Ron Lab


 

Mouse PERK, K618A, 9E10-tagged at C-terminus, mammalian expression vector pCDNAamp

The K->A mutation introduces a unique Nru1 site at position 5070. An EcoR1-Nru1 digest thus readily distinguishes the wild-type and mutant plasmids

Note on growth of PERK plasmids: We have found that the PERK cDNA confers instability (low replication ability) to plasmids. The plasmid will be provided in the STBL2 bacterial strain (gibco) which helps alleviate this problem. To recover the plasmid, stick a sterile toothpick into the stab and streak on an LB amp plate and grow at 30C. Pick an individual colony and grow in LB amp at 30C for the highest yield of plasmid.

PERK.K618A.9E10.pCDNAamp (8345nt, predicted). Insert between EcoR1 and Xho1:

GGCGTAATCT GCTGCTTGCA AACAAAAAAA CCACCGCTAC CAGCGGTGGT TTGTTTGCCG GATCAAGAGC TACCAACTCT TTTTCCGAAG GTAACTGGCT TCAGCAGAGC GCAGATACCA AATACTGTCC TTCTAGTGTA GCCGTAGTTA GGCCACCACT TCAAGAACTC TGTAGCACCG CCTACATACC TCGCTCTGCT AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG TGTCTTACCG GGTTGGACTC AAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA ACGGGGGGTT CGTGCACACA GCCCAGCTTG GAGCGAACGA CCTACACCGA ACTGAGATAC CTACAGCGTG AGCATTGAGA AAGCGCCACG CTTCCCGAAG GGAGAAAGGC GGACAGGTAT CCGGTAAGCG GCAGGGTCGG AACAGGAGAG CGCACGAGGG AGCTTCCAGG GGGAAACGCC TGGTATCTTT ATAGTCCTGT CGGGTTTCGC CACCTCTGAC TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG GGGGGCGGAG CCTATGGAAA AACGCCAGCA ACGCAAGCTA GCTTCTAGCT AGAAATTGTA AACGTTAATA TTTTGTTAAA ATTCGCGTTA AATTTTTGTT AAATCAGCTC ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT AAATCAAAAG AATAGCCCGA GATAGGGTTG AGTGTTGTTC CAGTTTGGAA CAAGAGTCCA CTATTAAAGA ACGTGGACTC CAACGTCAAA GGGCGAAAAA CCGTCTATCA GGGCGATGGC CGCCCACTAC GTGAACCATC ACCCAAATCA AGTTTTTTGG GGTCGAGGTG CCGTAAAGCA CTAAATCGGA ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT GACGGGGAAA GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA GCGGTCACGC TGCGCGTAAC CACCACACCC GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG