Design+SMC1fs+primers+to+determine+orientation+and+sequence+6-14-13

I need to design primers so that I can do a PCR that amplifies from the vector to the frameshift region of the SMC1fs sequence so that I can determine the orientation of the sequence in the plasmid as well as sequence the fragment to determine whether or not it is in frame.

see also SMC1fs

Shen sent me some sequences of the wild type SMC1fs sequence and the frameshift SMC1fs sequence for human and mouse. These sequences are found here "F:\kurt\storage\CIM Research Folder\DR\2013\6-14-13\SMC1fs\raw sequences"

I aligned these two sequences and saved the blast output page here "F:\kurt\storage\CIM Research Folder\DR\2013\6-14-13\SMC1fs\wt and smc1fs sequence aligned 6-14-13\wt and fs smc1a sequence aligned 6-14-13.htm"

The wild type smc1a mRNA can be found here http://www.ncbi.nlm.nih.gov/nucleotide/258613891?report=genbank&log$=nucltop&blast_rank=1&RID=VR3DTB3301R

From my analysis the fs sequence has all of exon1, two base pairs of exon 2, and then the sequence cuts into exon 4 with the first two base pairs of exon 4 missing. Here are my annotated ape files

"F:\kurt\storage\CIM Research Folder\DR\2013\6-14-13\SMC1fs\SMC1A 6-14-13.ape" "F:\kurt\storage\CIM Research Folder\DR\2013\6-14-13\SMC1fs\SMC1Afs 6-14-13.ape"

However, this is not how Shen remembers that it should be so he said that he would send me an annotated file.

-- 6-16-13

Shen sent me an annotated file. here

"F:\kurt\storage\CIM Research Folder\DR\2013\6-16-13\SMC1fs\Human and mouse SMC1A FS rna.doc"

email here https://mail.google.com/mail/u/0/?ui=2&shva=1#sent/13f49833251822d5

He said that the frameshift sequence is simply exon1 spliced together with exon4. see Mouse SMC1a sequence information

Now that I know the sequence, I can design primers that will amplify from the vector to the sequence. Note that the mRNA could insert into the vector in either orientation. cDNA library was constructed with pSMART2IF vector

primer designed can be found here smc_fus primer