D-Zyvec step-by-step tutorial

This step-by-step tutorial will show you how to use the e-Zyvec plasmid drawing interface from A to Z in less than 10 minutes. After this, you will be able to draw a lot of plasmids really fast. If you have any questions, To contact us, please fill out the form at www.e-zyvec.com,. or send an email to support@polyplus-transfection.com and mention that you would like to discuss with a member of e-zyvec services by Polyplus. We will answer quickly..

I. Create your first plasmid II. Master the plasmid drawing III. Submit and manage your projects

Create your first plasmid

In D-Zyvec, all the plasmid are grouped in projects. Each user manage its own projects that he can share with his registered colleagues belonging to the same research team.

To be able to start drawing plasmids, you must start by creating a new project. Creating a project is pretty straightforward: simply go to "Your projects" and click the "+" button. You are then asked some basic questions about your project in a pop-up window. Once you filled the empty fields, click on the "New project" button:

Congratulations ! You just created your first e-Zyvec's project.

Creating a project does not commit you to anything. As long as a project remains unsubmitted we will not issue any quote. However, if you decide to order the plasmids you draw by submitting the project to us, the description you provided will be recalled on your specification requirements file. So, the more specific you are the better.

If your project is based on DNA plasmids you previously ordered from us, please indicate it here. As a result, any quote requested using the D-Zyvec will be calculated with the discount fee applying to follow up projects.

You are now on the page in which you will design your plasmids for this project. This page is made up of two main parts :

1 The project-related fields required to submit it. We will come back on it later. This part will stay the same whatever the plasmid you are working on within a given project.
2 The plasmid you are currently working on. This second part will change each time you want to work on a different plasmid. On the screen below, the plain grey circle you see is your first plasmid waiting to be drawn.

The first thing you must do is to give a name to your plasmid using the purple blinking field in the small pop-up that appeared on your screen. You can also provide here more input about the purpose of this plasmid within the project. This will help us better understand your needs and possibly share relevant advises at a later stage. Both name and description can be edited later.

Once done, this window will be replaced by the backbone suggestion menu: by filling up a small series of questions, the system will automatically provide you a starting scaffold for your plasmid, with prefilled genetic features where it applies!

The expression system and the plasmid type are mandatory fields. You won't be able to save your plasmid if you don't fill them.

When you finished filling the questions, click on "Suggest me a backbone".

The corresponding genomic features appear on the plasmid !

The backbone is only a suggestion. You will be free to change anything you want on your plasmid : sequences, order, orientation and more !

Now you can save your plasmid by clicking on e-Zyvec

. Your plasmid ID tag now appears on the project plasmid list (left of the screen) :

Saving a plasmid with e-Zyvec

will not automatically submit the project and request for a quote. This can only be achieved by clicking on e-Zyvec

that will remain below the plasmids ID tags, on the left of the screen.

As you can see, there are a couple more buttons you can now use form the plasmid ID Tag: you can remove your plasmid, duplicate it and mutate it (we will see the latter in the next part). Now we will duplicate your first plasmid. Simply click on "duplicate". It created, and automatically saved an exact copy of your original plasmid. Now you can modify your two plasmids separately by clicking on the Tag.

Congratulations ! you created your two first plasmids. You may leave here without losing any data. Everything is safely saved on our servers. Now we will learn how to entirely customize and manipulate them in the second part of the tutorial.

Master the plasmid drawing

After doing the first part of the tutorial we obtain the following plasmid displayed at the right of your screen (or a similar one depending on your backbone choice) :

An e-Zyvec plasmid is compound of features. Each small block you see on the image corresponds to a feature. Features are regrouped in cassettes that you can manipulate at once. All features belonging to the same cassettes automatically share a shade of the same color. This example is a plasmid composed of 4 cassettes (blue, purple, green, pink), for a total of 8 features (1 blue, 1purple, 3 green and 3 pink).

A cassette doesn't necessarily corresponds to an expression cassette. The cassettes on the drawing are customizable at will and their only purpose is readability and ease of manipulation. The features are what's matter.

Manipulate features

You can entirely modify any feature, even the ones that were suggested in the backbone. Try to hover a feature with your cursor :

The feature options appear. The button inside the plasmid act on the feature :

	Reverse the orientation of the feature.
	Permanently remove the feature from the plasmid.
	Pull back the feature from its current cassette and put it alone in a new cassette. If the feature was surrounded by features of the same cassette, it is then moved outside its previous cassette.

The green transparent arc outside the plasmid underlines all the features belonging to the same cassette. We will come back to the padlock button later.

Ok but how do I put my own sequences in my plasmid ? and what's this blank feature ?

The blank features are features for which the system does not have a nucleotidic sequence yet. In case of our backbone example, every feature but the ORF already have a sequence, even though you cannot access it at this stage.

