“open” button – opens files with the extensions: .pdb. “save” button – saves files with the extensions: .pdf. “close” button – closes file and clean all related windows. “print” buttons – print the screenshot content of the 3D view.
3D Protein/DNA/RNA view
Creating the database for protein secondary structures prediction
Open a protein sequence in the Protein Tab
3D Protein/DNA/RNA view There are three view options – “ball & stick”, “wire” and “space fill”. To open a protein chain select the corresponding row in the table “Present chains”. To see the protein sequence select the corresponding row in the table “Selected chains”. The sequence will be displayed in the sequence window. To remove the selected row in the table “Selected chains” use the button “remove selected protein chain”. To remove a particular region of the protein from the tertiary structure view, first, select the corresponding region of the protein sequence in the sequence view. Second, set to “on” the checkboxes “backbone chain”, “side chain” or “AA name/position” to remove the corresponding parts. Then, press “remove” button. To view other (not protein, DNA, RNA) molecules and atoms select the corresponding row in the table “Other atoms/molecules”. To remove the selected molecules and atoms select the corresponding row in the table “Selected atoms/molecules” and press the button “remove selected atoms/molecules”. To save the image of the tertiary structures as .pdf file use the “save” button. To print the image of the tertiary structures, use the “print” button. Press the “colors / non-canonical AA” button to see the color schemes for atoms and secondary structures, as well as the list of displayed side chains of non-canonical amino asides. Note. The protein subsequences are marked as corresponding secondary structures according to the SHEET, HELIX definitions present in the PDB file.
Creating the database for protein secondary structures prediction The default database is for mesophilic organisms (cytoplasm and membrane locations). To create a custom database, open the .pdb file, press “add SS motifs to DB (all chains)” button, select the database(s) where you wish to add the motifs, and then press the “add” button.
Open a selected protein sequence in the Alignment Tab The sequence of selected protein can be transferred to the Protein Tab for further analysis. For this, press the “Protein” button after the selection of protein in the “Selected chains” table.
“open” button – opens files with the extensions: .txt, .rtf, .fasta (.fas, .faa), .seq, .gp, .blprotein. “save” button – saves files with the extensions: .txt, .rtf, .fasta (.fas, .faa), .seq, .gp, .blprotein. “close” button – closes file and clean all related windows. “print” buttons – print the content of the main window or the peptides table.
The buttons “SS Prediction/PDB”, “Hydropathicity/pI Plots”, “Features”, “Annotation”, “Limited Proteolysis”, “SS database” open pop up windows. A mouse click outside of the popup window will close the window, and a click and drag action will generate a separate window..
Protein parameters
Prediction of protein secondary structures
Reverse translation
Hydropathicity/pI plots
Features and annotation
Limited proteolysis
“text colour” and “background colour” buttons
Mark and unmark amino acid residues
Mark and unmark protease sites
Mark and unmark peptides
Open a selected protein sequence in the Alignment Tab
Format protein sequence
Protein parameters The protein parameters: pI value, MW, net charge will be automatically displayed for the whole protein or for its part after the corresponding sequence selection. The amino acid composition of protein is displayed automatically for the whole protein in the “Amino Acids” table.
Prediction of protein secondary structures Press the “SS Prediction/PDB” button to perform the prediction or to see the protein secondary structures from a PDB file, and to display the coloured protein sequence in the popup window. Before, press the “SS database” button to open the popup window to set the protein secondary structures databases. The default database used for secondary structure prediction is that for mesophilic organisms (cytoplasm and membrane locations), but other databases can be available that may be more appropriate (e.g. for halophilic proteins). These databases should generated by user from the pdb files and may be useful for the analysis of particular types of proteins, e.g. when proteins belonging to the same protein family, or proteins in the same cellular location (membrane/cytoplasme) or environment (halophilic, mesophilic, thermophilic). To delete a custom database, set the corresponding checkbox on and press the “delete” button. The databases can be exported as text files. The created file of the custom databases can be imported. For this, use the “export”, “import” buttons. Add the custom databases for analysis by setting the corresponding checkboxes.
