pET-33b(+)

The pET-33b(+) vector (Cat. No. 69054-3) is derived from pET-28b(+) and carries a 15bp sequence encoding the protein kinase A (PKA) site RRASV, located between the thrombin cleavage and Nde I sites (1). Proteins expressed in pET-33b(+) can be easily purified by metal chelation chromatography (via either N- or C-terminal His?Tag? sequences) and efficiently labeled with 32P- or 33P-gATP and the catalytic subunit of cAMP-dependent protein kinase from heart muscle. Labeled proteins can be used as direct probes in protein-protein interaction studies. Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the circle map. The cloning/ expression region of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage will produce virions containing single-stranded DNA that corresponds to the coding strand. Therefore, single-stranded sequencing should be performed using the T7 terminator primer (Cat. No. 69337-3).

0001 ATCCGGATAT AGTTCCTCCT TTCAGCAAAA AACCCCTCAA GACCCGTTTA GAGGCCCCAA 
0061 GGGGTTATGC TAGTTATTGC TCAGCGGTGG CAGCAGCCAA CTCAGCTTCC TTTCGGGCTT 
0121 TGTTAGCAGC CGGATCTCAG TGGTGGTGGT GGTGGTGCTC GAGTGCGGCC GCAAGCTTGT 
0181 CGACGGAGCT CGAATTCGGA TCCCGACCCA TTTGCTGTCC ACCAGTCATG CTAGCCATAT 
0241 GAACAGATGC ACGACGGCTG CCGCGCGGCA CCAGGCCGCT GCTGTGATGA TGATGATGAT 
0301 GGCTGCTGCC CATGGTATAT CTCCTTCTTA AAGTTAAACA AAATTATTTC TAGAGGGGAA 
0361 TTGTTATCCG CTCACAATTC CCCTATAGTG AGTCGTATTA ATTTCGCGGG ATCGAGATCT 
0421 CGATCCTCTA CGCCGGACGC ATCGTGGCCG GCATCACCGG CGCCACAGGT GCGGTTGCTG 
0481 GCGCCTATAT CGCCGACATC ACCGATGGGG AAGATCGGGC TCGCCACTTC GGGCTCATGA 
0541 GCGCTTGTTT CGGCGTGGGT ATGGTGGCAG GCCCCGTGGC CGGGGGACTG TTGGGCGCCA 
0601 TCTCCTTGCA TGCACCATTC CTTGCGGCGG CGGTGCTCAA CGGCCTCAAC CTACTACTGG 
0661 GCTGCTTCCT AATGCAGGAG TCGCATAAGG GAGAGCGTCG AGATCCCGGA CACCATCGAA 
0721 TGGCGCAAAA CCTTTCGCGG TATGGCATGA TAGCGCCCGG AAGAGAGTCA ATTCAGGGTG 
0781 GTGAATGTGA AACCAGTAAC GTTATACGAT GTCGCAGAGT ATGCCGGTGT CTCTTATCAG 
0841 ACCGTTTCCC GCGTGGTGAA CCAGGCCAGC CACGTTTCTG CGAAAACGCG GGAAAAAGTG 
0901 GAAGCGGCGA TGGCGGAGCT GAATTACATT CCCAACCGCG TGGCACAACA ACTGGCGGGC 
0961 AAACAGTCGT TGCTGATTGG CGTTGCCACC TCCAGTCTGG CCCTGCACGC GCCGTCGCAA 
1021 ATTGTCGCGG CGATTAAATC TCGCGCCGAT CAACTGGGTG CCAGCGTGGT GGTGTCGATG 
1081 GTAGAACGAA GCGGCGTCGA AGCCTGTAAA GCGGCGGTGC ACAATCTTCT CGCGCAACGC 
1141 GTCAGTGGGC TGATCATTAA CTATCCGCTG GATGACCAGG ATGCCATTGC TGTGGAAGCT 
1201 GCCTGCACTA ATGTTCCGGC GTTATTTCTT GATGTCTCTG ACCAGACACC CATCAACAGT 
1261 ATTATTTTCT CCCATGAAGA CGGTACGCGA CTGGGCGTGG AGCATCTGGT CGCATTGGGT 
