to update the predefined boxes. You can select additional appropriate enzymes manually in the Dynamo Box by opening the Restriction Enzyme box editor (Menu option Boxes->Enzyme Boxes). For example, the site/cut TT^CGAA is achieved by a group of 8 isoschizomers, with AsuII being the prototype. If you would prefer BstBI to be listed on the graphic maps, select BstBI in the Dynamo Enzyme box (Menu option Boxes->Enzyme Boxes - selected enzyme box -> 'Dynamo' --> select BstBI).

By default, only unique restriction enzyme sites are displayed. To add sites that appear twice in the sequence, select the 'Double Cutter' checkbox.

You can also opt to only display sites in the insert sequence outside the currently set ORF, using the 'outside orf' control.

2) Selecting the Restriction Sites to Join

First mouse over an enzyme name, and then click and hold the mouse button. Note that the enzyme is underlined as the mouse button is held and that any enzyme with a compatible cohesive end in the vector is also underlined.

Now drag the mouse, with the button still held down towards the vector. Note that a line appears between the selected enzyme and your mouse position. Move the mouse to the enzyme in the vector that you wish to ligate to. Note that this does not need to be a compatible cohesive end. When the vector site is selected, release the mouse leaving a line joining the two enzyme sites.

Repeat the procedure for the second pair of enzymes

. When two joints have been defined, an illustration of the resulting construct is displayed. Finally, press the create button to open new window sequence window containing the insert sequence placed into the vector sequence.

Note that if the ends to be joined do not represent two compatible cohesive ends or two blunt ends, DNADynamo fills in 5' overhangs to create a blunt end or chews back 3' overhangs to the blunt end and joins the two blunt ends together. If other behaviour is required you must copy and paste the DNA sequences yourself as required.

If the same site is used twice in the vector sequence (eg an insert flanked by two BamH1 sites placed into a single BamH1 site in a vector), the orientation of the insert is determined by the order in which you join the inserts sites. Connecting the 5' insert site first places in insert in the forward direction. Connecting the 3' insert site first places the insert sequence in the reverse orientation. In the tube of course you would have a mixture of the two orientations

The 'Vector Map' of the DNA Sequence will show the insert subcloned into the Vector. Selecting the 'Diagnostic' checkbox in the restriction enzyme site display table will show enzymes that cut at least once within the insert and once within the vector sequence. Pressing digest will give a comparison of fragment sizes that would result from digestion with the selected enzymes of the vector sequence alone or the vector and insert sequence.

'Topo' Mode



Select to show TOPO sites 'CCCTT^AAGGG' in vector sequences - can be used together with the 'TA' mode button for topo TA cloning

Drag the the blunt ends of an insert to the TOPO site in the vector

Results in...
VECTOR-CCCTT -BLUNTPCRPRODUCT- AAGGG-VECTOR
VECTOR-GGGAA -BLUNTPCRPRODUCT- TTCCC-VECTOR
topo TA
VECTOR-CCCTT  -TAILEDPCRPRODUCT-A  AGGG-VECTOR
VECTOR-GGGA  A-TAILEDPCRPRODUCT-  TTCCC-VECTOR
the orientation of the insert is determined by the order in which you join the inserts sites. Connecting the 5' insert blunt site first places in insert in the forward direction. Connecting the 3' insert blunt site first places the insert sequence in the reverse orientation.

Also shows directional topo sites CCCTTCACCAAGGG
VECTOR-CCCTT      -CACCBLUNTPCRPRODUCT- AAGGG-VECTOR
VECTOR-GGGAAGTGG  -GTGGBLUNTPCRPRODUCT- TTCCC-VECTOR
for directional topo - 5' insert blunt ends start CACC - can't be used with the 'TA' checkbox selected

The presence of prepared vector is required for TOPO reactions - refer to your vector and kit specifications


'TA-Cloning' Mode



Drag A-tailed 'insert' ends to a T-tailed 'vector' site with the mouse

Results in...

TAILEDVECTOR-T   TAILEDPCRPRODUCT-A   TAILEDVECTOR
TAILEDVECTOR   A-TAILEDPCRPRODUCT   T-TAILEDVECTOR

the orientation of the insert is determined by the order in which you join the inserts sites. Connecting the 5' insert blunt site first places in insert in the forward direction. Connecting the 3' insert blunt site first places the insert sequence in the reverse orientation. The presence of prepared vector and insert is required for TA cloning.

if used in conjunction with the TOPO cloning option the following prepared topo vector site is expected...
CCCTT   TAILEDPCRPRODUCT-A  AGGG
GGGA  A-TAILEDPCRPRODUCT   TTCCC


'Gateway Cloning' Mode



Select to show GateWay joints

GateWayJoint 1 - TTT^GTACAAAAAA
GateWayJoint 2 - TTT^CTTGTACAAA

You can join a GateWayJoint1 to another GateWayJoint1
TTT             GTACAAAAAA    >>>>     TTTGTACAAAAAA
AAACATGTTT             TTT    >>>>     AAACATGTTTTTT
You can join a GateWayJoint2 to another GateWayJoint2
TTT             CTTGTACAAA   >>>>      TTTCTTGTACAAA
AAAGAACATG             TTT   >>>>      AAAGAACATGTTT

The presence of additional sequences are required for GateWay reactions - refer to your vectors specifications to determine if a GateWay reaction is possible

'Add Restriction Sites' Mode



Drag 'insert' blunt ends to required 'vector' restriction sites with the mouse to specify joints as illustrated below



If the insert is annotated, you can select an annotation within the insert by clicking it with the mouse

After connection of both ends - the oligo design and display window will open where you can visualise the PCR primers on the sequence and save the designed PCR primers in the master oligo box

Press the 'Create Clone' button to open a new window containing the resulting construct

'Gibson Cloning' Mode



Gibson Assembly joins two or more DNA fragments when they have overlapping end sequences. In the assembly reaction, T5 exonuclease chews back DNA from the 5' end of fragments resulting in single-stranded regions on adjacent DNA fragments that can anneal. A DNA polymerase incorporates nucleotides to fill in any gaps and a DNA ligase covalently joins the DNA of adjacent segments

How to use...

