pET11a-link-NGFP, 6181 bp pET11a-link-NGFP is a plasmid vector designed for fusion of a target protein to the N-terminal fragment of GFP sg100 (residues 1-157). The construct contains a linker for the convenient subcloning of the target between XhoI and BamHI. There is an XmaI site between those two sites, as well. The protein has a hexahistidine tag fused to its N-terminus. With the linker, the protein sequence is: MASHHHHHHGASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYG KLTLKFICTTGKLPVPWPTLVTTLCYGVQCFSRYPDHMKRHDFFKSAMPE GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHK LEYNYNSHNVPIMADKQGGSGSGSSNPGN* The sequence GGSGSGSS is intended as a spacer between the NGFP fragment and the target protein. The target must be cloned in frame for proper fusion. XhoI XmaI BamHI ...caaggtggctctggctctggCTCGAGcaatcccgggaattaaGGATCCattatt ...Q G G S G S G S S N P G N * The first residue of the target protein should be inserted at the first AAT (Asn) codon above. TAA is a stop codon. 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-link-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) 1 - 543 NGFP+linker 1054 - 1914 ampicillin resistance 2144 - 2544 ColE1 origin 3008 - 2817 rop 5708 - 4626 lacI Thomas J. Magliery, Ph.D., July 20, 2004 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 CTCGAGCAAT CCCGGGAATT AAGGATCCGG 551 CTGCTAACAA AGCCCGAAAG GAAGCTGAGT TGGCTGCTGC CACCGCTGAG 601 CAATAACTAG CATAACCCCT TGGGGCCTCT AAACGGGTCT TGAGGGGTTT 651 TTTGCTGAAA GGAGGAACTA TATCCGGATA TCCCGCAAGA GGCCCGGCAG 701 TACCGGCATA ACCAAGCCTA TGCCTACAGC ATCCAGGGTG ACGGTGCCGA 751 GGATGACGAT GAGCGCATTG TTAGATTTCA TACACGGTGC CTGACTGCGT 801 TAGCAATTTA ACTGTGATAA ACTACCGCAT TAAAGCTTAT CGATGATAAG 851 CTGTCAAACA TGAGAATTCT TGAAGACGAA AGGGCCTCGT GATACGCCTA 901 TTTTTATAGG TTAATGTCAT GATAATAATG GTTTCTTAGA CGTCAGGTGG 951 CACTTTTCGG GGAAATGTGC GCGGAACCCC TATTTGTTTA TTTTTCTAAA 1001 TACATTCAAA TATGTATCCG CTCATGAGAC AATAACCCTG ATAAATGCTT 1051 CAATAATATT GAAAAAGGAA GAGTATGAGT ATTCAACATT TCCGTGTCGC 1101 CCTTATTCCC TTTTTTGCGG CATTTTGCCT TCCTGTTTTT GCTCACCCAG 1151 AAACGCTGGT GAAAGTAAAA GATGCTGAAG ATCAGTTGGG TGCACGAGTG 1201 GGTTACATCG AACTGGATCT CAACAGCGGT AAGATCCTTG AGAGTTTTCG 1251 CCCCGAAGAA CGTTTTCCAA TGATGAGCAC TTTTAAAGTT CTGCTATGTG 1301 GCGCGGTATT ATCCCGTGTT GACGCCGGGC AAGAGCAACT CGGTCGCCGC 1351 ATACACTATT CTCAGAATGA CTTGGTTGAG TACTCACCAG TCACAGAAAA 1401 GCATCTTACG GATGGCATGA CAGTAAGAGA ATTATGCAGT GCTGCCATAA 1451 CCATGAGTGA