primer design - please help

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monkey07
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primer design - please help

Post by monkey07 » Tue May 05, 2009 4:15 pm

Ive been given the sequence for a Human Sox4 protein..need to design primers to amplify DNA sequences encoding the HMG box only

HMG box : IK RPMNAFMVWS QIERRKIMEQ SPDMHNAEIS KRLGKRWKLL KDSDKIPFIR EAERLRLKHM ADYPDYDYRP


Does anyone know how to do this??

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MrMistery
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Post by MrMistery » Tue May 05, 2009 11:59 pm

you could blast it and find out what the dna sequence for it is, assuming you want to amplify it directly from a person.
In any way, you do need the dna sequence
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monkey07
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Re: primer design - please help

Post by monkey07 » Wed May 06, 2009 4:18 pm

Ive got the full amino acid sequence for Sox4:
MVQQTNNAENTEALLAGESSDSGAGLELGIASSPTPGSTASTGG
KADDPSWCKTPSGHIKRPMNAFMVWSQIERRKIMEQSPDMHNAEISKRLGKRWKLLKD
SDKIPFIREAERLRLKHMADYPDYKYRP
RKKVKSGNANSSSSAAASSKPGEKGDKVGG
SGGGGHGGGGGGGSSNAGGGGGGASGGGANSKPAQKKSCGSKVAGGAGGGVSKPHAKL
ILAGGGGGGKAAAAAAASFAAEQAGAAALLPLGAAADHHSLYKARTPSASASASSAAS
ASAALAAPGKHLAEKKVKRVYLFGGLGTSSSPVGGVGAGADPSDPLGLYEEEGAGCSP
DAPSLSGRSSAASSPAAGRSPADHRGYASLRAASPAPSSAPSHASSSASSHSSSSSSS
GSSSSDDEFEDDLLDLNPSSNFESMSLGSFSSSSALDRDLDFNFEPGSGSHFEFPDYC
TPEVSEMISGDWLESSISNLVFTY

I have highlighted the start codon for translated bases in red (the methionine which is the start codon) and the HMG box that I need to design primers for is highlighted in blue.

The mRNA sequence:

