Serial Cloner 1.2
Contents
1. Available in the Sequence window this allows to undo the last action deletion insertion upper lowercase 5 levels of undo are available Open C Y Redo and action that has just been undone Similarly 5 levels of redo are available e Select all CA Whenever the active field 1s containing text this command allows to select all this text e Cut C X Delete the selected text and store 1t 1n the clipboard Copy C C Copy the selected text and store it in the clipboard When copied from a sequence window the text is formatted as 80 nucleotide long lines before storing e Copy as Fasta CK Available in the Sequence window this command allows to copy the portion of the sequence selected and store it 1n the clipboard after formatting 1n Fasta format Header starting with a gt and 80 nucleotide long lines e Paste CV Paste the text contained in the clipboard at the insertion point Note that in sequence fields non ACGT letters are filtered out In case non ACGT letters are filtered out a message window will pop up to indicate how many letters were filtered out Direct parsing of certain format is provided by the Paste function if the sequence in the clipboard is in a FASTA format then the first line starting by a gt is pasted in the comment field and the rest of the sequence is pasted in the sequence field In case the sequence in the clipboard is a complete HTML page copied from a NCBI Genbank page usi2. 79 Pabi 205 gt Bsp24i 547 sw 726 gt amp co57Mi ME 537 coNI 942 Msll TRIS Jac D Gco 145 sra 290 estyi 477 Sphi 652 lt 8bs 854 Eag 1054 Mspan EL LH LN MN Hi Eu gd HPE dH E gd I DXILI S DUI Zz d Tetracyclin Gene 1191 nt To select a fragment one needs to select a BEGINNING and an END restriction site that will be the beginning and end of the produced fragment The BEGINNING site is selected clicking on restriction site name box When selected the site 1s shown in a different color green by default customizable using the preference window The END restriction site is selected by double clicking or by shift clicking of the site name box Again when selected a different color is displayed red by default To cancel a selection either press on Delete on the keyboard or use the contextual menu Some differences between linear or circular maps Circular One should decide whether the selection is done in a clockwise or counterclockwise CW CCW way between the Beginning and End restriction sites To change it either press on the Spacebar or use the contextual menu In a circular vector selecting only one site Beginning or End is enough to extract a fragment The resulting fragment is then the complete linearized vector with cohesive ends corresponding to the chosen site Linear the CW CCW option is replace by Sense antisense In this case the Beginning and End
3. Serial Cloner 1 2 User Manual Part Basic functions SenalCloner 1 2 File Edi Sequence Restriction Cenerate Window 9 9 uw 235 1 2 A P F0 Sel 2938 bel Crash Man of pBluescript wn KS pBluescript II KS 2961 nt Show vV unns Sow Detur iw Perteslar sites Chess ome i r Mutuo FE TL m 4 pee aa one gace Fax gE mg ag tjm are eel me ay gerer caneret garri gerer t goa nat ommy xc qoc nam OKO staat pee l hd A SE i DE Di E TA ma a a iip i SESE Ist IHEIT I TE Enter nere Pe actus veQuence of Pe fagment Typen or pase ATGC ene ody are accepted ofer are nec ar Crt Cace or Fagt che Sapuys a corta mens j Built In Help Window You will find in this manual the description of the different windows and functions of Serial Cloner as well as some useful tips A general description is also available inside Serial Cloner using the built in Help window You can show or hide the Help window using a menu command available in the Window menu and on the Toolbar window o Help Make the pointer fly over a button Help on active window or a field of the front most and To display Help and Hints just point on an element on the active window active window to dynamically obtain help and hints Il Differences between the MacOs X and the Windows versions All the functions of Serial Cloner are available in both versions with only interface differences The Abou
4. e Antiparallel C Transform the sub sequence selected in the up most Sequence window into its anti parallel sequence e g AAGGAAGG becomes CCTTCCTT Identical to a Reverse Complement operation e Reverse Reverse the sub sequence selected in the up most Sequence window e g AAGGAAGG becomes GGAAGGAA e Complement Tranform the sub sequence selected in the up most Sequence window into its complementary sequence e g AAGGAAGG becomes TTCCTTCC Linearize or Circularize Modifies the topology of the sequence The menu indicates Linearize if the up most sequence is circular Circularize if it is linear Set Origin Sets the origin of the current sequence at the insertion point e Read selection Use the voice set up of the operating system to read the selected sequence Stop reading by pressing ESC D The Restriction Menu Restriction PRestriction Graphic Map Ctri G Graphic Map 36G Sequence Map Ctrl M Sequence Map 88M Site Usage interactive Site Usage interactive Site Usage table Site Usage table Enzyme library Enzyme library e Graphic Map C G Available with a Sequence Window and in the Adaptor window this command will open a new window and display the Graphic map of the sequence Options are available in the Graphic Map window see underneath e Sequence Map C M Available with a Sequence Window this command will open a new window and display the textual