TAACACTGCG GCCAACTTAC TTCTGACAAC GATCGGAGGA 1501 CCGAAGGAGC TAACCGCTTT TTTGCACAAC ATGGGGGATC ATGTAACTCG 1551 CCTTGATCGT TGGGAACCGG AGCTGAATGA AGCCATACCA AACGACGAGC 1601 GTGACACCAC GATGCCTGCA GCAATGGCAA CAACGTTGCG CAAACTATTA 1651 ACTGGCGAAC TACTTACTCT AGCTTCCCGG CAACAATTAA TAGACTGGAT 1701 GGAGGCGGAT AAAGTTGCAG GACCACTTCT GCGCTCGGCC CTTCCGGCTG 1751 GCTGGTTTAT TGCTGATAAA TCTGGAGCCG GTGAGCGTGG GTCTCGCGGT 1801 ATCATTGCAG CACTGGGGCC AGATGGTAAG CCCTCCCGTA TCGTAGTTAT 1851 CTACACGACG GGGAGTCAGG CAACTATGGA TGAACGAAAT AGACAGATCG 1901 CTGAGATAGG TGCCTCACTG ATTAAGCATT GGTAACTGTC AGACCAAGTT 1951 TACTCATATA TACTTTAGAT TGATTTAAAA CTTCATTTTT AATTTAAAAG 2001 GATCTAGGTG AAGATCCTTT TTGATAATCT CATGACCAAA ATCCCTTAAC 2051 GTGAGTTTTC GTTCCACTGA GCGTCAGACC CCGTAGAAAA GATCAAAGGA 2101 TCTTCTTGAG ATCCTTTTTT TCTGCGCGTA ATCTGCTGCT TGCAAACAAA 2151 AAAACCACCG CTACCAGCGG TGGTTTGTTT GCCGGATCAA GAGCTACCAA 2201 CTCTTTTTCC GAAGGTAACT GGCTTCAGCA GAGCGCAGAT ACCAAATACT 2251 GTCCTTCTAG TGTAGCCGTA GTTAGGCCAC CACTTCAAGA ACTCTGTAGC 2301 ACCGCCTACA TACCTCGCTC TGCTAATCCT GTTACCAGTG GCTGCTGCCA 2351 GTGGCGATAA GTCGTGTCTT ACCGGGTTGG ACTCAAGACG ATAGTTACCG 2401 GATAAGGCGC AGCGGTCGGG CTGAACGGGG GGTTCGTGCA CACAGCCCAG 2451 CTTGGAGCGA ACGACCTACA CCGAACTGAG ATACCTACAG CGTGAGCTAT 2501 GAGAAAGCGC CACGCTTCCC GAAGGGAGAA AGGCGGACAG GTATCCGGTA 2551 AGCGGCAGGG TCGGAACAGG AGAGCGCACG AGGGAGCTTC CAGGGGGAAA 2601 CGCCTGGTAT CTTTATAGTC CTGTCGGGTT TCGCCACCTC TGACTTGAGC 2651 GTCGATTTTT GTGATGCTCG TCAGGGGGGC GGAGCCTATG GAAAAACGCC 2701 AGCAACGCGG CCTTTTTACG GTTCCTGGCC TTTTGCTGGC CTTTTGCTCA 2751 CATGTTCTTT CCTGCGTTAT CCCCTGATTC TGTGGATAAC CGTATTACCG 2801 CCTTTGAGTG AGCTGATACC GCTCGCCGCA GCCGAACGAC CGAGCGCAGC 2851 GAGTCAGTGA GCGAGGAAGC GGAAGAGCGC CTGATGCGGT ATTTTCTCCT 2901 TACGCATCTG TGCGGTATTT CACACCGCAT ATATGGTGCA CTCTCAGTAC 2951 AATCTGCTCT GATGCCGCAT AGTTAAGCCA GTATACACTC CGCTATCGCT 3001 ACGTGACTGG GTCATGGCTG CGCCCCGACA CCCGCCAACA CCCGCTGACG 3051 CGCCCTGACG GGCTTGTCTG CTCCCGGCAT CCGCTTACAG ACAAGCTGTG 3101 ACCGTCTCCG GGAGCTGCAT GTGTCAGAGG TTTTCACCGT CATCACCGAA 3151 ACGCGCGAGG CAGCTGCGGT AAAGCTCATC AGCGTGGTCG TGAAGCGATT 3201 CACAGATGTC TGCCTGTTCA TCCGCGTCCA GCTCGTTGAG TTTCTCCAGA 3251 AGCGTTAATG TCTGGCTTCT GATAAAGCGG GCCATGTTAA GGGCGGTTTT 3301 TTCCTGTTTG GTCACTGATG CCTCCGTGTA AGGGGGATTT CTGTTCATGG 3351 GGGTAATGAT ACCGATGAAA CGAGAGAGGA TGCTCACGAT ACGGGTTACT 3401 GATGATGAAC ATGCCCGGTT ACTGGAACGT TGTGAGGGTA AACAACTGGC 3451 GGTATGGATG CGGCGGGACC AGAGAAAAAT CACTCAGGGT