pET11a-Z-NGFP, 6256 bp pET11a-Z-NGFP is a plasmid vector that expresses a fusion of an antiparallel leucine zipper peptide to the N-terminal fragment of GFP sg100 (residues 1-157). The plasmid can be used as a positive control for the GFP reassembly screen in conjunction with pMRBAD-Z-CGFP, or it can be used as a negative control with a protein of choice cloned into pMRBAD-link-CGFP. The protein has a hexahistidine tag fused to its N-terminus. The protein sequence is: MASHHHHHHGASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYG KLTLKFICTTGKLPVPWPTLVTTLCYGVQCFSRYPDHMKRHDFFKSAMPE GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHK LEYNYNSHNVPIMADKQGGSGSGSSALKKELQANKKELAQLKWELQALKK ELAQ* The sequence GGSGSGSS is intended as a spacer between the NGFP fragment and the leucine zipper peptide. The NGFP fusion is under the control of the T7 promoter. Therefore, expression is only possible in a strain expressing T7 RNA Polymerase. We typically use BL21(DE3), but you could lysogenize any strain with lambdaDE3. For use with pMRBAD-Z-CGFP, we recommend growth on LB agar supplemented with 100 ug/mL ampicillin, 35 ug/mL kanamycin, 10 uM IPTG and 0.02-0.2% arabinose. Grow the cells overnight at 30C and transfer them to room temperature for 2 days. Plasmid features (approx) 3 - 618 NGFP+Z fusion 1129 - 1989 ampicillin resistance 2219 - 2619 ColE1 origin 3083 - 2892 rop 5783 - 4701 lacI Thomas J. Magliery, Ph.D., September 28, 2004 (Note that the DNA sequence below is now correct. The 10 Sept. 2004 version actually had the sequence of pET11a-link-NGFP below.) References: Magliery TJ, Wilson CGM, Pan W, Mishler D, Ghosh I, Hamilton AD, Regan L. 2004. Detecting protein-protein interactions with a GFP-fragment reassembly trap: scope and mechanism. J Am Chem Soc, submitted. Ghosh I, Hamilton AD, Regan L. 2000. Antiparallel leucine zipper-directed protein reassembly: application to the green fluorscent protein. J Am Chem Soc 122: 5658. References can be downloaded from: http://www.csb.yale.edu/people/regan/publications.html 1 ATATGGCTAG CCATCACCAC CATCACCATG GCGCGAGCAA AGGAGAAGAA 51 CTCTTCACTG GAGTTGTCCC AATTCTTGTT GAATTAGATG GTGATGTTAA 101 CGGCCACAAG TTCTCTGTCA GTGGAGAGGG TGAAGGTGAT GCAACATACG 151 GAAAACTTAC CCTGAAGTTC ATCTGCACTA CTGGCAAACT GCCTGTTCCA 201 TGGCCAACAC TAGTCACTAC TCTGTGCTAT GGTGTTCAAT GCTTTTCAAG 251 ATACCCGGAT CATATGAAAC GGCATGACTT TTTCAAGAGT