Reverse translation Pressing the “RevTran” button opens a popup window with a reverse translated gene sequence optimised for highest codon usage frequency.
Hydropathicity/pI plots Pressing the “Hydropathicity/pI Plots” button opens a popup window with hydropathicity and pI plots for the opened protein sequence. Set the “Hydrophobicity”, “Hydrophilicity” and “pI value” checkboxes to see the corresponding plots. To view the protein secondary structures underneath these plots, set the “SS prediction” checkbox to “on”, or the “SS from PDB” checkbox to “on” when a PDB file is opened. To change the “window size” of the plots use the corresponding slider. To save the plots as a .pdf file, use the “save” button, or to print – use the “print” button.
Features and annotation Click on the featured region of a protein and its features will be displayed in the “features” text field. Features Use the “Features” button to display, edit and add protein features. To select the features and colors to display in the protein sequence, use the corresponding check-boxes and color wells, then press the “selected” button. To remove or to edit a feature, delete or change the corresponding text in the features window and press “save” button. Alternatively to delete a feature, select a text inside the feature and press the “delete” button. If the features were incorrectly edited, to restore all initial features press the restore features button. For the selected protein part a new feature can be added from the features list or as a custom feature. Feature locations can be typed or will be set automatically for the selected part of the protein sequence. Use the “add feature” button to add the feature. The edited features are not saved until the “save” button is pressed. Annotation In the “Annotation” window, an annotation from .gp, .blprotein, or .fasta files can be displayed, edited and added. To save the annotation use the “save” button
Limited proteolysis Press the “Limited Proteolysis” button to see the list of peptides resulting from limited proteolysis for a protease selected in the table “Present Enzymes Sites”. The list contains the peptides sequences and their masses in parenthesis. To see the location of a peptide in the protein sequence and a peptide pI value, select the peptide sequence. To unmark the peptide sequence cancel the selection.
“text colour” and “background colour” wells Use these color wells placed in the bottom left corner to color the text and the text background.
Mark and unmark amino acid residues To mark amino acid residues in a protein sequence, select the corresponding row in the “Amino Acids” table. To unmark all amino acid residues use the “all” button. To unmark particular amino acid residues, select the corresponding amino acid residue in the “Amino Acids” table and then press “selected” button. To change the color of the marked amino acid residues, first unmark them, change color in the corresponding color well and then mark them.
Mark and unmark protease sites To mark protease cut sites in a protein sequence, select the corresponding row in the table “Present ES”. The selected protease name will appear in the table “Selected ES”. To unmark all protease sites use the “all” button. To unmark particular protease sites, select the corresponding protease in the table “Selected ES” and then press “selected” button. To change the color of the marked protease sites use the corresponding color well.
Mark and unmark peptides To mark the peptides, select the corresponding row in the peptides table. To unmark the selected peptide use the button “unmark”.
Open a selected protein sequence in the Alignment Tab To open the selected part of protein sequence in the “Alignment” tab select the corresponding sequence and press the “Alignment” button.
Format protein sequence Use the “format” button to remove colors, line breaks and white spaces in a protein sequence.
“open” button – opens files with extensions: .gb, .gbk. “save” button – saves files with extensions: .gb, .gbk. “close” button – closes file, clean the graphical and sequence views. “print” buttons – print the content of graphical or sequence view.
Graphical view
Sequence view
DNA features and annotation
DNA features databases and find features
Find motifs in DNA sequence
Find ORFs in DNA sequence
Transcribe, reverse complement and translate DNA sequence
GC content, CpG islands and stem loops
“text colour” and “background colour” buttons
Copy/paste the selected genome sequence with features
Open a protein from CDS in the Protein Tab or Alignment Tab
Open the selected genome sequence in the DNA or Alignment Tab
Graphical view If the genome size is more than 1000 000 bp (1 Mbp), the genome graphical view is displayed as a scale without features. To see the features reduce the genome scale below the 1000 000 bp by clicking and dragging in the corresponding region of genome. To zoom in or out, set the zooming size (top right of window) and press the “+” or “-” buttons. To shift the view frame to the left or right side use the “<” and “>” buttons. To return to whole genome view use the “return” button . To search for a word present in the features descriptions use search field and the “<” and “>” buttons. The graphical view and the sequence view will automatically be changed according to the feature position where this word is found.