5. enzyme list As with other enzyme list enter the first 2 or 3 letters to directly scroll the list to the corresponding name Double click on the name select it and press Return on the keyboard or select it and click on the magnifying tool icon to start the search Another click will find the 19 next occurrence If the end of the map is reach the search will start again from the top Modify the display of sequence translation If translate all is unselected it is then possible to determine the nucleotide between which translation should be displayed If a segment was selected in the Sequence Window like an ORF when the Sequence map was created then the coordinate of this segment will be already pasted here see the screenshot above for example The Frame checkboxes allow to select the translation frames to display If only one frame is selected it is possible to display the map highlighting the codons or not Modify the display of the restriction map All restriction sites can be hidden when selecting Do not show RE sites If Show all sites 1s unselected then only RE cutting more than 1 unique sites 2 or x times will be shown depending on the value entered You can modify here the display of the map and whether you want to show the antisense strand or the nucleotide coordinates Character size as well as the number of nucleotide per line can be modified here The sequence map is displayed here and is updated automatically wh
6. 27 Nm 421 54 421 54 466 9 466 9 471 519 471 519 488 27 v is m s I amp Close Drawer e Close Drawer Ps Close Drawer 14 Sequence l Site ORFs Select an enzyme Aarl p Aatll tran Accl Acco5l Acelll Acil Acll Acul Afel 4 ARI x C Graphic Map Note To select the restriction site to be looked at either scroll in the list or enter the first 2 or 3 letters of the enzyme name to directly scroll to that position The name of the selected enzyme is then indicated in the Selected Enzyme box and the recognition site recalled bellow the enzyme list Click on find site to find the next occurrence of the site The search is only done in the portion of sequence that is after the insertion point When a non palindromic restriction site is found that the orientation of site is indicated by an arrow A new graphic map of the sequence contained in the currently active Sequence Window can be displayed pressing the Graphic Map button in the Sequence Window using the Restriction Graphic Map C G menu or pressing the corresponding button in the Toolbar Note that the Graphic Map also constitutes the left part of the Build Construct window A contextual menu can be displayed by Ctrl right clicking on the map It allows to Linearize circularize the map also available using the Sequence menu and to save the map as a picture When a selection has been done see bellow it ca
7. C Hh v E E EK YR De A E BD Q L A TCATACTCATARGITGTARAGGCACAGCOOGAATAAGGGARARAACGCCOTARAACGGAAGGACARAAACGAGTOGGICTTTGCGACCACTPTPCATITSCTACGACTHTCTABICAACCCACGNGC 0 130 140 150 160 170 180 190 200 210 220 230 Chal DpnI gt BpuEI MboI AlwI BStKTI BStKTI HpyCE4IV gt Ppil Dpni Tail lt Acil ESaBC3I Chai Acli Msei Sell TadI gt Alwl BstYI XmnI Dral BstUI gt Hgal gt Cdil BstYI lt Acil Smli 2MboII BSiEKAI HinP1I BsaHI MaeIIli gt Bsri MSpA1I Mboi Sth1321 BSp12861 Hhal 2Sthi321 A Rx a l LI AGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTG lt 375 Y 6 Page Setup Prnt 8 1 Find a sub sequence or a restriction site in the map A nucleotide or an amino acid subsequence can be entered in the field box Amino acid should be separated by 2 spaces 3 1n the codon based display to be found for example V A L F G should be entered to find the first five amino acid sequence Note also that if a sequence is interrupted by a carriage return continued to next line then it will not be found Press Return on the keyboard or click on the magnifying tool 1con to start the search Another click will find the next occurrence If the end of the map is reach the search will start again from the top A restriction site can also be searched Either enter directly the name or part of it in the box or click on the pop up menu to display the restriction
