Click and drag the icon to move the insert box to its correct position. Hover your mouse above the insert box you wish to move and two new icons will appear. You can also rearrange or delete your insert boxes.The insert box will remain, but the sequence will be deleted. Click on the trash can icon that appears. If you need to remove an incorrect insert sequence, hover your mouse over the right side of the insert box.Repeat as necessary for each additional insert you want to clone into your vector. Enter your insert sequence as described in Step 1.For multiple-insert cloning projects, click "+ Add another insert".NOTE: If you are cloning just one insert into your vector, skip to Step 4. You can also drag and drop vector files into the text box. Clicking on the up arrow icon ( ) to upload a vector file (.fasta or.Clicking on 'Input insert sequence' and pasting your nucleotide sequence into the text box.Add your first insert sequence to the "Add Inserts" box by either:.The map will display the vector in linear format. To select a prelinearized vector, click the 'Already Linearized' option.You can also select the insert site by typing in the appropriate nucleotide numbers in the boxes below the map. Click on the vector to indicate the point at which the insert should be placed, or click and drag on the vector to indicate the region which should be replaced by the insert.NOTE: If you want to generate primers that preserve the restriction site(s) in your final construct, click on the 'Include restriction site in final product' checkbox. The selected enzyme names will populate at the bottom of the window with their cut sites indicated. Choose your restriction site(s) in the vector map by clicking on each desired enzyme.NOTE: To see enzymes with different numbers of cut sites, or to choose enzymes by name, click on the down arrow icon ( ) in the "Search cut sites" text box. Available restriction enzyme sites will be displayed on the vector map to the right. Click on the 'Restriction Digest' option.Use the "Linearize by" box to select one of three options for linearizing your destination vector: Restriction Digest, PCR, Already Linearized.
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