Ok. First I pasted the sequence above into http://www.ncbi.nlm.nih.gov/orffinder/ and found the longest open reading frame.
Then I googled "C.perfringens alpha toxin" , and from the wikipedia page for it followed the entrez link to here: https://www.ncbi.nlm...ids=988262&rn=1, and from there I clicked on the FASTA link to get the nucleotide sequence here: https://www.ncbi.nlm...=48590&to=49786
Then I compared both the DNA nucleotide sequences of these two pieces of information and the translated protein sequences (http://web.expasy.or...ranslate/dna_aa or via the orffinder tool linked above). They are not identical, but are very close (you can do this with BLAST and get exact alignments here: https://blast.ncbi.n...h.gov/Blast.cgi - choose the align two sequences option). So I assume that the longest open reading frame in your sequence of interest it what you want to amplify.
That is this (in bold and underlined):
AAGCTTGATATGGACTTTTTAGCAATATTAACAGCCTCATCTAGGTTAGGATTTAGAAGAGGAAGTAGTG CTTCTGAGAATCTAGCTAAGTTCCATAAGGCCATATTAGGTTGATTTCCATAGGCATATCTACCAGCATA ATCAATGGAACTAAATACTGTGTTTGTATCATAAGTATCCATAAATGCACATGGTCCATAATCTATAGTT TCTCCTGAGATTACCATATTATCAGTGTTCATAACCCCATGAATGAATCCAACACTTTGCCACTTAACTA TAAGTTCAGCTTGACGATTTATTACCTCTTCAAGAAATAAAATATATTTATTTTCACTATTAGCTATATT AGGAAAGTGTCTTTTAATAGTGTAGTCAGCAAGACTTTTAAGATCCTCTAAAGTACCCCATTGAGCTGCA TAAGCAAAAGTTCCAACTCTAATGTGACTAGAAGCTATTCTTGTTAATATAGCACCTTGTTCAAATTTTT CTCTTAAAACTTCCTCACCAGTACTTACTACAGCAAGGCTTCTAGTTGTAGGAATTCCAAGACTATGCAT ACCTTCACTTATTATATATTCTCTAAGCATAGGTGCAAGGGCAGCCTTTCCATCTCCACCTCTTGAATAT ATTGTTCTACCAGACCCTTTTAATTGAACATCATATCTTTTACCATCTTTAGTTACATGTTCTCCTAATA AGAGTGCTCTACCATCTCCTAGCATTGTAAAATGGCCAAATTGGTGACCGGCATAAGCCTGTGCAATTGG GGTAATACCTGGGAAAGTTTCATTTCCTGCAAATATATTAAGTCCAAAATCGCTGTTTAAAATTTCTTCA TTAAGCCCAAGTTCTTTAGCAAGAGAAGTATTAAACTTAATAAGTTTAGGATTTTTTGAACCCTTTGGAT TTTGTTCACTAAAGAATATATTTGGAAGAGTTAAATAAGTATTTTCTAGGTTAAAACCTGTTTTTGATTG AAAATTTTTATTATCCATATTAAAATCCTTTGCCTTATAATTTATTTCAAATTTTATTCCATCCCTTATA TTATGAGTAAAAATTCTTATTAAATTAAAAAACAATATTTAACTTATTATAACACTAATAATTGTAAATT TTCATATTAAAAATAAGTTTAATAATTTAGAGTGGGTGAGTTTAGATATTTTAAATTAAAATTTGAATTT TATTAAAAAATATTTAAAAAATATTCAAAAGTTTAGTGAGGTTATGTTAATTATATGGTATAATTTCAAT GCGAGTGTTAATCGTTATCAAAAAGGGGAGATTAATACTTGAAAAAAATTAACGGGGGATATAAAAATGA AAAGAAAGATTTGTAAGGCGCTTGTTTGTGCCACGCTAGTAACTAGCCTATGGGCTGGGGTATCAACTAA AGTCTACGCTTGGGATGGAAAAATTGATGGAACAGGAACTCATGCTATGATTGTAACTCAAGGTGTTTCA ATCTTAGAAAATGATATGTCCAAAAATGAACCAGAAAGTGTAAGAAAAAACTTAGAGATTTTAAAAGATA ACATGCATGAGCTTCAATTAGGTTCTACTTATCCAGATTATGATAAGAATGCATATGATCTATATCAAGA TCATTTCTGGGATCCTGATACAAATAATAATTTCTCAAAGGATAATAGTTGGTATTTAGCTTATTCTATA CCTGACACAGGGGAATCACAAATAAGAAAATTTTCAGCATTAGCTAGATATGAATGGCAAAGAGGAAATT ATAAACAAGCTACATTCTATCTTGGAGAAGCTATGCACTATTTTGGAGATATAGATACTCCATATCATCC TGCTAATGTTACTGCCGTTGATAGCGCAGGACATGTTAAGTTTGAGACTTTTGCAGAAGAAAGGAAAGAA CAGTATAAAATAAACACAGTAGGTTGCAAAACTAATGAGGATTTTTATGCTGATATCTTAAAAAACAAAG ATTTTAATGCATGGTCAAAAGAATATGCAAGAGGTTTTGCTAAAACAGGGAAATCAATATACTATAGTCA TGCTAGCATGAGTCATAGTTGGGATGATTGGGATTATGCAGCAAAGGTAACTTTAGCTAACTCTCAAAAA GGAACAGCAGGATATATTTATAGATTCTTACACGATGTATCAGAGGGTAATGATCCATCAGTTGGAAATA ATGTAAAAGAACTAGTAGCTTACATATCAACTAGTGGTGAAAAAGATGCTGGAACAGATGACTACATGTA TTTTGGAATCAAAACAAAGGATGGAAAAACTCAAGAATGGGAAATGGACAACCCAGGAAATGATTTTATG GCTGGAAGCAAAGACACTTATACTTTCAAATTAAAAGATGAAAATCTAAAAATTGATGATATACAAAATA TGTGGATTAGAAAAAGAAAATATACAGCATTCCCAGATGCTTATAAGCCAGAAAACATAAAGGTAATAGC AAATGGAAAAGTTGTAGTTGACAAGGATATAAATGAGTGGATTTCAGGAAATTCAACTTATAATATAAAA TAATAAAAGTAAAAAAATAATTATTGGTTTTGGTGGTATTTACAAAATAAAAGCTT