GCCATGCCCG 301 AAGGTTATGT ACAGGAAAGG ACCATCTTCT TCAAAGATGA CGGCAACTAC 351 AAGACACGTG CTGAAGTCAA GTTTGAAGGT GATACCCTTG TTAATAGAAT 401 CGAGTTAAAA GGTATTGACT TCAAGGAAGA TGGCAACATT CTGGGACACA 451 AATTGGAATA CAACTATAAC TCACACAACG TTCCCATCAT GGCAGACAAA 501 CAAGGTGGCT CTGGCTCTGG CTCGAGCGCC CTCAAAAAAG AATTGCAGGC 551 AAACAAAAAA GAACTTGCGC AGCTGAAGTG GGAGTTACAA GCTCTGAAAA 601 AGGAACTGGC GCAGTAAGGA TCCGGCTGCT AACAAAGCCC GAAAGGAAGC 651 TGAGTTGGCT GCTGCCACCG CTGAGCAATA ACTAGCATAA CCCCTTGGGG 701 CCTCTAAACG GGTCTTGAGG GGTTTTTTGC TGAAAGGAGG AACTATATCC 751 GGATATCCCG CAAGAGGCCC GGCAGTACCG GCATAACCAA GCCTATGCCT 801 ACAGCATCCA GGGTGACGGT GCCGAGGATG ACGATGAGCG CATTGTTAGA 851 TTTCATACAC GGTGCCTGAC TGCGTTAGCA ATTTAACTGT GATAAACTAC 901 CGCATTAAAG CTTATCGATG ATAAGCTGTC AAACATGAGA ATTCTTGAAG 951 ACGAAAGGGC CTCGTGATAC GCCTATTTTT ATAGGTTAAT GTCATGATAA 1001 TAATGGTTTC TTAGACGTCA GGTGGCACTT TTCGGGGAAA TGTGCGCGGA 1051 ACCCCTATTT GTTTATTTTT CTAAATACAT TCAAATATGT ATCCGCTCAT 1101 GAGACAATAA CCCTGATAAA TGCTTCAATA ATATTGAAAA AGGAAGAGTA 1151 TGAGTATTCA ACATTTCCGT GTCGCCCTTA TTCCCTTTTT TGCGGCATTT 1201 TGCCTTCCTG TTTTTGCTCA CCCAGAAACG CTGGTGAAAG TAAAAGATGC 1251 TGAAGATCAG TTGGGTGCAC GAGTGGGTTA CATCGAACTG GATCTCAACA 1301 GCGGTAAGAT CCTTGAGAGT TTTCGCCCCG AAGAACGTTT TCCAATGATG 1351 AGCACTTTTA AAGTTCTGCT ATGTGGCGCG GTATTATCCC GTGTTGACGC 1401 CGGGCAAGAG CAACTCGGTC GCCGCATACA CTATTCTCAG AATGACTTGG 1451 TTGAGTACTC ACCAGTCACA GAAAAGCATC TTACGGATGG CATGACAGTA 1501 AGAGAATTAT GCAGTGCTGC CATAACCATG AGTGATAACA CTGCGGCCAA 1551 CTTACTTCTG ACAACGATCG GAGGACCGAA GGAGCTAACC GCTTTTTTGC 1601 ACAACATGGG GGATCATGTA ACTCGCCTTG ATCGTTGGGA ACCGGAGCTG 1651 AATGAAGCCA TACCAAACGA CGAGCGTGAC ACCACGATGC CTGCAGCAAT 1701 GGCAACAACG TTGCGCAAAC TATTAACTGG CGAACTACTT ACTCTAGCTT 1751 CCCGGCAACA ATTAATAGAC TGGATGGAGG CGGATAAAGT TGCAGGACCA 1801 CTTCTGCGCT CGGCCCTTCC GGCTGGCTGG TTTATTGCTG ATAAATCTGG 1851 AGCCGGTGAG CGTGGGTCTC GCGGTATCAT TGCAGCACTG GGGCCAGATG 1901 GTAAGCCCTC CCGTATCGTA GTTATCTACA CGACGGGGAG TCAGGCAACT 1951 ATGGATGAAC GAAATAGACA GATCGCTGAG ATAGGTGCCT CACTGATTAA 2001 GCATTGGTAA CTGTCAGACC AAGTTTACTC ATATATACTT TAGATTGATT 2051 TAAAACTTCA TTTTTAATTT AAAAGGATCT AGGTGAAGAT CCTTTTTGAT 2101 AATCTCATGA CCAAAATCCC TTAACGTGAG TTTTCGTTCC ACTGAGCGTC 2151 AGACCCCGTA GAAAAGATCA AAGGATCTTC TTGAGATCCT TTTTTTCTGC 2201 GCGTAATCTG CTGCTTGCAA ACAAAAAAAC CACCGCTACC AGCGGTGGTT 2251 TGTTTGCCGG ATCAAGAGCT ACCAACTCTT TTTCCGAAGG