8. change the name of the modified sequence When the sequence window contains a new sequence then Save As 1s indicated and a new name will be asked for 17 Shows the textual sequence map restriction map and translation in a separate window If a selection has been made the coordinate of the selection will be exported in the sequence map and used to translate only this selection 1f needed 18 Shows the Graphic map of the sequence in a separate window 19 Opens the Find window Under MacOSX it opens a drawer Window Under Windows this 1s a separate window that behaves as a drawer window 20 Shows additional data bout the selection translation Tm GC etc When a selection is made one can extract the fragment using either the corresponding Toolbar button the Generate Extract Fragment C 6 menu or using the contextual menu The selected fragment is then pasted in a new Sequence Window and the comments associated with it summarizes how the fragment was obtained The fragment is blunt ended It can be saved or used directly in the Build a Construct window Selection Information and Translation The sequence window is extended when pressing the Show button in the Sequence Window or using the Sequence Show Info C menu 12 mug ocu HUNA c 1 arrsrauur Selected DNA 1 Tm 79 7 A 179 C 157 6 176 T 154 SC 50 096 Translation j 3 Size aa 221 MW 24 12 kDa Show only 1st ORF IMMSTFKVLL
9. restriction map of the sequence Options are available in the Sequence Map window see underneath Using these option one can use the Sequence Map not only to display the restriction map but also the multiframe translation of the sequence Site Usage interactive Available with a Sequence Window this command will open a new window and display a dynamic table representing the sequence site usage frequency of restriction sites This table cannot be printed but allows to quickly find absent unique sites or other sites quickly and compare to filter the site usage using another sequence site usage see underneath Site Usage table Available with a Sequence Window this command will open a new window and display a table representing the sequence site usage frequency of restriction sites This table can be printed Three tables are actually displayed the complete site usage table the unique site table and the absent site table Enzyme Library This command will open the Enzyme Library window This window allows to display select and modify the enzyme libraries used by Serial Cloner E The Generate Menu Build a construct Ctrl 1 Build a construct g l Create an adaptor Ctrl 2 Create an adaptor 3 2 Run a PCR Ctrl 3 Run a PCR 3 3 Make shRNA Ctrl 4 Make shRNA 4 Gateway Recomb cloning Ctrl 5 Gateway Recomb cloning 3 5 Extract Fragment Ctrl 6 Extract Fragment 986 Align two seque
10. restriction sites are logically swapped by pressing on the spacebar In addition one can Select all the linear fragment To do so press A on the keyboard After selecting the sites one can extract the fragment using either the corresponding Toolbar button the Generate Extract Fragment C 6 menu or using the contextual menu The selected fragment is then pasted in a new Sequence Window and the comments associated with it summarizes how the fragment was obtained Depending on the restriction site used the fragment may display cohesive ends It can be saved or used directly in the Build a Construct window 18 D Sequence Map A new Sequence map of the sequence contained in the currently active Sequence Window can be displayed pressing the Sequence Map button in the Sequence Window using the Restriction Sequence Map C M menu or pressing the corresponding button in the Toolbar Most of the default settings of the Sequence Map can be modified in the Preference Window eo060 Restriction Map of pBluescript Il KS Translation 2 Restriction sites 3 Display 4 A e v Translate all Translate To 989 Do not show RE sites vi Show all sites v Show Reverse strand v Coord S M Frame 1 V Frame 2 r Frame3 V Codons Show RE cutting maximum times Character size 12 pts 125 ntline Serial Cloner V1 2 lt 15 avr 07 23 59 5 Restriction map of pBluescript II KS Showing restriction enzymes cutting max