Sequence view The genome sequence with features will be displayed when the genomic scale is less the 50 000 bp (50 kb).
DNA features and annotation (Note. The graphical user interface is the same as for the DNA Tab) Click on the featured region of the genome sequence and its features will be displayed in the “features” text field. To see the complete feature description, place the mouse pointer over the corresponding graphic element in the genome graphical view. Click on this element to highlight the corresponding sequence in the sequence view. Use the “Features” button to display, edit and add DNA features. To select the features and colors to display in the genome sequence and Map, use the corresponding check-boxes and color wells, then press the “selected” button. To remove or to edit a feature, delete or change the corresponding text in the features window and press “save” button. Alternatively to delete a feature, select a text inside the feature and press the “delete” button. If the features were incorrectly edited, to restore all initial features press the restore features button . For the selected part of the genome, a new feature can be added from the features list or as a custom feature. Feature location can be typed or will be set automatically for the selected part of genome sequence. To mark the DNA strand as complementary use the checkbox “complementary strand”. Use the “add feature” button to add the feature. Press the “features table” button to open the website with a complete list of genbank features. The edited features are not saved until the “save” button is pressed. Use the “Annotation” button to display and edit the anannotation. To save the annotation use the “save” button.
DNA features databases and find features (Note. The graphical user interface is the same as for the DNA Tab) Use the “Features Databases/Find” button to set the features databases and to find features in the DNA sequence from the main window. There are two custom databases: “Vectors” and “Genomes”. Features can be added to the databases from a genbank file when the corresponding checkboxes are On. The features of the same origin selected in the tables can be deleted by pressing the “delete” button. To find features, set the checkboxes and press the button “Genome”. The find function searches for exact matches of databases features and sequences. The databases can be exported and imported as text (.txt) files.
Find motifs in a DNA sequence (Note. The graphical user interface is the same as for the DNA Tab) Use the “Find” button to search for repeats, palindromes, stem loops, “start…end” and arbitrary sequences. Searches will be applied to the selected part of DNA sequence. To start a search press the “find” button and to cancel an active search use “cancel” button. To clear the windows use the “clear” button. To save the found motifs as a text file, use the “save” button. When the motif is selected it will be marked in the main DNA window. The selected motif in the DNA window will be unmarked when the selection in the “Palindromes”, “Repeats” or “Found Stem Loops / Start-End sequences” windows is cancelled. Use the color well to change the color of the marked motif in the main DNA window. Palindromes and repeats Use the “length (bp): minimum” and “maximum” to set the minimal and maximal sizes of the palindromes and repeats to be located. When the checkbox “inverted” is set to “on”, the program will search for both direct and inverted repeats. If the checkbox “inverted” is “off”, then the program will search only for direct repeats. Any sequence To find, mark and unmark any DNA sequence in the main DNA window, use the “find”, “mark” and “unmark” buttons. To clear the search DNA window, use the “clear” button. The positions of the found DNA sequence will be displayed in the “Found positions” window. Start-End sequences To locate a sequence using only starting and ending sequences, use the “start-end sequence” button and provide a “start” sequence and an “end” sequence in the corresponding windows. Also provide the maximal distance between the “start” and end” sequences. Stem Loops To find stem loops in the selected DNA sequence choose the “stem loops” segmented button, set the parameters of stem loop and press the “find” button.
