ApE Window

ApE Window
New window
Shift: Duplicate selection

Save window
Shift: Save asOpen FileShortcuts
CommandFunction

OOpenN

New window

D

Duplicate selected region

Shift-D

Duplicate whole sequence

SSave sequence

Shift-S

Save sequence as…

ApE Window
Sequence length

Cursor location

Circular/ Linear

Edit lock
Dam/Dcm status

ApE Window

Selection end
Selection start Selection length
double click to set
double click to set

ApE Window
ORF state

Selection melting
temperature%GC

Selection translation
The translation ofthe selectedregion is shownhere

Selection translation

Right click hereto set thetranslation
directionPreferences

ApE Window
Cut
Shift: Cut rev-com

Paste
Shift: Paste rev com

Copy
Shift: Copy rev-com

Shortcuts
CommandFunction

X

Cut

Shift-X

Cut rev-com

C

Copy

Shift-C

Copy rev-com

V

Paste

Shift-V

Paste rev-com

Copy Special

ApE Window
Find
Shift: Clear find highlightShortcuts
CommandFunction

F

Find

Shift-F

Clear find highlight

GDo find again

Shift-GNew feature from find highlightFind dialog

Find dialog
You can search for multiple
patterns with a comma orbar

ApE Window

Open this toshow the filecomment

Shortcuts
CommandFunction.

New FeatureK

Add features using feature library

Shift-K

Clear all features

Enzymes
Select Enzymes

Select an enzyme tohighlightEnzyme highlights
Enzyme nameand sequenceshows up here
when you put themouse over thesequence

Enzymes can behighlighted

X-ray window

spacebar turns
on the X-raywindow

X-ray window
The X-raywindow shows
all the featuresand enzyme sites
in a row

Side maps
First map shows
the features in
the current lines

Second map
shows thefeatures in thewhole sequence

Side maps
Side mapfeatures underthe pointer are
listed here

Click on any ofthese to select afeature in thefeature table andset the selection
to that featureVirtual Gel
Select Enzymes

Virtual Gel

Click here to digest the sequencewith each enzyme individuallyVirtual Gel

Virtual Gel

Click here to digest the sequencewith each enzyme at one timeVirtual Gel

Virtual Gel

Mouse here to highlight the band
Double-click to select that region
in the sequenceVirtual Gel

Mass % shows how much of thetotal DNA each band represents
Use this to decide how much DNA
to load on a gel. 50 ng is about theleast DNA you can see on a gel.

Virtual Gel

Click here to see a list of digestlanes

Virtual Gel

Click and drag lanes to rearrange

Shortcuts
CommandFunction

RDigest with currently selected enzymes

Shift-R

Digest Dialog

EEnzyme selection dialog

Shift-E

Set all functions to apply to selection

Digestion dialog

Digestion dialog

Each gel lane is a tablerow
Add and remove rows
with these buttons
Rows can be dragged
and duplicatedDigestion dialog
Add enzyme columns
with this button

Enzymes for digestions
are added here
Show partial digest
options with thisbutton

Digestion dialog

Change what is in thelane with this menu

Digestion dialog

Change the enzyme
with this menu

Digestion Dialog

Select the DNA or ladder for eachlane

Digestion Dialog
Add an Enzyme Column
Select EcoRI and SpeI

Digestion Dialog

You can also just create a set of
new lanes with each open DNA

Digestion Dialog

For convenience, you can digest asingle DNA with all current
enzymesDigestion Dialog
Press this to do partial digests