11. AT CCGCTCATGAGAC AAT AACCCTGAT 41 TTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATG 97 AAATGCTT CAAT AAT ATT GAAAAAGGAAGAGT ATOAGTATTCAACATT 81 AGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAA 145 TCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTT 121 GGAAGACTATGAGTATTCAACATTTCCGTGTCGCCCTTAT 192 TTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGT 161 TCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCAC 241 TGGGTGCACGAGTGGGTT ACATCGAACTGGATCTCAACAGCGGTAAGA 201 CCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGT 289 TCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTT 241 TGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAG 227 TTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGC 281 CGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTT 285 AAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTG 321 ORKAAN SA OROCO edita OAA 422 AGTACTCACCAGTCACAGAAAAGCATCTT ACGGATGGCATGACAGTAA 361 T ARAM CGTATT GGGCAAGAGCAACT G 451 GAGAATTATGCAGTGCTGCCATAACCATGAGTGAT AACACTGCGGCCA 401 CC AARAA eire resco 529 ACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTT 441 poe TCA apnea E Meere SAESASADA 577 TGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG 481 a EE es UE i A pide dien 3 E25 AGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTG 521 B us pere ves TC eT E eus aA 672 TAGCAATGGCAACAACGTTGCGCAAACT ATT AACTGGCGAACTACTTA 561 ere mes PS peus an TGC Dem eee ias GG paisg 72 CTCTAGCTTCCCGGCAACAATT AAT AGACTGGATGGAGGCGGAT AAAG 601 ieee aoara Annas p are A p mere 269 TTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTG 641 Crane COELAN EANN diei a W 817 CTGATAA
12. ATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAG 681 TTGCGCAAACTATTAACTGGCGAAC t a 16 17 18 EnA Zz T pve I Show Selection Information amp Translation 20 Show Selection Information amp Translation 20 7 This window allows sequence visualization manipulation and modification From the sequence window other windows can be opened find info The sequence opened in this window is available for other actions like Graphic and Text restriction maps Site usage or directly usable in the different action windows 1 The name of the opened sequence 2 The size of the sequence in nucleotide 3 The type as define by the DNA strider format essentially So far Serial Cloner only manipulates DNA and degenerate DNA sequences 4 The type of format Serial Cloner recognize the DNA strider Serial Cloner modified Strider type pDRAW32 and FASTA formats 5 Topology of the sequence Right Click or CTRL Click displays a contextual menu allowing to change the topology 6 Use this checkbox to lock or unlock the sequence A locked sequence cannot be inadvertently modified Newly opened sequences can be automatically locked See Preference Window 7 Usethese arrows to go directly at the beginning or at the end of the sequence 8 When a selection is made it indicates the position of the first nucleotide selected Can also be use to set the b
13. CGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEK HLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPEELTAFLHNM GDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLREKLLTGELL ITLASRQQLIDWMEADKVAGPLLRSALPAGUFIADKSGAGERGSR GIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKH mae Selecion inrormauon amp ITansiauon Tm 797 A479 157 G 176 T454 Translaton Size aa 221 2 MW 24 12 kDa pi GC 50 0 MMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSP VTEKELTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKE LTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMA TTLRKLLTGELLTLASRQOLIDWMEADKVAGPLLRSALPA GWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQ D e Show only 1st ORF W 4 ATMDERNROIAEIGASLIKEW 4 7 ee 1 Displays information about the selected DNA Tm number of A C G and T and of GC The Tm 1s calculated as follows For fragments 14 nt Tm A T 2 G C 4 16 6 log10 0 050 16 6 log10 Na For fragments gt 51 nt Tm 100 5 41 G C A T G C 820 A T G C 16 6 log10 Nat For larger fragments Tm 81 5 41 G C A T G C 500 A T G C 16 6 log10 Na 2 Displays information concerning the translation of the selected sequence the size calculated MW and sequence of the protein 3 Determine whether translation should finish at the first in frame STOP codon encountered or should continue through the successive STOP codons 4 The sequence of the translated peptide is displayed in this box The full sequence or sub sequences can be selected A contextual menu 1s accessible here
14. and allows in particular to save the peptide sequence in FASTA format When the user clicks in the protein field then the File menu is modified to offer the possibility of saving the protein sequence in Strider or FASTA format ITAANLLLTTIGGPKE i re Select All Translation 1G Copy Translation Save Protein Sequence As Save Translation Export Protein Sequence To Fasta B Find Window This window is attached to the Sequence Windows it is referring to This is a drawer window under MacOSX that is displayed when pressing the Find button in the Sequence Window using the Edit Find C F menu or pressing the corresponding button on the toolbar To close the Find drawer window either press again the Find button of the Sequence Window the Close Drawer of the Find Window or the Find button in the Toolbar Under Windows only one Find Window can be displayed to attach it to a particular Sequence window press the Find button of that window or use the Edit Find C F 13 menu or the toolbar when this sequence window is the front most one To close the info window under Windows click the Close Window button in the find window or click on the top right of the window ud Find 1 2 3 E mE ry coy Position Size Strand 6 TCA amp Close Drawer O Close Window 1 Click on this tab if