If you need an N-term his tag you want to make a primer with a start (ATG) plus a 6xHis (e.g. CACCACCACCACCACCAT) plus some of the sequence that comes immediately after the very first ATG codon in the above bolded sequence (e.g. part of this: AAAAGAAAGATTTGTAAGGCGCTTGTTTGTGCCACG). You may also need to add some extra bases prior to the ATG and the his tag for cloning into your pET vector. This may be some dummy bases and a restriction site if you're doing ligation-based cloning (google for NEB's traditional cloning guide). It may be sequence homologous to the cloning site of pET28+ for Gibson or some other chewback assembly technique. Read the manual of those kits on how to make this sequence. This extra sequence is indicated with X's below.
You'll end up with an N-term primer that looks something like this:
For the part in bold and underlined you'll need to calculate an annealing temperature for your first PCR cycles. You can add or subtract gene-specific homologous bases from it as necessary to get an appropriate annealing temperature. Go to NEB, Thermo, or wherever for a Tm calculator appropriate to your polymerase.
For the 3' end of the gene you do the same, but you only need the pET28+ cloning sequence and the C-term sequence of the gene. Tack them together like this: GGATATAAATGAGTGGATTTCAGGAAATTCAACTTATAATATAAAATAAXXXXXXXXXXXXXXX and then cut and paste into a program to generate the reverse complement sequence (google to find). This reverse complement sequence will be what you order as an oligo, after you have modified the gene specific sequence to get an appropriate annealing temperature as above.
If you need a C-term his tag make your primers like this:
N-term primer: XXXXXXXXXXXXXXXXATGAAAAGAAAGATTTGTAAGGCGCTTG (notice that the ATG start codon is now part of the bold and underlined gene specific sequence, and the his tag is gone).
C-term primer: Reverse complement of: AGGAAATTCAACTTATAATATAAAACACCACCACCACCACCATTAAXXXXXXXXXXXXXXX (notice the the TAA stop codon is no longer part of the bold and underlined gene specific sequence, and there is a his tag between it and the rest of the ORF).
Do the same annealing temperature calculations on the gene specific portions of these primers.
Pro-tip: You can often increase the annealing temperature to be the annealing temperature (or 72C, whichever is less) of the total oligo (including the sequences which are not homologous to the gene you are PCRing) and get just as good, if not better, amplification.
I would try to clone your gene close to the RBS of the pET vector so that it will express well. Or you can incorporate your own RBS into the N-term primer.
Note: I just noticed that pET28+ already has his tags in it. You can clone directly into these his tag with the above sort of primer design procedures, but not including a his tag in the primer. Beware of trying to clone using ligation into the NdeI site as your gene has an internal NdeI site. This will not work. If you clone it this way make sure your gene is missing the initial start codon (ATG) and is cloned in-frame to the start codon of the pET vector. Your gene will be expressed as a fusion protein to the peptide that is already in the pET vector (which contains the his tags and contains a start ATG in the NcoI site).
Edited by 827753, 05 September 2016 - 12:07 AM.