TACTATGGTT GCTTTGACGA GACCGTATAA CGTGCTTTCC TCGTTGGAAT CAGAGCGGGA GCTAAACAGG AGGCCGATTA AAGGGATTTT AGACAGGAAC GGTACGCCAG CTGGATTACC AAAGGGCCTC GTGATACGCC TATTTTTATA GGTTAATGTC ATGATAATAA TGGTTTCTTA GACGTCAGGT GGCACTTTTC GGGGAAATGT GCGCGGAACC CCTATTTGTT TATTTTTCTA AATACATTCA AATATGTATC CGCTCATGAG ACAATAACCC TGATAAATGC TTCAATAATA TTGAAAAAGG AAGAGTATGA GTATTCAACA TTTCCGTGTC GCCCTTATTC CCTTTTTTGC GGCATTTTGC CTTCCTGTTT TTGCTCACCC AGAAACGCTG GTGAAAGTAA AAGATGCTGA AGATCAGTTG GGTGCACGAG TGGGTTACAT CGAACTGGAT CTCAACAGCG GTAAGATCCT TGAGAGTTTT CGCCCCGAAG AACGTTTTCC AATGATGAGC ACTTTTAAAG TTCTGCTATG TGGCGCGGTA TTATCCCGTG TTGACGCCGG GCAAGAGCAA CTCGGTCGCC GCATACACTA TTCTCAGAAT GACTTGGTTG AGTACTCACC AGTCACAGAA AAGCATCTTA CGGATGGCAT GACAGTAAGA GAATTATGCA GTGCTGCCAT AACCATGAGT GATAACACTG CGGCCAACTT ACTTCTGACA ACGATCGGAG GACCGAAGGA GCTAACCGCT TTTTTGCACA ACATGGGGGA TCATGTAACT CGCCTTGATC GTTGGGAACC GGAGCTGAAT GAAGCCATAC CAAACGACGA GCGTGACACC ACGATGCCTG CAGCAATGGC AACAACGTTG CGCAAACTAT TAACTGGCGA ACTACTTACT CTAGCTTCCC GGCAACAATT AATAGACTGG ATGGAGGCGG ATAAAGTTGC AGGACCACTT CTGCGCTCGG CCCTTCCGGC TGGCTGGTTT ATTGCTGATA AATCTGGAGC CGGTGAGCGT GGGTCTCGCG GTATCATTGC AGCACTGGGG CCAGATGGTA AGCCCTCCCG TATCGTAGTT ATCTACACGA CGGGGAGTCA GGCAACTATG GATGAACGAA ATAGACAGAT CGCTGAGATA GGTGCCTCAC TGATTAAGCA TTGGTAACTG TCAGACCAAG TTTACTCATA TATACTTTAG ATTGATTTAA AACTTCATTT TTAATTTCTC TAGCGCGTTG ACATTGATTA TTGACTAGTT ATTAATAGTA ATCAATTACG GGGTCATTAG TTCATAGCCC ATATATGGAG TTCCGCGTTA CATAACTTAC GGTAAATGGC CCGCCTGGCT GACCGCCCAA CGACCCCCGC CCATTGACGT CAATAATGAC GTATGTTCCC ATAGTAACGC CAATAGGGAC TTTCCATTGA CGTCAATGGG TGGACTATTT ACGGTAAACT GCCCACTTGG CAGTACATCA AGTGTATCAT ATGCCAAGTA CGCCCCCTAT TGACGTCAAT GACGGTAAAT GGCCCGCCTG GCATTATGCC CAGTACATGA CCTTATGGGA CTTTCCTACT TGGCAGTACA TCTACGTATT AGTCATCGCT ATTACCATGG TGATGCGGTT TTGGCAGTAC ATCAATGGGC GTGGATAGCG GTTTGACTCA CGGGGATTTC CAAGTCTCCA CCCCATTGAC GTCAATGGGA GTTTGTTTTG GCACCAAAAT CAACGGGACT TTCCAAAATG TCGTAACAAC TCCGCCCCAT TGACGCAAAT GGGCGGTAGG CGTGTACGGT GGGAGGTCTA