1 ttccccagca ttcgagaaac tcctctctac tttagcacgg tctccagact cagccgagag
61 acagcaaact gcagcgcggt gagagagcga gagagaggga gagagagact ctccagcctg
121 ggaactataa ctcctctgcg agaggcggag aactccttcc ccaaatcttt tggggacttt
181 tctctcttta cccacctccg cccctgcgag gagttgaggg gccagttcgg ccgccgcgcg
241 cgtcttcccg ttcggcgtgt gcttggcccg gggaaccggg agggcccggc gatcgcgcgg
301 cggccgccgc gagggtgtga gcgcgcgtgg gcgcccgccg agccgaggcc atggtgcagc
361 aaaccaacaa tgccgagaac acggaagcgc tgctggccgg cgagagctcg gactcgggcg
421 ccggcctcga gctgggaatc gcctcctccc ccacgcccgg ctccaccgcc tccacgggcg
481 gcaaggccga cgacccgagc tggtgcaaga ccccgagtgg gcacatcaag cgacccatga
541 acgccttcat ggtgtggtcg cagatcgagc ggcgcaagat catggagcag tcgcccgaca
601 tgcacaacgc cgagatctcc aagcggctgg gcaaacgctg gaagctgctc aaagacagcg
661 acaagatccc tttcattcga gaggcggagc ggctgcgcct caagcacatg gctgactacc
721 ccgactacaa gtaccggccc aggaagaagg tgaagtccgg caacgccaac tccagctcct
781 cggccgccgc ctcctccaag ccgggggaga agggagacaa ggtcggtggc agtggcgggg
841 gcggccatgg gggcggcggc ggcggcggga gcagcaacgc ggggggagga ggcggcggtg
901 cgagtggcgg cggcgccaac tccaaaccgg cgcagaaaaa gagctgcggc tccaaagtgg
961 cgggcggcgc gggcggtggg gttagcaaac cgcacgccaa gctcatcctg gcaggcggcg
1021 gcggcggcgg gaaagcagcg gctgccgccg ccgcctcctt cgccgccgaa caggcggggg
1081 ccgccgccct gctgcccctg ggcgccgccg ccgaccacca ctcgctgtac aaggcgcgga
1141 ctcccagcgc ctcggcctcc gcctcctcgg cagcctcggc ctccgcagcg ctcgcggccc
1201 cgggcaagca cctggcggag aagaaggtga agcgcgtcta cctgttcggc ggcctgggca
1261 cgtcgtcgtc gcccgtgggc ggcgtgggcg cgggagccga ccccagcgac cccctgggcc
1321 tgtacgagga ggagggcgcg ggctgctcgc ccgacgcgcc cagcctgagc ggccgcagca
1381 gcgccgcctc gtcccccgcc gccggccgct cgcccgccga ccaccgcggc tacgccagcc
1441 tgcgcgccgc ctcgcccgcc ccgtccagcg cgccctcgca cgcgtcctcc tcggcctcgt
1501 cccactcctc ctcttcctcc tcctcgggct cctcgtcctc cgacgacgag ttcgaagacg
1561 acctgctcga cctgaacccc agctcaaact ttgagagcat gtccctgggc agcttcagtt
1621 cgtcgtcggc gctcgaccgg gacctggatt ttaacttcga gcccggctcc ggctcgcact
1681 tcgagttccc ggactactgc acgcccgagg tgagcgagat gatctcggga gactggctcg
1741 agtccagcat ctccaacctg gttttcacct actgaagggc gcgcaggcag ggagaagggc
1801 cggggggggt aggagaggag aaaaaaaaag tgaaaaaaag aaacgaaaag gacagacgaa
1861 gagtttaaag agaaaaggga aaaaagaaag aaaaagtaag cagggctcgt tcgcccgcgt
1921 tctcgtcgtc ggatcaagga gcgcggcggc gttttggacc cgcgctccca tcccccacct
1981 tcccgggccg gggacccact ctgcccagcc ggagggacgc ggaggaggaa gagggtagac
2041 aggggcgacc tgtgattgtt gttattgatg ttgttgttga tggcaaaaaa aaaaagcgac
2101 ttcgagtttg ctcccctttg cttgaagaga ccccctcccc cttccaacga gcttccggac
2161 ttgtctgcac ccccagcaag aaggcgagtt agttttctag agacttgaag gagtctcccc
2221 cttcctgcat caccaccttg gttttgtttt attttgcttc ttggtcaaga aaggagggga
2281 gaacccagcg cacccctccc cccctttttt taaacgcgtg atgaagacag aaggctccgg
2341 ggtgacgaat ttggccgatg gcagatgttt tgggggaacg ccgggactga gagactccac
2401 gcaggcgaat tcccgtttgg ggcctttttt tcctccctct tttccccttg ccccctctgc
2461 agccggagga ggagatgttg aggggaggag gccagccagt gtgaccggcg ctaggaaatg
2521 acccgagaac cccgttggaa gcgcagcagc gggagctagg ggcgggggcg gaggaggaca
2581 cgaactggaa gggggttcac ggtcaaactg aaatggattt gcacgttggg gagctggcgg
2641 cggcggctgc tgggcctccg ccttcttttc tacgtgaaat cagtgaggtg agacttccca
2701 gaccccggag gcgtggagga gaggagactg tttgatgtgg tacaggggca gtcagtggag
2761 ggcgagtggt ttcggaaaaa aaaaaagaaa aaaaggg

So do I now find the bit of DNA sequence where the HMG box starts, by counting the amino acids and bases (3 bases for each aa?)

Any further help will be greatly appreciated!!!! Thanks so much

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jonmoulton
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Re: primer design - please help

Post by jonmoulton » Wed May 06, 2009 11:34 pm

Exactly right - count the codons. Then translate a section where you think your domain of interest starts to see whether you have correctly identified the place on the RNA that encodes those amino acids.

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