Find ORFs in DNA sequence (Note. The graphical user interface is the same as for the DNA Tab) The “ORFs” button opens a popup window with options for searching ORFs in the DNA sequence shown in the main window. The “ORFs” search is applied to the selected part of DNA sequence. (The same functions are used for DNA1 and DNA2). Type a minimal ORF length in the textfield “ORF minimal length(bp)” to limit the ORFs length. Set the “minimal pI” and “maximal pI” to search for ORFs encoding the proteins with pI values between these limits. To choose the start codons for forward and reverse ORFs, use the “Forward:” and “Reverse:” popup menus. Use the “find” button to start the ORFs search. If in the “Forward ORFs” or “Reverse ORFs” windows the ORF sequence is selected the corresponding translated protein sequence and its features will be displayed in the “Protein” window. To translate all ORFs, use the “translate all ORFs” button. To clear the windows, use the “clear” buttons. To save the found ORFs as a text file, or the graphic view of ORFs as a pdf file, use the “save” button. To print the graphic, use the “print” button.
Transcribe, reverse complement and translate DNA sequence (Note. The graphical user interface is the same as for the DNA Tab) Use the “Tran/RevCom/Tran” button to convert a DNA sequence in the main window into an RNA sequence, to perform DNA reverse-complement conversion and to translate the DNA. The “Tran/RevCom/Tran” functions will be applied to the selected part of DNA sequence. When the reverse-complement sequence is selected in the popup window the corresponding proteins will be displayed. When a protein sequence is selected, the protein properties will be displayed. To change the translation frame, use the appropriate selection of the DNA sequence.
GC content, CpG islands and stem loops Press the “GC/CpG/SL” button to open a popup window with a GC content, CpG islands and Stem Loops plots for the selected DNA sequence. To view the plot of stem loops, set the “Stem Loops content” checkbox on. Press the “SL settings” button to set the stem loop parameters. To change the window size of the plots use the corresponding slider. To save the plots as a .pdf file, use the “save” button, or to print – use the “print” button.
“text colour” and “background colour” wells Use these color wells placed in the bottom left corner to color the text and the text background.
Copy/paste the selected genome sequence with features The selected genome sequence can be copied together with features. For this, use the “copy” button. To paste this sequence with features, use the standard paste function. The alert window will ask if you would like to paste only the sequence or the sequence with features. To empty the buffer containing the selected sequence and its features use the “empty” button.
Open the protein from CDS in the Protein Tab or Alignment Tab To open the protein sequence in the “Protein” or “Alignment” tab, click on the corresponding CDS and press the buttons “Protein” or “Alignment”.
Open the selected genome sequence in the DNA or Alignment Tab To open the selected genome sequence in the “DNA1”, “DNA2” or “Alignment” tab, select the corresponding sequence and press the “DNA1”, “DNA2” or “Alignment” buttons.
“open” button – opens files with the extensions: .txt, .rtf, .fasta (.fas, .fa, .fsa, .ffn, .fna), .seq, .ape, .gb, .gbk, .ab1, .bldna, .xdna. BioLabDonkey reads files created by Serial Cloner(.xdna), DNA Strider(.xdna) and ApE (.ape) programs. “save” button – saves files with the extensions: .txt, .rtf, .fasta (.fas, .fa, .fsa, .ffn, .fna), .seq, .gb, .gbk, .ab1, .bldna. “close” button – closes file and clean all related windows. “print” buttons – print the content of the main window or the DNA fragments table.
The buttons “Tran/RevCom/Tran”, “ORFs”, “Find”, “Primers”, “SeqChrom”, “SilentMut”, “GC/CpG/SL”, “Plasmid”, “Linear Map” “Feature Databases/Find”, “Features”, “Annotation”, “make”, “show Gel” open popup windows. A mouse click outside of the popup window will close the window, and a click and drag action will generate a separate window.