15. ct all the Choose Site button This pops up a window Usa previo t ka where the different restriction site can be selected Aad by clicking on the checkbox or double clicking on v cc the name The list of particular site can be erased Acc85I double clicking on unselect all and clicking on Acelli use previous selection re uses the same list as the Aci E Ac one used in a precedent Graphic Map F Acu Just click outside from the pop up window to close L amp te it J m Jan lAnel ea M jna 2981 nt ud 16 ear Graphic Map of Tetracyclin Gene Tetracyclin Gene 1191 nt Show W Unique Sites W Double Site Particular sites Choose site 942 MslII 175 Fspl 174 FspAl 5 Hp 864 BseYl 167 Tsp5091 66 Sa 1054 MspAl ie Tai 600 gt Acelll 404 sspe Sz 386 mani 547 gt Pie 767 TA 106 _ lt Ciel 537 EcoNi 714 Avai 368 gni 566 Acc 726 gt EcoS7MI 3 ggcc 3 5 3 ccgg 290 8am Sees 1051 Avali ELE M EHEE M I HI 0 wg wopuugug dp ru a Hl 4 ze Tetracyclin Gene 1191 nt An example of linear map where unique and double sites have been displayed in yellow and orange respectively In addition a particular site the site AvallI present 3 times is also shown in blue The red stretch on both extremities indicate that the fragment is cohesive The cohesive end sequence is
16. eginning of the selection in this case a value can be directly entered and or the up and down arrows can be used 10 to modify the value If no selection is made indicates the coordinates of the insertion point 9 When a selection is made it indicates the position of the last nucleotide selected Can also be use to set the end of the selection in this case a value can be directly entered and or the up and down arrows can be used to modify the value Blank when no selection 1s made 10 When a selection is made it indicates the length of the selected fragment Can also be use to set the length of the selection in this case a value can be directly entered and or the up and down arrows can be used to modify the value Blank when no selection is made 11 Use this tab to show the nucleotide sequence 12 Use this tab to show the c DNA Strider format Locked Sequence Lomments Extremities gil58061 emb x52327 ARBL2KSP pBluescript II KS vector DNA phagemid excised from lambda ZAPII Limited to 255 characters by DNA Strider Number of Characters 96 omments associated to the sequence length lt gt 256 DNA Strider format IV Locked Sequence Comments Extremities bi 58061 emb X52327 ARBL2KSP pBluescript Il KS vector DNA phagemid excised from lambda ZAPII Limited to 255 characters by DNA Strider Number of Characters 96 The comments respects the Strider format and 1s thus li
17. enever a parameter 1s modified Using the Edit Menu or C C it is possible to copy all Select all C A or a part of the map Click here to display a window to get some hints about how to set up the print parameters Modify the page set up used to print the sequence map also available using the File menu Print the map also available using the File menu or C P 20
18. ide will be considered as unknown e Close C W Closes the top most window If this is a window were modification have not been saved a confirmation window will be shown Close All Closes all opened window If there are windows were modification have not been saved a confirmation window will be shown for each of them e Page Setup Defines the printing parameters e Print C P Prints the information contained in the top most window Available for the Graphic Map Sequence Map Build a Construct and Align Two Sequences windows e Save C S Saves modifications done to an opened Sequence Sequence Window or adaptor Save s Proposes to save under a new name When a Graphic Map or a Build a Construct is the top most window the menu reads Save Graphic Map As allowing to save a picture file e Export to Fasta Save the Sequence in a text file in a fasta format Preferences Windows under MacOSX this option is found in the SerialCloner menu C Opens the Preferences window e Quit C Q Windows under MacOSX this option is found in the SerialCloner menu Quit Serial Cloner If there are windows were modifications have not been saved a confirmation window will be shown for each of them B The Edit Menu Edit Edit Undo Z Select all Ctrl 4 Select all aA Paste Ctrl y Paste de V Find Ctrl F Fi nd a6 F Find next ORF Ctrl Find next ORF R e Undo C Z