TAACTGGCTT 2301 CAGCAGAGCG CAGATACCAA ATACTGTCCT TCTAGTGTAG CCGTAGTTAG 2351 GCCACCACTT CAAGAACTCT GTAGCACCGC CTACATACCT CGCTCTGCTA 2401 ATCCTGTTAC CAGTGGCTGC TGCCAGTGGC GATAAGTCGT GTCTTACCGG 2451 GTTGGACTCA AGACGATAGT TACCGGATAA GGCGCAGCGG TCGGGCTGAA 2501 CGGGGGGTTC GTGCACACAG CCCAGCTTGG AGCGAACGAC CTACACCGAA 2551 CTGAGATACC TACAGCGTGA GCTATGAGAA AGCGCCACGC TTCCCGAAGG 2601 GAGAAAGGCG GACAGGTATC CGGTAAGCGG CAGGGTCGGA ACAGGAGAGC 2651 GCACGAGGGA GCTTCCAGGG GGAAACGCCT GGTATCTTTA TAGTCCTGTC 2701 GGGTTTCGCC ACCTCTGACT TGAGCGTCGA TTTTTGTGAT GCTCGTCAGG 2751 GGGGCGGAGC CTATGGAAAA ACGCCAGCAA CGCGGCCTTT TTACGGTTCC 2801 TGGCCTTTTG CTGGCCTTTT GCTCACATGT TCTTTCCTGC GTTATCCCCT 2851 GATTCTGTGG ATAACCGTAT TACCGCCTTT GAGTGAGCTG ATACCGCTCG 2901 CCGCAGCCGA ACGACCGAGC GCAGCGAGTC AGTGAGCGAG GAAGCGGAAG 2951 AGCGCCTGAT GCGGTATTTT CTCCTTACGC ATCTGTGCGG TATTTCACAC 3001 CGCATATATG GTGCACTCTC AGTACAATCT GCTCTGATGC CGCATAGTTA 3051 AGCCAGTATA CACTCCGCTA TCGCTACGTG ACTGGGTCAT GGCTGCGCCC 3101 CGACACCCGC CAACACCCGC TGACGCGCCC TGACGGGCTT GTCTGCTCCC 3151 GGCATCCGCT TACAGACAAG CTGTGACCGT CTCCGGGAGC TGCATGTGTC 3201 AGAGGTTTTC ACCGTCATCA CCGAAACGCG CGAGGCAGCT GCGGTAAAGC 3251 TCATCAGCGT GGTCGTGAAG CGATTCACAG ATGTCTGCCT GTTCATCCGC 3301 GTCCAGCTCG TTGAGTTTCT CCAGAAGCGT TAATGTCTGG CTTCTGATAA 3351 AGCGGGCCAT GTTAAGGGCG GTTTTTTCCT GTTTGGTCAC TGATGCCTCC 3401 GTGTAAGGGG GATTTCTGTT CATGGGGGTA ATGATACCGA TGAAACGAGA 3451 GAGGATGCTC ACGATACGGG TTACTGATGA TGAACATGCC CGGTTACTGG 3501 AACGTTGTGA GGGTAAACAA CTGGCGGTAT GGATGCGGCG GGACCAGAGA 3551 AAAATCACTC AGGGTCAATG CCAGCGCTTC GTTAATACAG ATGTAGGTGT 3601 TCCACAGGGT AGCCAGCAGC ATCCTGCGAT GCAGATCCGG AACATAATGG 3651 TGCAGGGCGC TGACTTCCGC GTTTCCAGAC TTTACGAAAC ACGGAAACCG 3701 AAGACCATTC ATGTTGTTGC TCAGGTCGCA GACGTTTTGC AGCAGCAGTC 3751 GCTTCACGTT CGCTCGCGTA TCGGTGATTC ATTCTGCTAA CCAGTAAGGC 3801 AACCCCGCCA GCCTAGCCGG GTCCTCAACG ACAGGAGCAC GATCATGCGC 3851 ACCCGTGGCC AGGACCCAAC GCTGCCCGAG ATGCGCCGCG TGCGGCTGCT 3901 GGAGATGGCG GACGCGATGG ATATGTTCTG CCAAGGGTTG GTTTGCGCAT 3951 TCACAGTTCT CCGCAAGAAT TGATTGGCTC CAATTCTTGG AGTGGTGAAT 4001 CCGTTAGCGA GGTGCCGCCG GCTTCCATTC AGGTCGAGGT GGCCCGGCTC 4051 CATGCACCGC GACGCAACGC GGGGAGGCAG ACAAGGTATA GGGCGGCGCC 4101 TACAATCCAT GCCAACCCGT TCCATGTGCT CGCCGAGGCG GCATAAATCG 4151 CCGTGACGAT CAGCGGTCCA GTGATCGAAG TTAGGCTGGT AAGAGCCGCG 4201 AGCGATCCTT GAAGCTGTCC CTGATGGTCG TCATCTACCT GCCTGGACAG 4251 CATGGCCTGC AACGCGGGCA TCCCGATGCC