TATAAGCAGA GCTCTCTGGC TAACTAGAGA ACCCACTGCT TACTGGCTTA TCGAAATTAA TACGACTCAC TATAGGGAGA CCCAAGCTTG GTACCGAGCT CGGATCCACT AGTAACGGCC GCCAGTGTGC TGGAATTCGG CACGAGCGAT GTCTGCACAA GGCTGTCACT CAGGTGGCAG TGGCTGACAC GTGGCCGGGC AGCTCTGCTG CTGCGGCGCG AAGTCGAGAG GCGGCGGGGT CCGTGGCGCG CGCTCGCATT GCTCCGAGGC TCCGAGCGGC GATACGGGCG GGCGCCGACG GCAGGGTCTC CATGCCCGCG CGTGGGGCGG GCCGCTGATG GAGCGCGCCA CCCGGCCCGG GCCGCGCGCG CTGCTGCTGC TTCTGTTCCT GCTGCTGGGC TGCGCGGCGG GGATCTCTGC GGTCGCGCCC GCCCGCAGTT TGCTTGCTCC CGCGTCGGAG ACAGTGTTTG GCTTAGGGGC AGCGGCCGCC CCGACTTCGG CCGCGCGGGT GCCTGCGGTG GCAACGGCCG AAGTGACCGT GGAGGACGCC GAGGCATTGC CGGCTGCCGC TGGCGAACCG GAGTCACGCG CGACGGAGCC CGATGACGAC GTGGAACTGC GGCCTCGCGG CAGGTCCTTG GTAATCATCA GCACTTTAGA TGGACGAATC GCTGCACTGG ATGCCGAGAA TGATGGGAAA AAGCAGTGGG ATTTGGACGT GGGGTCTGGT TCCTTGGTTT CATCTAGCCT CAGCAAGCCA GAGGTGTTTG GGAACAAGAT GATCATCCCC TCCCTGGATG GAGACCTCTT CCAGTGGGAC CGGGACCGAG AGAGCATGGA GGCCGTCCCC TTCACGGTGG AGTCCCTGCT CGAATCTTCC TACAAGTTTG GAGATGATGT TGTTCTGGTT GGAGGGAAAT CTCTGATTAC ATACGGACTC AGTGCTTACA GTGGAAAGCT GAGGTATATC TGTTCTGCCT TGGGATGTCG CCGATGGGAT AGTGATGAAA TGGAAGAAGA GGAAGACATC TTGCTTCTGC AGCGTACGCA GAAGACTGTG CGAGCTGTCG GGCCTCGAAG CGGCAGTGAG AAGTGGAATT TCAGTGTTGG CCACTTTGAA CTTCGGTATA TTCCAGACAT GGAAACTAGA GCCGGATTCA TTGAAAGCAC CTTTAAACCG GGTGGAAACA AAGAAGACTC TAAAATTATT TCAGATGTGG AAGAACAAGA AGCCACCATG CTGGACACAG TGATAAAAGT TTCCGTTGCT GATTGGAAGG TCATGGCGTT TAGTAGGAAG GGAGGCCGCC TGGAATGGGA GTACCAGTTT TGTACTCCCA TCGCGTCCGC CTGGCTGGTG AGGGATGGCA AGGTCATCCC CATCAGCCTG TTTGATGATA CAAGTTACAC AGCCAGCGAA GAAGCCTTGG GAGACGAAGA AGACATTGTA GAGGCTGCTC GGGGAGCCAC AGAGAACAGC GTGTACTTAG GGATGTACAG AGGCCAGCTG TACCTGCAGT CGTCCGTCAG GGTCTCAGAA AAGTTCCCTA CAAGCCCAAA GGCCTTGGAG TCTGTAAATG GCGAAAATGC AATTATTCCT CTGCCGACGA TCAAATGGAA GCCCTTAATC CATTCTCCTT CTAGGACTCC TGTCTTGGTT GGGTCTGATG AATTTGACAA ATGTCTAAGT AATGATAAGT ATTCCCACGA AGAATACAGT AATGGTGCAC TTTCAATCCT CCAGTATCCA TACGATAACG GTTACTATCT GCCATACTAC AAGAGAGAAA GGAATAAGCG GAGCACGCAG ATCACAGTCA