19. imum 1 time using RELibrary as a Restriction Enzyme Library LEE NlaliI NlalIV Fati lt Acil Hpyl8811II Seli BspHI BstUI 2BSrBI HinPil gt Acil lt BsmAI gt MboII Sth1321 Ehai gt Bcivi CviAII SspI Eari I Ic ee GTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAG lt 125 WC K uu EU m UE UE JA dgESG B UIS Se V RO X 4o WX EROR D RC oU DUA WC D U BRE UO EK WEN AO JE Be WU OA Wo X BOG A Co A OX Gp POS ho GU OI CR 0d OQ OX OR NW 5 w BOACMH X Tod XT L EF UNS OA Bo ud do B m X BOE C UN 3 oW aae OW Er AE 4e Roc 9 RB SM Wm UE X WU qe WU D WX x WX is Q SW RE ES OA UK CACCGTGAAAAGCCCCTTTACACGCGCCTTGGGGATAAACAAATAAAAAGATTTATGTAAGTTTATACATAGGCGAGTACTCTGTTATTGGGACTATTTACGAAGTTATTATAACTTTTTCCTTC 10 20 30 40 60 70 80 90 100 110 20 50 1 BstKTI gt BssSI chal HpyCH4 v ACiIl gt MboII BSiEKAI Fnu4Hi 2ECO57MI Bspl2861i TauI gt Acul Bme1580I TSpGWI BthCI lt SspD5I gt SspD5I lt SfaNI DpnI ApaLI MslI BisI gt HpyAV lt Hphi Hphi lt BscAI MboI HpyBI LJ Li AGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACG lt 250 Wo K Uv Ge MSS oo Ww OM Lh OE o To Ao GO dL E M OX 44 UE B ho XOAE OK OR UM ES 4 0 V GA SX LN S ks BOO oY 4E x Ta 2 o LL F 2 j Ig Gs X O8 dye ow ve Sw H W CE A a R g i A E B GA amp HB C F ew Bo N E C 2 S M 2 x E HC Xi Xx c Eo E A Z
20. indicated in a gray box The map can be printed using the the File Print C P menu or saved as an image file using the File Save As that reads Save Graphic Map as when a graphic map is at the forefront Saving Printing the map Use the corresponding Toolbar button the File Save Graphic Map As or the contextual Menu to save the graphic map as a PICT file under Macintosh or as a BMP file under Windows Use the corresponding Toolbar button or the File Print to print the graphic map Under MacOSX it is then easy to save it as a pdf file Selecting and extracting a fragment In addition to display save and print graphic maps this window can be use to select and extract sub fragments delimited by two restriction sites This method will be similarly used in the Build a construct window to proceed to sub cloning and assembly of new constructs 17 S Sel 2878 bp Graphic Map of pBluescript II KS xdna eot Sel 860 bp Graphic Map of Tetracyclin Gene pBluescript II KS xdna 2961 nt Tetracyclin Gene 1191 nt Show Unique Sites Double Site Particular sites Choose site Show Unique Sites C Double Site Particular sites Choose site T zi Extract Fragment a Circularize Linearize E Save Map Save Map 695 Xmal spi S elect All CW CCW Selection 167 T lt 0505 Sens Antisense Selection Cancel Selection Cancel Selection x i 23 lt spHi 600 7 Ple 31S 96
21. mited to 255 nucleotides However Serial Cloner comment field accepts longer text to allow you to organize and modify it more easily The number of character in the field 1s indicated underneath If there are more than 255 character the comment text will be truncated to 255 when 13 Use this tab to access to the extremities of a linear insert Extremities can saving be directly modified here Serial Cloner Format Jength Locked lo Sequence Comments Extremities 5 3 ggcc letad H 1 Sequence Comments Extremities Cohesive 5 Cohesive 3 ggcc ctaq 14 Use this slider to scroll the sequence 15 The main sequence field The sequence can be directly entered and modified here A contextual menu can be displayed When a selection 1s made this menu brings some of the most used functions 11 Undo iCCCACTACGTGAACCATCACC T c Undo Select All Gt Cop E Select All i d Fast et Copy i a Copy as Fasta Cut G4 Cut Paste ice E Paste t GC Extract Fragment Extract Fragment E Copy Translation fo Copy Translation UPPERCASE UPPERCASE s lowercase lowercase Antiparallel Antiparallel Speak Speak 16 Save or Save As the sequence When the sequence has been opened from the disk the button is not enabled until the sequence is modified If pressing on the Save button the modified version will replace the previous version Press Alt before clicking on this button to Save as and
22. n be used to extract the fragment decide whether sense or antisense selection has to be done or to cancel the selection Arranging the map 15 O Graphic Map of pBluescript Il KS xdna y eo Graphic Map of pBluescript Il KS pBluescript II KS xdna 2961 nt pBluescript II KS 2961 nt Show Unique Sites Double Site Particular sites Choose site Show W Unique Sites Double Site Particularsites Choose site L zu 870 Eagl 689 BamHi 695 Smal 701 Psti ly 713 EcoRV i ns Tati 2524 O 725 Cla N E 734 Hin 734 Acc 734 Sal pe eve leJ im s ce o S ie J v w I1 Io po t us m 9 i m x OSE s wis o 3 9 la ml fe i a wu i 739 Pspxi pBluescript Il KS 2961 nt Z i E i Y E gt 1030 Sapl h3 The map can be save as a bitmap image using the File Save Graphic Map as or using the contextual menu It can also be printed using the File Print C P menu The name and size of the sequence is shown at the top Checkbox underneath can be used to display unique sites double sites or any particular set of sites These option are non exclusive Each category unique double or particular will be displayed in a different color that can be change using the Preference window The set of sites to be displayed when the Particular 7 Particular sites Choose site Site checkbox is selected is chosen by pressing on unsele