Digestion Dialog
Press this to select EcoRI in allcolumns

Digestion Dialog

This will digest pMLS280 andgfasPurple with EcoRI and SpeI

Digestion Dialog

This will digest pMLS280 andgfasPurple with EcoRI and SpeI

Restriction-Ligation
Assembler

Restriction-Ligation
Assembler

Click and drag a gel lane here

Restriction-Ligation
Assembler

Click and drag a gel lane here

Restriction-Ligation
Assembler

Restriction-Ligation
Assembler

Restriction-Ligation
Assembler

Restriction-Ligation
Assemblermouse here to see the featurenamesRestriction-Ligation
Assembler

click here to flip a fragment

Restriction-Ligation
Assembler

click here to delete a fragment

Restriction-Ligation
Assembler

When everything works, you canclick OK

Restriction-Ligation
Assembler

Restriction-Ligation
Assembler

Feature library

New Feature

Features
Current features
under the mousepointer are listedhere

Features
Click this to hide/show the featuretable

Features
Right-click here
to edit a feature

Features
Click this to sortthe feature table

Features
Command-click
here to selectmultiple features
Shift-click here
to select a rangeof features

Features
Right-click here
to edit a range offeatures

Features
Right-click here
to edit a feature’s
direction

Features
Right-click here
to edit a feature’stype, or to raise,
lower or hide allof a type offeature

Features
Right-click here
to edit a feature’srange

Features
Shift-click this tohide/show thefeature tablebuttons

Golden Gate Assembler

Golden Gate Assembler

Golden Gate Assembler

Golden Gate Assembler

Golden Gate Assembler

Golden Gate Assembler

Golden Gate Assembler

Golden Gate Assembler

Golden Gate Designer

Golden Gate Designer

Golden Gate Designer

Golden Gate Designer

You can’t use afragment that has asite already in itGolden Gate Designer

Golden Gate Designer

Add fragments usingthis button

Golden Gate Designer

Select these ranges

Then click “Next”

Golden Gate Designer
Calculates the totaleﬃciency (on-target tooﬀ target)

Each fragment and theprimers needed

You can set default
values here bases

Golden Gate Designer

Golden Gate Designer

Re-start thecalculation if it’s stuckin an unfavorable
searchDo a longer calculationfrom the current
search position

Golden Gate Designer

Generate the newproductGolden Gate Designer

Golden Gate Designer

The primers and PCR
conditions are in thecomments

Golden Gate Designer

The primers are newfeatures in the featuretable

Golden Gate DesignerThe primers are newfeatures in thesequenceText map dialog

Text map dialog
Each box is ananalysis line

Turn oﬀ the line byunchecking this box

Drag the frames tochange the orderText map dialog

ORF map dialog

ORF map dialog

Three forward frames

Three reverse framesLonger than this arehighlighted in color,and can be clickedSilent/ diagnostic sites dialog

Silent/ diagnostic sites dialog***Select a forward
ORF and a set of
restriction enzymes
before selecting themenu item

Silent/ diagnostic sites dialog

Silent/ diagnostic sites dialog

Select diagnostic site if
you are adding a sitethat is NOT in an ORF

dCAPS dialog

dCAPS dialog

Two orientations

WT or mutant cut
Red is the variant base

Green is a mutant
primer base

dCAPS dialog

Sequence of the DNA
Enzyme recognition
sequence

Primer sequencePrimers- hand designed

Drag the selection
where you want theprimer

Primers- hand designed

Drag the selection
until the Tm is what
you want

Primers- hand designed

Try to keep the %GC
near 50%, if possible

Primers- hand designed

Try to keep the %GC
near 50%, if possible

Primers- ApE designed

Primers- ApE designed

Primers- ApE designed

Primers- ApE designed

PCR reaction tool

PCR reaction tool

PCR reaction tool

PCR reaction tool

Active primers show up here

Select this to activate just unique primers

PCR reaction tool

Select primers to show
them in the sequencemap

PCR reaction tool

Click here to add
selected primers as
new primer_bindfeatures

PCR reaction tool

Modify the priming
search parameters
herePCR reaction tool

Select all of the class primers
and sequences then copy

PCR reaction tool
Primers can be:sequencename (tab) sequencename (tab) sequence (tab) notefeature library format

PCR reaction tool

PCR reaction tool

PCR reaction tool
Select oMBC22 and23 primers.
These are the aroundthe horn mutagenicprimers.

PCR reaction tool

Select this to do a new
PCR reaction

PCR reaction tool
In primer pair mode,
only one forward andone reverse primer canbe selected. Selectedprimers are in bold.

New PCR will become
active when a pair ofprimers is selected.