GGTTCCTGGA CAGCCCCCAC TACAGCAAGA ACATCCGCAA GAAGGACCCT ATCCTCCTGC TGCACTGGTG GAAGGAGATA TTCGGGACGA TCCTGCTTTG CATCGTAGCC ACGACCTTCA TCGTGCGCAG GCTTTTCCAT CCTCAGCCCC ACAGGCAGCG GAAGGAGTCT GAAACTCAGT GCCAGACTGA AAGTAAATAC GACTCCGTGA GTGCCGATGT CAGTGACAAC AGCTGGAATG ACATGAAGTA CTCAGGATAC GTATCCCGAT ATCTAACAGA TTTTGAGCCA ATTCAGTGCA TGGGTCGTGG TGGCTTTGGC GTTGTCTTTG AAGCTAAAAA CAAAGTAGAT GACTGCAATT ACGCTATCGC GAGGATCCGG CTCCCCAACA GGGAGTTGGC ACGGGAGAAG GTAATGCGGG AAGTTAAAGC CTTGGCTAAG CTGGAACACC CAGGCATTGT GAGGTATTTC AACGCCTGGC TGGAAACCCC ACCAGAGAAG TGGCAAGAAG AGATGGATGA GATCTGGCTC AAAGACGAAA GCACAGACTG GCCGCTCAGC TCCCCTAGCC CGATGGATGC CCCATCTGTT AAGATCCGAA GGATGGATCC YTTCTCTACA AAAGAGCAGA TCGAAGTCAT AGCTCCTTCT CCTGAAAGAA GTCGGTCTTT CTCGGTGGGC ATTTCCTGTG GCCAGACAAG CTCATCGGAG AGCCAGTTCT CTCCCCTGGA GTTCTCAGGG ACAGACTGCG GAGACAACAG TGACTCAGCG GACGCAGCCT ACAACCTCCA GGACAGTTGC CTGACGGACT GCGAGGACGT GGAAGATGGC ACCGTGGACG GCAATGACGA GGGACACTCC TTTGAACTTT GTCCGTCCGA AGCTTCTCCC TATACCCGGT CTAGGGAAGG AACGTCCTCC TCCATAGTGT TTGAGGACTC TGGCTGCGGC AACGCGTCCA GTAAGGAGGA GCCCAGAGGG AACCGGCTGC ATGATGGCAA CCATTATGTT AATAAGCTAA CTGATCTCAA GTGCTCCAGC AGCAGGTCTT CTTCAGAAGC CACCACCTTG TCTACCTCCC CTACCAGGCC AACCACTCTA AGCTTGGATT TCACCAAGAA CACTGTGGGC CAGCTCCAGC CCAGCTCCCC CAAGGTGTAT CTGTACATTC AGATGCAGCT GTGCAGGAAG GAGAACCTCA AAGACTGGAT GAACCGGCGC TGCAGCTTGG AGGACCGGGA GCACGGCGTG TGCCTGCACA TCTTCCTGCA GATCGCAGAG GCAGTGGAGT TCCTGCACAG CAAGGGACTC ATGCACAGGG ACCTCAAGCC TTCCAACATA TTCTTCACAA TGGATGATGT GGTCAAGGTT GGGGACTTTG GACTGGTGAC TGCTATGGAC CAAGATGAAG AAGAGCAGAC TGTTCTGACT CCAATGCCAG CCTATGCTAC GCACACGGGA CAAGTAGGGA CCAAGCTATA CATGAGCCCA GAGCAGATTC ATGGAAACAA CTACTCCCAT AAAGTGGACA TCTTCTCTTT AGGCTTGATT CTGTTTGAAC TCCTCTACCC ATTCAGCACC CAGATGGAAC GAGTCCGGAT TTTAACTGAT GTCAGAAATC TCAAGTTTCC TCTACTGTTC ACTCAGAAAT ATCCCCAAGA GCATATGATG GTTCAAGACA TGCTCTCTCC ATCCCCCACG GAGCGGCCTG AAGCCACAGA CATCATTGAA AATGCCATAT TTGAGAACTT GGAGTTTCCC GGGAAAACGG TTCTGAGACA GCGGTCCCGC TCCATGAGTT CATCTGGAAC AAAACATTCC