23. nces Ctrl Align two sequences 67 e Build a construct C1 This command will open the Build window This window allows the rapid construction of new vector or chimeric sequences based on graphical selection and ligation of fragments Create an adaptor C 2 This command will open the Create an adaptor window This window allows the rapid definition of the oligonucleotides necessary to construct an adaptor The adaptor 1s constructed by entering the core double stranded sequence and the optional cohesive extensions e RunaPCR C c3 This command will open the Run a PCR window This window allows the virtual amplification of a fragment from a defined matrix sequence and a couple of primer These primers may contain un annealed tails e Make shRNA C4 This command will open the Make shRNA window This window allows the rapid definition of the oligonucleotides necessary to construct a shRNA insert This shRNA insert can then be cloned in a specialized vector using the Build a construct window Multiple layout of shRNA can be entered and stored Based on a pre defined layout a shRNA is then constructed using the chosen siRNA core sequence e Gateway cloning C45 This command will open the Gateway cloning window This window automate the two basic Gateway reactions the LR and the BP reactions The att sites are looked at in the selected vector and the sub cloning reaction executed between do
24. ng any Web browser then a more complex parsing 1s executed where some comment information are extracted from the page and pasted in the comment field while the sequenceis directly extracted and pasted in the sequence field see examples e Find C F Available in Sequence windows this command opens the find window e Find ORF CR Available in Sequence windows this commands find the next Open Reading Frame 1 e a piece of sequence starting with an ATG and ending with an in frame STOP codons TAA TAG or TGA C The Sequence Menu Show Info Ctri I Show Info del Copy Translation Ctri T Copy Translation 38T UPPERCASE Ctri U UPPERCASE U lowercase Ctri L lowercase L Antiparallel Ctri Antiparallel 86 Reverse Reverse Complement Complement Linearize Linearize Set Origin Set Origin Read Selection Read Selection e Show Info CI Open an Information Window associated to the up most Sequence Window This Info Window provides information bout the of A C G T the Tm or the translation and translated peptide MW of the sub sequence selected in the associated Sequence window e Copy Translation C T Translates into protein the sub sequence selected in the up most Sequence window and copy it in the clipboard in FASTA format e UPPERCASE C U Put in uppercase the sub sequence selected in the up most Sequence window e Lowercase C L Put in lowercase the sub sequence selected in the up most Sequence window
25. nor and acceptor vectors Extract Fragment C 6 This command is available with the Sequence window when a sequence portion has been selected and with the Graphic Map window when a fragment has been selected between one circular sequence or two circular or linear sequence sites e Align two Sequences C 7 This command will open the Align two Sequences window This window allow to select to sequences from currently opened sequences submit them to the BLAST2Sequences server at the NCBI and display the obtained alignment The 3 frame translation of the aligned sequence can be additionally displayed F The Window Menu Bring All To front a B Show Toolbar Hide Help Window 26 Show Toolbar Hide Help Window Ctrl Standard Genetic Code DNA Alphabet Standard Genetic Code About Serial Cloner DNA Al D habet Register pBluescript Il KS pBluescript II KS xdna e Bring All To Front CB Only available under MacOSX this command will bring all Serial Cloner windows to front Sometimes necessary due to the MacOS X specific windows management system e Show Hide Toolbar C This command allows to show or hide the toolbar window e Show Hide Help Window C This command allows to show or hide the contextual help window The description function of most Serial Cloner windows elements is displayed in the help window when the element is pointed by the mouse arrow e Standard Genetic Code This c
