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in silico assembly of pYPKa_A_ScATF1

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The pYPKa_A_ATF1 is a cloning vector which has the S. cerevisiae alcohol acetyltransferase gene ATF1 / YOR377W. The purpose of this plasmid is to serve as template for a subsequent PCR reaction. The plasmid is made by cloning a PCR product containing the ATF1 gene into the AjiI restriction site.

The AjiI restriction site is blunt, so we do not need to digest the PCR product as it has blunt ends already.

Do the following in order to assemble the sequence for the pYPKa_A_ATF1 plasmid in-silico:

1. PCR simulation

  1. Copy the sequence the sequence of the ATF1 gene (CP046095.1 ) from Genbank. This is the template for the PCR simulation. Be careful to copy the complete Genbank sequence.
  2. The two primer sequences below (1795 & 1732), used to amplify the ATF1 gene from chromosomal DNA.
>1795_ATF1f CP072089 1046219 1047796
gcaataATGGGTAATGAAATCGATGAGAAAAATCA
	
>1732_ATF1r CP072089 1046219 1047796
TTAAGGGCCTAAAAGGA

Assemble the PCR product manually from the primer sequences and the gene sequence.

The PCR product should be 1587 bp long and have a short seguid: ldseguid=cVE4g6, check this by using the fingerprint button in ApE.

2. in-silico cloning

Get the pYPKa sequence file and open it in ApE. You can do this by saving the file to your computer and
dragging the sequence file to the empty ApE window. See the result below.
The pYPKa sequence should have 3128 bp and seguid cdseguid=IFLMpKpCGZio0R0YkGSfqPaKKiw.

Find the AjiI or BtrI or BmgBI restriction site (They have the same specificity) . Use the Enzymes>Enzyme selector option and try to find the AjiI restriction site.

You can also use the Edit>Find or CTRL-F search to find the recognition sequence of the enzyme (CACGTC).

If AjiI is not available in the enzyme selection of ApE, try to find BtrI instead.

Paste the PCR product sequence at the cut site of the pYPKa. See figure below.

IMPORTANT

Paste only the DNA sequence of the PCR product, do not include the FASTA header.

3. Analyze result

Calculate the size and complete seguid checksum of the resulting plasmid.

The expected size is 4715 bp and the short seguid checksum is cdseguid=EsiXAn.

Compare your result with that of your colleagues. Save the new sequence file on your computer under the name pYPKa_A_ScATF1.gb.

You can now continue to the assembly of the AFT1 TU-vector pTA1_TDH3_ScATF1_PGI1.

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