Transcribe, reverse complement and translate DNA sequence
Find open reading frames (ORFs) in DNA sequence
Find sequence motifs in DNA
Generate primers
ABI sequencing chromatogram
ORF silent mutations
GC content, CpG islands and Stem Loops
Plasmid map
Linear map
Text map
DNA features
DNA features databases and find features
DNA annotation
“text colour” and “background colour” buttons
Sort found restriction sites by names or numbers
Make custom list of restrictases
Choose restrictases list
Search for restriction sites in DNA sequence
Mark, show and unmark restriction sites
Mark and unmark DNA fragments
Cloning of a restricted DNA fragment
Virtual gel
Copy/paste the selected DNA sequence with features
Open the protein from CDS in the Protein Tab or Alignment Tab
Open the selected DNA sequence in the Alignment Tab
Format DNA sequence
Notes
Transcribe, reverse complement and translate DNA sequence Use “Tran/RevCom/Tran” button to to convert the DNA from main window into an RNA sequence, to perform DNA reverse-complement conversion and to translate the DNA. The “Tran/RevCom/Tran” functions will be applied to the selected part of DNA sequence. When the reverse-complement sequence is selected in the popup window the corresponding proteins will be displayed. When protein sequence is selected the protein properties will be displayed. To change the translation frame use the appropriate selection of DNA sequence in main window.
Find open reading frames (ORFs) in DNA sequence The “ORFs” button opens a popup window with options for identifying ORFs in the DNA sequence of the main window. The “ORFs” search is applied to the selected part of DNA sequence. Type a minimal ORF length in the textfield “minimal ORF (bp)” to ignore ORFs below the desired length. Set the “minimal pI” and “maximal pI” to search for ORFs encoding the proteins having pI values relative to these limits. The types of start codons for forward and reverse ORFs can be chosen using the “Forward” and “Reverse” poopup menus. Use “find” button to start the ORFs search. By selecting the ORF sequence in the “Forward ORFs” or “Reverse ORFs” windows, the corresponding protein sequence and its features will be displayed in the “Protein” window. To translate all ORFs use the “translate all ORFs” button. To clear the windows use “clear” buttons. To save the found ORFs as text file or the graphic view of ORFs as a pdf file use the “save” button. To print the graphic use the “print” button. When the found ORF is selected it will be marked in the main DNA window. The selected ORF in the main DNA window will be unmarked when the selection of found ORF is cancelled.
Find sequence motifs in DNA Use “Find” button to search for repeats, palindromes, stem loops, “start…end” and arbitrary sequences. The motif search will be applied to the selected part of DNA sequence. To start a search press the “find” button and to cancel an active search use “cancel” button. To clear the windows use the “clear” buttons. To save the found motifs as a text file, use the “save” buttons. When the motif is selected it will be marked in the main DNA window. The selected motif in the DNA window will be unmarked when the selection in the “Palindromes”, “Repeats” or “Found Stem Loops / Start-End sequences” windows is cancelled. Use the color well to change the color of the marked motif in the main DNA window. Palindromes and repeats Use “length (bp): minimum” and “maximum” to set the minimum and maximum sizes of the palindromes and repeats. When the “inverted” checkbox is ticked the program will search for both direct and inverted repeats. When the “inverted” checkbox is off the program will search only for the direct repeats. Any sequence To find, mark and unmark any DNA sequence in the main DNA window use the “find”, “mark” and “unmark” buttons. To clear the search DNA window use the “clear” button. The positions of the found DNA sequence will be displayed in the “Found positions” window. Start-End sequences To search for a sequence by its starting and ending sequences, provide the “start” sequence and the “end” sequence in the corresponding windows, as well as the maximum distance between the “start” and end” sequences. To start the search set the segmented button to “start-end sequences” and press the “find” button. Stem Loops To find stem loops in the selected DNA sequence choose the “stem loops” in the segmented button, set the parameters of stem loop and press the “find” button.