1321 CACCAGCAAA TCGCGCTGTT AGCGGGCCCA TTAAGTTCTG TCTCGGCGCG TCTGCGTCTG 
1381 GCTGGCTGGC ATAAATATCT CACTCGCAAT CAAATTCAGC CGATAGCGGA ACGGGAAGGC 
1441 GACTGGAGTG CCATGTCCGG TTTTCAACAA ACCATGCAAA TGCTGAATGA GGGCATCGTT 
1501 CCCACTGCGA TGCTGGTTGC CAACGATCAG ATGGCGCTGG GCGCAATGCG CGCCATTACC 
1561 GAGTCCGGGC TGCGCGTTGG TGCGGATATC TCGGTAGTGG GATACGACGA TACCGAAGAC 
1621 AGCTCATGTT ATATCCCGCC GTTAACCACC ATCAAACAGG ATTTTCGCCT GCTGGGGCAA 
1681 ACCAGCGTGG ACCGCTTGCT GCAACTCTCT CAGGGCCAGG CGGTGAAGGG CAATCAGCTG 
1741 TTGCCCGTCT CACTGGTGAA AAGAAAAACC ACCCTGGCGC CCAATACGCA AACCGCCTCT 
1801 CCCCGCGCGT TGGCCGATTC ATTAATGCAG CTGGCACGAC AGGTTTCCCG ACTGGAAAGC 
1861 GGGCAGTGAG CGCAACGCAA TTAATGTAAG TTAGCTCACT CATTAGGCAC CGGGATCTCG 
1921 ACCGATGCCC TTGAGAGCCT TCAACCCAGT CAGCTCCTTC CGGTGGGCGC GGGGCATGAC 
1981 TATCGTCGCC GCACTTATGA CTGTCTTCTT TATCATGCAA CTCGTAGGAC AGGTGCCGGC 
2041 AGCGCTCTGG GTCATTTTCG GCGAGGACCG CTTTCGCTGG AGCGCGACGA TGATCGGCCT 
2101 GTCGCTTGCG GTATTCGGAA TCTTGCACGC CCTCGCTCAA GCCTTCGTCA CTGGTCCCGC 
2161 CACCAAACGT TTCGGCGAGA AGCAGGCCAT TATCGCCGGC ATGGCGGCCC CACGGGTGCG 
2221 CATGATCGTG CTCCTGTCGT TGAGGACCCG GCTAGGCTGG CGGGGTTGCC TTACTGGTTA 
2281 GCAGAATGAA TCACCGATAC GCGAGCGAAC GTGAAGCGAC TGCTGCTGCA AAACGTCTGC 
2341 GACCTGAGCA ACAACATGAA TGGTCTTCGG TTTCCGTGTT TCGTAAAGTC TGGAAACGCG 
2401 GAAGTCAGCG CCCTGCACCA TTATGTTCCG GATCTGCATC GCAGGATGCT GCTGGCTACC 
2461 CTGTGGAACA CCTACATCTG TATTAACGAA GCGCTGGCAT TGACCCTGAG TGATTTTTCT 
2521 CTGGTCCCGC CGCATCCATA CCGCCAGTTG TTTACCCTCA CAACGTTCCA GTAACCGGGC 
2581 ATGTTCATCA TCAGTAACCC GTATCGTGAG CATCCTCTCT CGTTTCATCG GTATCATTAC 
2641 CCCCATGAAC AGAAATCCCC CTTACACGGA GGCATCAGTG ACCAAACAGG AAAAAACCGC 
2701 CCTTAACATG GCCCGCTTTA TCAGAAGCCA GACATTAACG CTTCTGGAGA AACTCAACGA 
2761 GCTGGACGCG GATGAACAGG CAGACATCTG TGAATCGCTT CACGACCACG CTGATGAGCT 
2821 TTACCGCAGC TGCCTCGCGC GTTTCGGTGA TGACGGTGAA AACCTCTGAC ACATGCAGCT 
2881 CCCGGAGACG GTCACAGCTT GTCTGTAAGC GGATGCCGGG AGCAGACAAG CCCGTCAGGG 
2941 CGCGTCAGCG GGTGTTGGCG GGTGTCGGGG CGCAGCCATG ACCCAGTCAC GTAGCGATAG 
3001 CGGAGTGTAT ACTGGCTTAA CTATGCGGCA TCAGAGCAGA TTGTACTGAG AGTGCACCAT 
3061 ATATGCGGTG TGAAATACCG CACAGATGCG TAAGGAGAAA ATACCGCATC AGGCGCTCTT 
3121 CCGCTTCCTC GCTCACTGAC TCGCTGCGCT CGGTCGTTCG GCTGCGGCGA GCGGTATCAG 
3181 CTCACTCAAA GGCGGTAATA CGGTTATCCA CAGAATCAGG GGATAACGCA GGAAAGAACA 
3241 TGTGAGCAAA AGGCCAGCAA AAGGCCAGGA ACCGTAAAAA GGCCGCGTTG CTGGCGTTTT 
3301 TCCATAGGCT CCGCCCCCCT GACGAGCATC ACAAAAATCG ACGCTCAAGT CAGAGGTGGC 