If generating linearised vector with restriction enzymes, drag the 5' and 3' inserts ends with the mouse to the required restriction sites in the vector map. If the insert is annotated, you can select an annotation within the insert by clicking it with the mouse, and drag the annotations ends to the vector. The 5' exonuclease removes 5' overhangs generated by restriction sites, while 3' overhangs are incorporated in the final construct.




If generating linearised vector by PCR, either...

a) select an annotation in the vector that will be replaced with the insert by moving your mouse over the annotation and clicking. You can reverse insert orientation by holding down the shift key


b) select the vectors 'Show Sequence' option and place the cursor at the insertion point or drag the cursor to select sequence to be replaced. Note that in 'show sequence' mode you can click the map to locate the sequence displayed




for a) and b) you can reverse insert orientation by holding down the shift key

The required overlapping regions can be added to the ends of DNA fragments using PCR.

DNADynamo suggests primers for fragment generation and uses Primer3 to calculate primer Tm and secondary structure predications. The length of the overlap and specific sections of each primer can be adjusted manually if necessary


PCR primers for Gibson Assembly have two sequence components

- an overlap sequence, for the assembly of adjacent fragments
- a gene-specific sequence, for template priming during PCR amplification

NEB, who supply a kit for gibson assembly, say that to achieve efficient assembly of PCR fragments into a vector use a 15-25 nt overlap with a Tm equal to or greater than 48°C (assuming A-T pair = 2°C and G-C pair = 4°C). The overlap sequence can be composed of nucleotides which belong to only one fragment or can be split between the two adjacent fragments in any combination.

The priming gene-specific sequence at the 3´-end of the primer should meet the criteria required for template annealing during PCR amplification. ie The Tm is calculated for the 3' (gene-specific) section, not the entire primer.

Complementarity within each primer should be avoided, to prevent hairpin structures, and between primer pairs, to avoid primer dimers.

With regard to Tm - if you're using NEBs Q5 or Phusion polymerase, note that the NEB Tm calculater suggests Tms several degrees higher than the Primer3 or DNADynamo Tm calculater. You may wish to manually adjust primer length to reflect this - or use the free on-line NEBuilder for gibson assembly web application and compare the results etc

As it says in the Primer3 manual, the actual tm of an oligo can only be determined experimentally. Given the wide range of calculations suggested in the literature and the varying results achieved it is fortunate that PCR seems quite robust for a variety of oligo melting temperatures

For a second opinion on secondary structure try the DNAMelt/UNAFold webserver.

'Infusion Cloning' Mode



Infusion Cloning joins two or more DNA fragments when they have overlapping end sequences.

If generating linearised vector with restriction enzymes, drag the 5' and 3' inserts ends with the mouse to the required restriction sites in the vector map. If the insert is annotated, you can select an annotation within the insert by clicking it with the mouse, and drag the annotations ends to the vector. Note that 3' overhangs are removed during the Infusion Cloning process, but 5' overhangs are incorporated into the final construct.



If generating linearised vector by PCR, either...

a) select an annotation in the vector that will be replaced with the insert by moving your mouse over the annotation and clicking, or


b) select the vectors 'Show Sequence' option and place the cursor at the insertion point or drag the cursor to select sequence to be replaced. Note that in 'show sequence' mode you can click the map to locate the sequence displayed



for a) and b) you can reverse insert orientation by holding down the shift key

DNADynamo suggests primers for fragment generation and uses Primer3 to calculate primer Tm and secondary structure predications. The length of the overlap and specific sections of each primer can be adjusted manually if necessary

Clontech, who supply the infusion kit, suggest the following should be considered when desiging In-Fusion PCR primers

The 5' end of the primer must contain 15 bases that are homologous to 15 bases at one end of the DNA fragment to which it will be joined (i.e., the vector or another insert).

The 3' end of the primer must contain sequence that is specific to the target gene.

The 3' gene-specific portion of each primer should bebetween 18-25 bases in length, have a GC-content between 40-60% and have a melting temperature (Tm) between 58-65°C. The Tm difference between the forward and reverse primers should be no more than +/- 4°C, or you will not get good amplification. The Tm should be calculated based upon the 3' (gene-specific) end of the primer, not the entire primer.

The 3' gene-specific portion of each primer should not contain identical runs of nucleotides. The last five nucleotides at the 3' end of each primer should contain no more than two guanines (G) or cytosines (C).
Complementarity within each primer should be avoided, to prevent hairpin structures, and between primer pairs, to avoid primer dimers.

Clontech offer a free on-line web application for infusion cloning if you want to compare the results

As it says in the Primer3 manual, the actual tm of an oligo can only be determined experimentally. Given the wide range of calculations suggested in the literature and the varying results achieved it is fortunate that PCR seems quite robust for a variety of oligo melting temperatures

For a second opinion on secondary structure try the DNAMelt/UNAFold webserver.