
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.