AGACAGCCCA GCTGCTCGTA CACGCCACTG CCTGGCGAGC AGAAGCTGAT CTCTGAGGAG GATCTGTGAC TCGAGCATGC ATCTAGAGGG CCCTATTCTA TAGTGTCACC TAAATGCTAG AGGATCTTTG TGAAGGAACC TTACTTCTGT GGTGTGACAT AATTGGACAA ACTACCTACA GAGATTTAAA GCTCTAAGGT AAATATAAAA TTTTTAAGTG TATAATGTGT TAAACTACTG ATTCTAATTG TTTGTGTATT TTAGATTCCA ACCTATGGAA CTGATGAATG GGAGCAGTGG TGGAATGCCT TTAATGAGGA AAACCTGTTT TGCTCAGAAG AAATGCCATC TAGTGATGAT GAGGCTACTG CTGACTCTCA ACATTCTACT CCTCCAAAAA AGAAGAGAAA GGTAGAAGAC CCCAAGGACT TTCCTTCAGA ATTGCTAAGT TTTTTGAGTC ATGCTGTGTT TAGTAATAGA ACTCTTGCTT GCTTTGCTAT TTACACCACA AAGGAAAAAG CTGCACTGCT ATACAAGAAA ATTATGGAAA AATATTTGAT GTATAGTGCC TTGACTAGAG ATCATAATCA GCCATACCAC ATTTGTAGAG GTTTTACTTG CTTTAAAAAA CCTCCCACAC CTCCCCCTGA ACCTGAAACA TAAAATGAAT GCAATTGTTG TTGTTAACTT GTTTATTGCA GCTTATAATG GTTACAAATA AAGCAATAGC ATCACAAATT TCACAAATAA AGCATTTTTT TCACTGCATT CTAGTTGTGG TTTGTCCAAA CTCATCAATG TATCTTATCA TGTCTGGATC ATCCCGCCAT GGTATCAACG CCATATTTCT ATTTACAGTA GGGACCTCTT CGTTGTGTAG GTACCGCTGT ATTCCTAGGG AAATAGTAGA GGCACCTTGA ACTGTCTGCA TCAGCCATAT AGCCCCCGCT GTTCGACTTA CAAACACAGG CACAGTACTG ACAAACCCAT ACACCTCCTC TGAAATACCC ATAGTTGCTA GGGCTGTCTC CGAACTCATT ACACCCTCCA AAGTCAGAGC TGTAATTTCG CCATCAAGGG CAGCGAGGGC TTCTCCAGAT AAAATAGCTT CTGCCGAGAG TCCCGTAAGG GTAGACACTT CAGCTAATCC CTCGATGAGG TCTACTAGAA TAGTCAGTGC GGCTCCCATT TTGAAAATTC ACTTACTTGA TCAGCTTCAG AAGATGGCGG AGGGCCTCCA ACACAGTAAT TTTCCTCCCG ACTCTTAAAA TAGAAAATGT CAAGTCAGTT AAGCAGGAAG TGGACTAACT GACGCAGCTG GCCGTGCGAC ATCCTCTTTT AATTAGTTGC TAGGCAACGC CCTCCAGAGG GCGTGTGGTT TTGCAAGAGG AAGCAAAAGC CTCTCCACCC AGGCCTAGAA TGTTTCCACC CAATCATTAC TATGACAACA GCTGTTTTTT TTAGTATTAA GCAGAGGCCG GGGACCCCTG GGCCCGCTTA CTCTGGAGAA AAAGAAGAGA GGCATTGTAG AGGCTTCCAG AGGCAACTTG TCAAAACAGG ACTGCTTCTA TTTCTGTCAC ACTGTCTGGC CCTGTCACAA GGTCCAGCAC CTCCATACCC CCTTTAATAA GCAGTTTGGG AACGGGTGCG GGTCTTACTC CGCCCATCCC GCCCCTAACT CCGCCCAGTT CCGCCCATTC TCCGCCCCAT GGCTGACTAA TTTTTTTTAT TTATGCAGAG GCCGAGGCCG CCTCGGCCTC TGAGCTATTC CAGAAGTAGT GAGGAGGCTT TTTTGGAGGC CTAGGCTTTT GCAAAAAGCT AATTC