1. Set Assembly Parameters
Set your assembly’s name, choose the Type IIS restriction enzyme you will be using, and indicate the number of inserts for your construct. When done, select “Next Step”. (If building upon a previously designed assembly, select “Existing Assembly”.)
2. Provide Sequence Information
Click on the “destination vector” field and use one of the four provided methods to up-load your destination vector. Then click on the “Insert #1” field and similarly use one of the four provided methods to up-load your insert sequence. Repeat this process for all inserts for your assembly. (If your insert is contained within a larger sequence, you can enter the larger sequence and in a later step, indicate the subset of the loaded sequence to be used as the insert.) When done, select “Create Assembly”.
Assembly Workscreen Overview
Now you will be at the main assembly working split screen, where the sequence being manipulated is on the left, and a pictorial diagram is on the right. An on-screen tutorial will introduce the different informational windows available on the screen. It is a good idea to become familiar with this layout, as the presented options provide a great deal of control regarding your assembly. The sliders at the top left of each split screen window allow control of the zoom feature for the displayed sequence, and rotation of the plasmid diagram on the right panel. The gear symbol brings up a submenu of what features will be displayed. The diagram tabs at the top of the right panel allow viewing a linear or circular plasmid map, while the description tab allows text notes regarding your assembly. To change the left split screen sequence panel to a full view, click the bottom right “Split workspace” option.
By default the destination vector is displayed first, identifiable by the tabs across the top of the screen that display all your entered sequence files. The bottom part of the screen has two horizontal ribbons; the upper contains options about the current sequence selection while the lower schematically shows the order of assembly units, which can be changed if desired.
Set Sequences Involved In Your Assembly
3. Specify what part of the up-loaded destination vector sequence will be in your assembly
First, make sure your vector was up-loaded as a circular, not linear, form. To check this, select the “I” (information) icon on the right split screen. In the drop down menu for the Topology field, select “circular”, then “update information”. Next, select the sequence of the destination vector that will function as the vector backbone either by selecting the sequence from the left upper window or by choosing Type IIS cut sites pre-existing in the sequence. The cut site locations will be listed below this option in numerical order; if the fragment defined by the site’s numerical order defines the excluded sequence as opposed to the desired destination vector sequence, reverse the BsaI cut site numbers indicated in the sequence number boxes located directly under the “BsaI Cut Sites” option by clicking on the small triangles. When done, select “set fragment”. Notice a schematic of your chosen fragment will appear in a preview window in the bottom upper ribbon panel.
4. Similarly, specify what parts of your up-loaded insert sequences will be in your assembly
In the bottom lower ribbon panel select “Insert #1” and the uploaded sequence in the desired insert tab across the top of the screenthat displays all your entered sequence files (tab selection will become highlighted in blue). Select the part of the insert sequence that will function as the assembly insert either by selecting the sequence form the left upper window or by choosing the designed Type IIS cut sites flanking the sequence. Note that in many cases where new inserts are being designed, no existing Type IIS cut sites will be present and your selected sequence will comprise the entire uploaded insert sequence file. When done, select “set fragment”. Notice a schematic of your chosen insert will appear in a preview window in the bottom upper ribbon panel, indicating the fragment size and ends.Repeat this process with each desired insert/module.
Golden Gate Assembly/Summary
The bottom lower ribbon options allow rearrangements of inserts or adding spacer sequences, and will display either a green check mark indicating the elements of the assembly have been correctly entered, or red error messages if they have not. Any errors listed should be addressed. Then select highlighted orange “Assemble” option in right bottom corner.
Your Golden Gate assembly is now complete. The assembly sequence file will be created and displayed on the left. An on-screen, brief tutorial will illustrate different aspects of this assembly summary. Check all sequence junctions and proposed primer sequences. On the right will be a summary of your assembly elements, with tabs (blue link) to further information options, and a new right vertical panel for further sequence display options. Overall design parameters for the assembly are listed at the bottom of the summary. Primer sequences can be copied in tab-delimited format for direct pasting into oligonucleotide supplier’s order forms by selecting “copy primers”. Lastly, to print a complete assembly summary, select the “print” icon under the assembly tab, or select "PDF."
- How can I see my linear destination vector sequence displayed as a circular plasmid?
On the right split screen select the “I” (information) icon. In the drop down menu for the Topology field, select “circular”, then “update information”. Your linear sequence will be used to generate a circular map accessible by a new“plasmid” tab.
- What is the easiest way to designate the sequences involved in assembly from my uploaded sequence files?
There are many ways to do this. Sequences can be selected by highlighting the part of the uploaded sequence of choice, or selecting the beginning position and shift-selecting the end position, or using the right split screen to approximate the chosen sequence before more specifically defining the ends on the left split screen. For selecting sequence that bridges the beginning and ending of a linear sequence, it is easier to create a circular representation (see Q1), and approximate the region of interest on that circular map on the right split screen. Then more specifically define the selected sequence on the left linear sequence split screen display. Another approach with circular sequence files containing pre-existing Type IIS sites is to select the defined excluded sequence, then reverse the listed beginning and end numerical values to select the included sequence.
- How can I display restriction enzyme cut sites, annotations, ORFs and axis rulers for my sequences?
Select the gear option at the top of the left or right displayed split screen to access a drop down menu with selectable generally desired features.
- How can I specify a specific restriction enzyme(s) of interest in my displayed sequences or assembly?
Select the “scissors” icon on the extreme right options panel to display both location information in your sequences for that enzyme, and a full listing of the enzyme’s characteristics from major commercial enzyme suppliers, all without leaving the assembly tool.
- How can I introduce mutations into my generated primer sequences?
After your assembly has been made, select the base position in the sequence of interest in the left split screen panel that you wish to change. Type in your desired changes and a pop up window will display the corresponding base positions; press “enter”, and your suggested primer sequences will be automatically updated.