Generate primers Use the “Primers” button to search for primers. The primers will be generated for the selected part of DNA sequence. To clear windows and to save the primers as a text file use the “clear” and “save” buttons. Select the primer sequence after the restriction site to see this sequence in the main DNA window. Deselect the DNA sequence to unmark this sequence in the main DNA window. The primers search algorithm filter out the DNA oligonucleotides with: – subsequences of 4 or more of one base, – dinucleotide repeats, – self-dimers/hairpins (with stem length more than 4 bp and loop length less than 10 bp). Primers for cloning and sequencing Set the “primers length (after restriction site) (bp)”, the 5′ end sequence and the restriction site sequence in the corresponding fields. Then, use “forward primers” or “reverse primers” buttons to generate the primers. To test for cross-dimer formation by the selected forward and reverse primers pair use the “cross dimer test” button. Primers for ORF cloning Set minimum and maximum “length (bp)” of primers, the 5′ end sequence and the restriction site sequence in the corresponding fields. Use “forward primers” and “reverse primers” buttons to generate the primers. To test the cross dimer formation by the selected forward and reverse primers pair use the “cross dimer test” button. Primer pairs for qPCR Set the “length (bp)” of forward and reverse primers in the corresponding fields. Then, use the “primer pairs” button to generate the primers. The forward and reverse primers of a pair have the same number in the corresponding windows. The generated primers pairs will be automatically tested for the cross-dimer formation.
The generated primers can be stored inside the program as text file wich can be exported/imported/deleted. To add primer press “add” button and then type or copy/paste a primer name and a primer sequence in the corresponding fields. To delete the selected primer use “delete” button. The stored list of primers will be displayed automatically with opening “Primers” window. Use search field to search for a primer name/sequence in the stored primers list.
ABI sequencing chromatogram To open an .ab1 file use the “open” button. The ABI sequencing chromatogram will be opened in a popup window, while the corresponding DNA sequence from the ABI file will be opened in the main window. The distance between peaks and their heights can be scaled using the vertical and horisontal sliders. The DNA sequence letters in the popup window can be removed or substituted. To save these sequence changes use the “save sequence changes” button in the sequencing chromatogram popup window. After the saving these sequence changes will appear in the main DNA window. To reopen the popup window with the ABI Sequencing Chromatogram use the “SeqChrom” button.
ORF silent mutations Use the “SilentMut” button to open a popup window with options to make silent mutations in ORF. When an ORF sequence is selected in the main window, press the “SilentMut” button. The ORF sequence will appear in the “Initial ORF” window. Alternatively, the ORF sequence can be pasted or typed directly in the “Initial ORF” window. The tables “Absent RS to insert” and “Present RS to eliminate” will be generated automatically for the sequence in the “Initial ORF” window. There are options to insert or to delete the restriction sites, as well as to maximize the ORF codon usage in Escherichia coli or in Saccharomyces cerevisiae. To insert or to delete the restriction sites first choose the genetic code. To delete a restriction site select the restrictase name in the table “Present RS to eliminate” and the modified ORF sequence with corresponding mutations will appear in the “Modified ORF” window. The mutated codon will be marked in orange while the rest of the restriction site will be coloured in green. To insert a restriction site select the corresponding name in the table of “Absent RS to eliminate”. The positions of new restriction sites will appear in the table “RS position to insert”. Select a RS position and the mutated ORF will appear in the window “Modified ORF”. The subsequence containing the restriction site will be marked in orange in the window “Modified ORF” and in green in the “Initial ORF” window. To maximize the ORF codon usage in Escherichia coli or in Saccharomyces cerevisiae select the organism and press the “codon usage maximization” button. The programm will use the standard genetic code and the codons of initial ORF will be exchanged to the codons of maximal usage. The modified ORF sequence will appear in the window “Modified ORF”.
GC content, CpG islands and Stem Loops Press the “GC/CpG/SL” button to open a popup window with a GC content, CpG islands and Stem Loops plots for the DNA sequence selected in the main DNA window. To view the plot of stem loops, set the “Stem Loops content” checkbox to “on”. Press the “SL settings” button to set the stem loop parameters. To change the window size of the plots use the “window size” slider. To save the graph s as a .pdf file, use the “save” button, or to print – use the “print” button.