In order to customize a feature, simply click on it. The following window appears on the screen :

This is the Feature Window. You can either :

- Paste your own sequence (left)
- Choose a sequence in the database (right)

Don't forget to save by clicking on the save button below the plasmid from time to time (ideally after every crucial modification).

Now we will learn how to move a feature. For this, hold your left click on the feature you want to move. You can drag it anywhere on the plasmid. When you release your click, your feature will drop between the two features that are highlighted in blue :

This is it! you know how to manipulate features.

If dropped in the middle of a cassette, the feature will go inside this cassette-unless locked. If dropped between two different cassettes, it will go inside the nearest open cassette, or between the two if both are locked (see below).

Manipulate cassettes

Remember the padlock button displayed on the cassette arc?

By clicking on it, you can lock or unlock the cassette depending on its current state.
You can visually differentiate locked and unlocked cassettes by the appearance of the its arc: plain when locked, and hollow when unlocked (or open).

In a locked cassette, the two button (rotate and remove) in the center of the plasmid will now act on the entire cassette. Same goes for the drag'n drop system:

If you want to individually manipulate features again, re-click on the padlock. The cassette is now unlocked and you can manipulate features individually !

You can't drop features inside locked cassettes. If you wish to do so, simply unlock the cassette first.

Add new features and cassettes

One last thing : we will learn how to add new empty features and cassettes in our plasmid. At the bottom of the screen you should see these two buttons :

The green button allows you to add a new feature in the plasmid, the blue one an entire empty cassette. Let's try to click on the green button :

Small "+" buttons appear everywhere you can add a new feature in your plasmid. Click on the place you want to add your new feature, choose a feature category and that's it ! You just added a new feature in your plasmid. A new feature will always go in the cassette of the feature preceeding it.

You can't add new features inside locked cassettes.

The blue button works exactly the same but adds an entire preconstructed expression cassette : promoter + ORF + terminator.

Congratulations ! you know how to draw plasmid on D-Zyvec. Now we will submit the project and talk about project management in the last part of the tutorial.

Mutate plasmids

Not every plasmids of your project need to be entirely drawn for project submission. Indeed, many project consists on closely related plasmids that will differ only from one to few nucleotides introduced by point mutations, small deletion and truncation of sequences.
In order to create close mutant plasmids, click on e-Zyvec

on the plasmid you want to mutate. A pop-up window appears :

You simply need to fill the desired mutations using one line for each alternate plasmids (mutant). This is by typing free text so the more specific you can be the better. You can create as much mutant plasmids as you want by clicking on e-Zyvec

You can only mutate sequences that you pasted in the Feature Window, not sequences coming from e-Zyvec's database.

Once done, click on e-Zyvec

. The mutant plasmids will be generated. On the mutated plasmid ID tag, you can see the number of corresponding mutated plasmids :

Submit your project: what's next?

Once you finished drawing your plasmids, you can submit your project by clicking on e-Zyvec

. Your project will be summarized and you will get a price estimation.

Do not forget to define all relevant control plasmid as well as the production format (required).

You can't edit a submitted project !

A new window opens with a summary of your project as well as an estimate, solely based on what was communicated in this project. If everything sounds good, e-Zyvec

again, otherwise proceed to changes by clicking e-Zyvec

.

Our staff will then review your project and get back to you depending on the following:

1 Everything in the project is biologically consistent, our staff will make a quote for your project and send it to you.
2 There are some inconsistencies in your project (for example an ORF without a promoter in a mammalian expression system). Our staff will access and correct your project. Once this is done, you will receive an e-mail telling your to check it again. If everything is ok for you, you have to re-submit your project.

We may also try to reach you after reading your submission, to discuss more widely about your project. Indeed, we pride ourselves not to be simple product provider but rather scientific partners contributing to your success. So, we may have a couple of suggestions to pass on you if we think it would help.

Manage your projects

Click on "My projects", on the top-right of your screen. You are redirected on the project management page. This is the page where you created your first project. On this page, you can see all the projects you created on D-Zyvec.

You can also see all the projects created by your team members by clicking the checkbox, but only if your account was validated by e-Zyvec's staff.

If you followed the tutorial, you should see only one project on this page :

As you can see, you can easily edit and remove your project (unless already submitted). You can also manage the permissions: letting your team members be able to edit or delete your project. You can also check the modification history.

By default, only you can edit and delete a project you created.

If our staff modified your project, you will see a "reviewed" label at the top right of your project. It means that you should check your project and submit it again. While reviewing we also sent you a mail which explains the eventual changes we made. You can also view the content of this mail by clicking the dialog icon at the top left of your project :

You completed the tutorial. You can start to design your plasmids ! create your project here.

Any unanswered questions ? check our FAQ or directly contact us : To contact us, please fill out the form at www.e-zyvec.com,. or send an email to support@polyplus-transfection.com and mention that you would like to discuss with a member of e-zyvec services by Polyplus. We will answer quickly.