GCCGGAAGCG AGAAGAATCA 4301 TAATGGGGAA GGCCATCCAG CCTCGCGTCG CGAACGCCAG CAAGACGTAG 4351 CCCAGCGCGT CGGCCGCCAT GCCGGCGATA ATGGCCTGCT TCTCGCCGAA 4401 ACGTTTGGTG GCGGGACCAG TGACGAAGGC TTGAGCGAGG GCGTGCAAGA 4451 TTCCGAATAC CGCAAGCGAC AGGCCGATCA TCGTCGCGCT CCAGCGAAAG 4501 CGGTCCTCGC CGAAAATGAC CCAGAGCGCT GCCGGCACCT GTCCTACGAG 4551 TTGCATGATA AAGAAGACAG TCATAAGTGC GGCGACGATA GTCATGCCCC 4601 GCGCCCACCG GAAGGAGCTG ACTGGGTTGA AGGCTCTCAA GGGCATCGGT 4651 CGAGATCCCG GTGCCTAATG AGTGAGCTAA CTTACATTAA TTGCGTTGCG 4701 CTCACTGCCC GCTTTCCAGT CGGGAAACCT GTCGTGCCAG CTGCATTAAT 4751 GAATCGGCCA ACGCGCGGGG AGAGGCGGTT TGCGTATTGG GCGCCAGGGT 4801 GGTTTTTCTT TTCACCAGTG AGACGGGCAA CAGCTGATTG CCCTTCACCG 4851 CCTGGCCCTG AGAGAGTTGC AGCAAGCGGT CCACGCTGGT TTGCCCCAGC 4901 AGGCGAAAAT CCTGTTTGAT GGTGGTTAAC GGCGGGATAT AACATGAGCT 4951 GTCTTCGGTA TCGTCGTATC CCACTACCGA GATATCCGCA CCAACGCGCA 5001 GCCCGGACTC GGTAATGGCG CGCATTGCGC CCAGCGCCAT CTGATCGTTG 5051 GCAACCAGCA TCGCAGTGGG AACGATGCCC TCATTCAGCA TTTGCATGGT 5101 TTGTTGAAAA CCGGACATGG CACTCCAGTC GCCTTCCCGT TCCGCTATCG 5151 GCTGAATTTG ATTGCGAGTG AGATATTTAT GCCAGCCAGC CAGACGCAGA 5201 CGCGCCGAGA CAGAACTTAA TGGGCCCGCT AACAGCGCGA TTTGCTGGTG 5251 ACCCAATGCG ACCAGATGCT CCACGCCCAG TCGCGTACCG TCTTCATGGG 5301 AGAAAATAAT ACTGTTGATG GGTGTCTGGT CAGAGACATC AAGAAATAAC 5351 GCCGGAACAT TAGTGCAGGC AGCTTCCACA GCAATGGCAT CCTGGTCATC 5401 CAGCGGATAG TTAATGATCA GCCCACTGAC GCGTTGCGCG AGAAGATTGT 5451 GCACCGCCGC TTTACAGGCT TCGACGCCGC TTCGTTCTAC CATCGACACC 5501 ACCACGCTGG CACCCAGTTG ATCGGCGCGA GATTTAATCG CCGCGACAAT 5551 TTGCGACGGC GCGTGCAGGG CCAGACTGGA GGTGGCAACG CCAATCAGCA 5601 ACGACTGTTT GCCCGCCAGT TGTTGTGCCA CGCGGTTGGG AATGTAATTC 5651 AGCTCCGCCA TCGCCGCTTC CACTTTTTCC CGCGTTTTCG CAGAAACGTG 5701 GCTGGCCTGG TTCACCACGC GGGAAACGGT CTGATAAGAG ACACCGGCAT 5751 ACTCTGCGAC ATCGTATAAC GTTACTGGTT TCACATTCAC CACCCTGAAT 5801 TGACTCTCTT CCGGGCGCTA TCATGCCATA CCGCGAAAGG TTTTGCGCCA 5851 TTCGATGGTG TCCGGGATCT CGACGCTCTC CCTTATGCGA CTCCTGCATT 5901 AGGAAGCAGC CCAGTAGTAG GTTGAGGCCG TTGAGCACCG CCGCCGCAAG 5951 GAATGGTGCA TGCAAGGAGA TGGCGCCCAA CAGTCCCCCG GCCACGGGGC 6001 CTGCCACCAT ACCCACGCCG AAACAAGCGC TCATGAGCCC GAAGTGGCGA 6051 GCCCGATCTT CCCCATCGGT GATGTCGGCG ATATAGGCGC CAGCAACCGC 6101 ACCTGTGGCG CCGGTGATGC CGGCCACGAT GCGTCCGGCG TAGAGGATCG 6151 AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG 6201 CGGATAACAA TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG 6251 ATATAC