Plasmid map Press the “Plasmid” button to open a popup window with a plasmid map of the DNA sequence. Use the “add” button to add the featured region of DNA sequence to the map when the DNA sequence is selected or the I-cursor is placed in the featured region of DNA sequence. If no features are added, all features will be on the plasmid map. Use the “remove” button to remove all selected features for the map. After the removal all features will be on the plasmid map if the “Plasmid” button is pressed. The scale of the map can be adjusted in the the “Plasmid” window using the slider at the top. When the mouse cursor is placed on the plasmid feature its description will appear in popup note window. One mouse click on the plasmid feature will: 1. scroll to and highlight the corresponding sequence in the main window 2. scroll to and highlight the corresponding feature in the popup “feature” window when the “feature” window is open. A double mouse click on the plasmid feature will remove this feature from the map. To restore all features on the map use the restore button. Use the checkbox to turn on/off feature names on the map. Use the “print” button to print the map and to save it as pdf file. Use the “save” button to save the map as pdf, jpg(jpeg), png or eps file.
Linear map The linear map has the same functionality as the circular Plasmid Map, except for the map scaling.
Text map The text map represents the direct and the reverse complement strand sequences together with six (+1,+2,+3,-1,-2,-3) translation frames. The selected restriction sites are displayed above the direct strand, and the list of restriction sites is placed at the end of text map. For the direct and reverse complement ORFs the corresponding direct and reverse complement strand sequences are coloured. The genbank features can be displayed at the end of the text map, after the list of restriction sites.
DNA features Click on the featured region of DNA and its feature names, position, orientation will be displayed in the “features” text field. Use the “Features” button to display, edit and add DNA features for saving as genbank or bldna files. To select the features and colors to display in the DNA sequence and Maps use corresponding check-boxes and color wells, then press the “selected” button. To remove or to edit a feature, delete or change the corresponding text in the features window and press “save” button. Alternatively to delete a feature, select a text inside the feature and press the “delete” button. If the features were incorrectly edited, to restore all initial features press the restore features button. For the selected DNA part a new feature can be added from the features list or as a custom feature. Feature locations can be typed or will be set automatically for the selected part of the DNA sequence. To mark the DNA strand as complementary use the checkbox “complementary strand”. Use the “add feature” button to add the feature. Press the “features table” button to open the website with a complete list of genbank features. The edited features are not saved until the “save” button is pressed. Use the “unmark selected regions” button to unmark all region corresponding to the selected ORFs, repeats, palindromes or stem loops.
DNA feature databases and find features Use the “Feature Databases/Find” button to set the features databases and to find features in the DNA sequence from main DNA window. There are two custom databases: “Vectors” and “Genomes”. To add the features to the database, open the genbank file, set “On” the corresponding checkboxes and press the “add to DB” button. The features of the same origin selected in the table can be deleted by pressing the “delete” button. To find the features, set the checkboxes and press the button “DNA1”, “DNA2” or “Genome”. The find function will search for exact matches from the annotated features of the database sequences. The databases can be exported and imported as txt files.
DNA annotation Use the “Annotation” button to show, edit and add DNA annotations for genbank and bldna files, or a description line for fasta files. To save the annotation use the “save” button.
“text colour” and “background colour” wells Use these color wells placed in the bottom left corner to color the text and the text background.
Sort found restriction sites by names or numbers The list of restriction sites present in a sequence can be sorted. Select the corresponding section to sort by names of the restriction sites in alphabetic order, or by numbers of present restriction sites.
Make custom list of restrictases Press “make” to set a custom list of restrictases for DNA1 and DNA2 tabs. Select a restrictase by name from the complete list of restrictases and then use the “add ->” button to add it to the custom list. To remove a restrictase from the custom list, select the restrictase and press the “delete” button. The multiple rows can also be selected and deleted.
Choose restrictases list Select the “short” section to use the list of commonly used restrictases, the section “complete” to use the complete list of restrictase available on the market or “custom” to use a preselected custom list of restrictases.