26. ommand opens the Standard Genetic Code window DNA Alphabet This command opens the DNA Alphabet window e About Serial Cloner accessible in the Apple window under MacOSX This command opens the About window This window displays information about the current Serial Cloner version acknowledgment an registration information e Register accessible in the SerialCloner menu under MacOSX This command opens the Register window allowing to enter a serial number and have the pleasure to be using a registered Serial Cloner software e Opened windows names All opened windows sequence windows Generate Menu windows or Preference windows will be listed at the end of this menu Selecting one of them will bring the corresponding window to the front IV A The Sequence Window Serial Cloner Windows Main windows 3 CS pBluescript II KS xdna ws eno pBluescript Il KS File Name F sName Name _ eee 120 pBluescript II KS xdna 4 pBluescript II KS 1 cai 8 Total length Type Topology Total length Type Topology NEL 23289 2961 2 DNA Circular 2961 2 DNA 3 Circular BE 79 9 DNA Strider format 4 length DNA Strider format 4 i v Locked 6 7 S61 M Locked 6 U L bal aaa 10 12 43 Sequence Comments Extremities Comments Extremities ia T BM EXNSILINZTUUTYDLUEUUTUNSTTUDTN Baa 1 GTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGITTATTTT yy 49 TCTAAATACATTCAAAT AT GT
27. ort Close Ctrl im po rt hs ecce Close a6 W Close All Close All Save Ctrl 5 Save As Save 26S Export to Fasta Save As Preferences Chri Export to Fasta Quit Ctrl Q e New C N Displays a new blank Sequence Window Open C O Browses available storage devices and opens a Sequence file into a Sequence Window Accepted format file are Strider extension xdna pDRAW32 extension pdw fasta extension fasta or fsta or raw text extension txt text asc or ascii Serial Cloner cannot work with rtf formatted files Note that fasta or pDRAW formatted files can be opened using the raw text format Only ACGT letters are accepted If other characters are filtered out a message will be displayed reporting the total number of excluded characters e Open Recent A hierarchical menu displays recently opened or saved files for easy opening of current projects The hierarchical menu proposes an option to erase the recent list The number of recent files memorized is adjustable in the preference window Max 25 e Revert To Save Cancels any modification done to a sequenced saved on disk A message will be shown to confirm the reversion e New Degenerate DNA Displays a new blank Sequence Window allowing the entry of degenerate DNA Degenerate DNA can then be used in any window and function Note however that restriction maps site usage alignment etc should be taken with care as ay no ACGT nucleot
28. t Serial Cloner Quit and Preferences windows are located in the SerialCloner menu under MacOSX and are found respectively in the Windows about and File Pref and Quit menu under Windows The Find and Info windows are shown as drawer windows under MacOS X and as global floating windows under Windows Hence while one such a window can be attached to each Sequence window under MacOSX only one can be shown under Windows However the behavior of these windows mimics MacOS X s drawer windows as they follow the movement of the sequence Window they are linked to Pressing the info or find button in a Sequence Window link them to that Sequence Window The Bring All to Front in the Window menu is only available under MacOSX as this deals with MacOSX windows management As usually the shortcut are available using the Command apple key under MacOSX and through the CTRL key under Windows Both are noted C underneath You will frequently find contextual menu As usually they are accessible under Windows using a Right click while under MacOSX using single button mouse contextual menus are displayed using CTRL associated to Left click IIl Serial Cloner Menus A he File Menu File Eg New Ctrl h New N Open Ctrl o Open 50 Open Recent Open Recent Revert to Saved Revert to Saved New Degenerate DNA New Degenerate DNA Imp
29. you want to search for a sub sequence string 2 Click on this tab if you want to search for a restriction site 3 Click on this tab if you want to search for an Open Reading frame ORF 4 Enter here the nucleotide string you want to look for The search is case insensitive Search will be done on both strand 5 Click here to start searching 6 All the occurrences of the sequence you look for will be displayed here Click on an occurrence to localize on the sequence window The strand where the sequence has been found is indicated or strand The occurrence can be sorted according to their position to their size or to the strand where they where found by clicking on the corresponding header of the column 7 Close the drawer MacOSX or the window Windows Find L Find Find EJ Find Find i Find i Find Find Find Sequence Site ORFs Sequence Site ORFs Sequence Site ORFs Sequence Site ORFs Sequence MINIM ORFs i Sequence Site ORFs reATCGTTGGGAACCOG 99 C eea O BseM Gey Find ORFs CQ ctcag 10 8 e prii ctcag 10 8 J 7 Position Size Strand Position Size Strand Position Size Strand Position Size Strand Position poeton Sizo Strand 600 25 415 5 19 63 son 955 5 66 24 i x 1121 5 78 12 1121 5 78 12 1530 5 99 12 aara E s t 129 300 T 3t gS 226 102 226 102 324 666 E 324 666 3237 6693 HELL LEE 350
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