PCR reaction toolGibson Designer

Gibson Designer

Do oMBC26-oMBC27
PCR on pMLS280Gibson Designer

Do oMBC28-oMBC29
PCR on amilCPGibson Designer

Do oMBC30-oMBC31
PCR on amilCPGibson Designer

Gibson Designer
add the three new
DNA sequences inorder

In this case, we don’t
need new primers. We
are doing a Gibson
using someone else’s
designGibson Designer
First fragment

Second fragment

Press next to see eachof the three junctions

Gibson Designer
Second fragment

Final fragment

Generate productGibson Designer
The first template sequence
The reverse primer sequence
The forward primer sequence
The second template sequenceGibson DesignerDesign a new Gibson reaction

Gibson Designer
Use the open plasmids
with these selectionranges

Gibson Designer
First fragment
Reverse primer

Foreward primer

Second fragment

Gibson Designer
priming hybridization

Gibson hybridization

Gibson Designer

Adjust the Tm
Adjust the Tm
If there is already overlap
between the templates, you
can use that

Adjust the overlap Tm

Gibson Designer

You can also add nontemplated sequence here

Set which fragment gets theoverlap

Add overlap to the other side

Gibson Designer

You have to examine eachjunction

Gibson Designer

Gibson DesignerGibson PCR products,primers etc. are in the filecomment

Gibson Designer

Gibson PCR primers are inthe file features

Translation toolTranslation toolTranslate all of asequence, selected
text, or any “CDS”
type featureTranslation tool

Format the output

Translation tool

Enzyme Selection

Shift-click to change
all tools to apply toselection rather thansequence

You can select thisoption to change alltools to apply toselection rather thansequenceEnzyme Selection

This toolbar iconchanges when you
have “selection only”
applied

You can select thisoption to change alltools to apply toselection rather thansequenceEnzyme Selection

Current window tosearch
Apply search toselection
Current selection inwindow (can bechanged while thedialog is open)Enzyme Selection

All enzymes are shownhere

Enzyme Selection
The current enzymes
can be changed byloading new ones
using the file menu
WHILE THE DIALOG ISOPEN

Enzyme Selection

Click on any enzyme toselect it
Shift-click on any
enzyme to select onlythat enzyme
The enzyme currently
under the mouse isshown here

Enzyme Selection

The selection criteria
are shown here

Enzyme Selection

Any enzyme matchingboth the count AND
the group areunderlined

This is the countThis is the group

Enzyme Selection

Click here to select allunderlined enzymes

Click here to DE-selectall underlined enzymes

Click here to de-selectALL enzymes

Enzyme Selection

Click here to select
only enzymes that are
currently selected AND
underlined

Enzyme Selection

Right-click to searchfor enzyme info

Enzyme Selection

Click here to do
analysis functions

Enzyme Selection

The diﬀerence group isspecial

Enzyme Selection• Select the MCS in pMLS280• Open the enzyme selector• Select all unique enzymes in pMLS280• Chose the diﬀerence group andpMLS280+selection within thediﬀerence selector• Deselect the diﬀerent enzymes• Do a graphic map• What do you see?

Enzyme Selection

Enzyme Selection

Find enzymes to clone amilCP
into pMLS280

Enzyme Selection• Select from 25 to 751 in amilCPEnzyme Selection• select all enzymes that are absent fromthe selection in amilCPEnzyme Selection• select all enzymes that are ALSO present

somewhere in the plasmid, and in the Jorg collection

Enzyme Selection• do a graphic map

Enzyme Selection• do a graphic map

Enzyme Selection• select all enzymes that are ALSO unique in the pMLSMCS

Enzyme Selection• select all enzymes that are ALSO unique in pMLS

Enzyme SelectionPreferences dialog

Preferences dialog

Recombination tool

Recombination tool

Recombination tool

Recombination toolGraphic Map

Graphic Map

Graphic Map

Graphic Map

Graphic Map

Graphic Map

Graphic Map

ABI files

Drag this to a sequence
window to add the abi file
to a sequence fileScale

ABI files

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