Search for restriction sites in DNA sequence To find restriction sites in the whole DNA sequence use “whole” checkbox. In a search for restriction sites in the selected part of DNA sequence use “range” checkbox. The range of the selected DNA sequence can be defined by the corresponding selection of DNA or by typing the start and the end of the range.
Mark, show and unmark restriction sites To mark the restriction sites in a DNA sequence select the corresponding row in the table “Present RS”. The selected restrictase name will appear in the table “Selected RS”. To see the marked sites of the selected restrictase, use the “<” and “>” buttons. To refresh the positions of the marked sites of the selected restrictase, for instance after the sequence changes, use the refresh button. To unmark all restriction sites, press the “all” button. To unmark particular restriction sites, select the corresponding restrictase in the table “Selected RS” and press the “selected” button. To change the color of the marked restriction sites, use the corresponding color well.
Mark and unmark DNA fragments To mark the restricted DNA fragment, select the corresponding row in the table of restricted DNA fragments. To unmark the selected DNA fragment use the button “unmark”.
Cloning of a restricted DNA fragment To clone a restricted DNA fragment from a sequence in DNA1 (DNA2) Tab into the corresponding unique restriction sites of a vector sequence in DNA2 (DNA1) Tab, select the restricted DNA fragment from the table in DNA1 (DNA2) Tab and press the “DNA2” (“DNA1”) button. The restricted DNA fragment (with GenBank features) will be automatically inserted into the corresponding restriction sites of the vector. The fragment will not be be inserted if there are no suitable restriction sites in the vector. Press the “CS” button to see the cloning schemes and the table of cloning history.
Select a row in the table to see the corresponding cloning scheme. To chanage the vector, insert or product names double click in the corresponding fields of the table. The cloning scheme can be printed or saved to the image file. The table of cloning history can be printed.
Virtual gel Use the “show Gel” button to see a virtual agarose gel of the DNA restriction fragments. Beforehand, press the “add” button to add a line of DNA restriction fragments for the selected restrictases from the “Selected RS” table. The number of the line and the file name of restricted DNA sequence will appear in the table of lines. The line number is combined with C or L letter defining the circular or linear form of the restricted DNA sequence. To change the DNA form select the corresponding section in the “circular/linear” segmented button. The file name in the table can be substituted to any custom name. A complete description of the line is presented in the text field below the table of lines. It includes the line number, line name and the sizes of the fragments with the names of restrictases. To add a new line to the gel open a new file and repeat the described procedure. To clear the gell use “clear” button. To change the run-time use corresponding slider. To change the DNA ladder use corresponding popup button.
Copy/paste the selected DNA sequence with features The selected DNA sequence can be copied together with features. For this, use the “copy” button. To paste this DNA sequence with features, use the standard paste function. The alert window will ask if you want to paste only the sequence or the sequence with features. To empty the buffer containing the selected sequence and its features use the “empty” button.
Open the protein from CDS in the Protein Tab or Alignment Tab To open the protein sequence in the “Protein” or “Alignment” tab, click on the corresponding CDS and press the buttons “Protein” or “Alignment”.
Open the selected DNA sequence in the Alignment Tab To open the selected DNA sequence in the “Alignment” tab, select the corresponding sequence and press the “Alignment” button.
Format DNA sequence Use this button to remove colors, line breaks and white spaces in the DNA sequence.
Notes The melting temperature of a DNA molecule, Tm, is calculated using the following equation: Tm (°C) = 64.9 + 0.41(%GC) – (500/N) which came from Tm (°C) = 81.5 + 16.6(log[Na+]) + 0.41(%GC) – (500/N), for [Na+] = 100mM where: %GC is the percentage of G and C, N is the DNA length, [Na+] is the molar concentration (moles/L)
The annealing temperature for a primer, Ta, is calculated using the following equation: Ta (°C) = 81.5 + 0.41(%GC) – (675/N) (Reference: Oligonucleotide Melting Temperatures … )
placed in the bottom left corner to color the text and the text background.