3361 GAAACCCGAC AGGACTATAA AGATACCAGG CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT 
3421 CTCCTGTTCC GACCCTGCCG CTTACCGGAT ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG 
3481 TGGCGCTTTC TCATAGCTCA CGCTGTAGGT ATCTCAGTTC GGTGTAGGTC GTTCGCTCCA 
3541 AGCTGGGCTG TGTGCACGAA CCCCCCGTTC AGCCCGACCG CTGCGCCTTA TCCGGTAACT 
3601 ATCGTCTTGA GTCCAACCCG GTAAGACACG ACTTATCGCC ACTGGCAGCA GCCACTGGTA 
3661 ACAGGATTAG CAGAGCGAGG TATGTAGGCG GTGCTACAGA GTTCTTGAAG TGGTGGCCTA 
3721 ACTACGGCTA CACTAGAAGG ACAGTATTTG GTATCTGCGC TCTGCTGAAG CCAGTTACCT 
3781 TCGGAAAAAG AGTTGGTAGC TCTTGATCCG GCAAACAAAC CACCGCTGGT AGCGGTGGTT 
3841 TTTTTGTTTG CAAGCAGCAG ATTACGCGCA GAAAAAAAGG ATCTCAAGAA GATCCTTTGA 
3901 TCTTTTCTAC GGGGTCTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGGG ATTTTGGTCA 
3961 TGAACAATAA AACTGTCTGC TTACATAAAC AGTAATACAA GGGGTGTTAT GAGCCATATT 
4021 CAACGGGAAA CGTCTTGCTC TAGGCCGCGA TTAAATTCCA ACATGGATGC TGATTTATAT 
4081 GGGTATAAAT GGGCTCGCGA TAATGTCGGG CAATCAGGTG CGACAATCTA TCGATTGTAT 
4141 GGGAAGCCCG ATGCGCCAGA GTTGTTTCTG AAACATGGCA AAGGTAGCGT TGCCAATGAT 
4201 GTTACAGATG AGATGGTCAG ACTAAACTGG CTGACGGAAT TTATGCCTCT TCCGACCATC 
4261 AAGCATTTTA TCCGTACTCC TGATGATGCA TGGTTACTCA CCACTGCGAT CCCCGGGAAA 
4321 ACAGCATTCC AGGTATTAGA AGAATATCCT GATTCAGGTG AAAATATTGT TGATGCGCTG 
4381 GCAGTGTTCC TGCGCCGGTT GCATTCGATT CCTGTTTGTA ATTGTCCTTT TAACAGCGAT 
4441 CGCGTATTTC GTCTCGCTCA GGCGCAATCA CGAATGAATA ACGGTTTGGT TGATGCGAGT 
4501 GATTTTGATG ACGAGCGTAA TGGCTGGCCT GTTGAACAAG TCTGGAAAGA AATGCATAAA 
4561 CTTTTGCCAT TCTCACCGGA TTCAGTCGTC ACTCATGGTG ATTTCTCACT TGATAACCTT 
4621 ATTTTTGACG AGGGGAAATT AATAGGTTGT ATTGATGTTG GACGAGTCGG AATCGCAGAC 
4681 CGATACCAGG ATCTTGCCAT CCTATGGAAC TGCCTCGGTG AGTTTTCTCC TTCATTACAG 
4741 AAACGGCTTT TTCAAAAATA TGGTATTGAT AATCCTGATA TGAATAAATT GCAGTTTCAT 
4801 TTGATGCTCG ATGAGTTTTT CTAAGAATTA ATTCATGAGC GGATACATAT TTGAATGTAT 
4861 TTAGAAAAAT AAACAAATAG GGGTTCCGCG CACATTTCCC CGAAAAGTGC CACCTGAAAT 
4921 TGTAAACGTT AATATTTTGT TAAAATTCGC GTTAAATTTT TGTTAAATCA GCTCATTTTT 
4981 TAACCAATAG GCCGAAATCG GCAAAATCCC TTATAAATCA AAAGAATAGA CCGAGATAGG 
5041 GTTGAGTGTT GTTCCAGTTT GGAACAAGAG TCCACTATTA AAGAACGTGG ACTCCAACGT 
5101 CAAAGGGCGA AAAACCGTCT ATCAGGGCGA TGGCCCACTA CGTGAACCAT CACCCTAATC 
5161 AAGTTTTTTG GGGTCGAGGT GCCGTAAAGC ACTAAATCGG AACCCTAAAG GGAGCCCCCG 
5221 ATTTAGAGCT TGACGGGGAA AGCCGGCGAA CGTGGCGAGA AAGGAAGGGA AGAAAGCGAA 
5281 AGGAGCGGGC GCTAGGGCGC TGGCAAGTGT AGCGGTCACG CTGCGCGTAA CCACCACACC 
5341 CGCCGCGCTT AATGCGCCGC TACAGGGCGC GTCCCATTCG CCA