CAATGCCAGC 3501 GCTTCGTTAA TACAGATGTA GGTGTTCCAC AGGGTAGCCA GCAGCATCCT 3551 GCGATGCAGA TCCGGAACAT AATGGTGCAG GGCGCTGACT TCCGCGTTTC 3601 CAGACTTTAC GAAACACGGA AACCGAAGAC CATTCATGTT GTTGCTCAGG 3651 TCGCAGACGT TTTGCAGCAG CAGTCGCTTC ACGTTCGCTC GCGTATCGGT 3701 GATTCATTCT GCTAACCAGT AAGGCAACCC CGCCAGCCTA GCCGGGTCCT 3751 CAACGACAGG AGCACGATCA TGCGCACCCG TGGCCAGGAC CCAACGCTGC 3801 CCGAGATGCG CCGCGTGCGG CTGCTGGAGA TGGCGGACGC GATGGATATG 3851 TTCTGCCAAG GGTTGGTTTG CGCATTCACA GTTCTCCGCA AGAATTGATT 3901 GGCTCCAATT CTTGGAGTGG TGAATCCGTT AGCGAGGTGC CGCCGGCTTC 3951 CATTCAGGTC GAGGTGGCCC GGCTCCATGC ACCGCGACGC AACGCGGGGA 4001 GGCAGACAAG GTATAGGGCG GCGCCTACAA TCCATGCCAA CCCGTTCCAT 4051 GTGCTCGCCG AGGCGGCATA AATCGCCGTG ACGATCAGCG GTCCAGTGAT 4101 CGAAGTTAGG CTGGTAAGAG CCGCGAGCGA TCCTTGAAGC TGTCCCTGAT 4151 GGTCGTCATC TACCTGCCTG GACAGCATGG CCTGCAACGC GGGCATCCCG 4201 ATGCCGCCGG AAGCGAGAAG AATCATAATG GGGAAGGCCA TCCAGCCTCG 4251 CGTCGCGAAC GCCAGCAAGA CGTAGCCCAG CGCGTCGGCC GCCATGCCGG 4301 CGATAATGGC CTGCTTCTCG CCGAAACGTT TGGTGGCGGG ACCAGTGACG 4351 AAGGCTTGAG CGAGGGCGTG CAAGATTCCG AATACCGCAA GCGACAGGCC 4401 GATCATCGTC GCGCTCCAGC GAAAGCGGTC CTCGCCGAAA ATGACCCAGA 4451 GCGCTGCCGG CACCTGTCCT ACGAGTTGCA TGATAAAGAA GACAGTCATA 4501 AGTGCGGCGA CGATAGTCAT GCCCCGCGCC CACCGGAAGG AGCTGACTGG 4551 GTTGAAGGCT CTCAAGGGCA TCGGTCGAGA TCCCGGTGCC TAATGAGTGA 4601 GCTAACTTAC ATTAATTGCG TTGCGCTCAC TGCCCGCTTT CCAGTCGGGA 4651 AACCTGTCGT GCCAGCTGCA TTAATGAATC GGCCAACGCG CGGGGAGAGG 4701 CGGTTTGCGT ATTGGGCGCC AGGGTGGTTT TTCTTTTCAC CAGTGAGACG 4751 GGCAACAGCT GATTGCCCTT CACCGCCTGG CCCTGAGAGA GTTGCAGCAA 4801 GCGGTCCACG CTGGTTTGCC CCAGCAGGCG AAAATCCTGT TTGATGGTGG 4851 TTAACGGCGG GATATAACAT GAGCTGTCTT CGGTATCGTC GTATCCCACT 4901 ACCGAGATAT CCGCACCAAC GCGCAGCCCG GACTCGGTAA TGGCGCGCAT 4951 TGCGCCCAGC GCCATCTGAT CGTTGGCAAC CAGCATCGCA GTGGGAACGA 5001 TGCCCTCATT CAGCATTTGC ATGGTTTGTT GAAAACCGGA CATGGCACTC 5051 CAGTCGCCTT CCCGTTCCGC TATCGGCTGA ATTTGATTGC GAGTGAGATA 5101 TTTATGCCAG CCAGCCAGAC GCAGACGCGC CGAGACAGAA CTTAATGGGC 5151 CCGCTAACAG CGCGATTTGC TGGTGACCCA ATGCGACCAG ATGCTCCACG 5201 CCCAGTCGCG TACCGTCTTC ATGGGAGAAA ATAATACTGT TGATGGGTGT 5251 CTGGTCAGAG ACATCAAGAA ATAACGCCGG AACATTAGTG CAGGCAGCTT 5301 CCACAGCAAT GGCATCCTGG TCATCCAGCG GATAGTTAAT GATCAGCCCA 5351 CTGACGCGTT GCGCGAGAAG ATTGTGCACC GCCGCTTTAC AGGCTTCGAC 5401 GCCGCTTCGT TCTACCATCG ACACCACCAC GCTGGCACCC AGTTGATCGG 5451 CGCGAGATTT AATCGCCGCG ACAATTTGCG ACGGCGCGTG CAGGGCCAGA 5501 CTGGAGGTGG CAACGCCAAT CAGCAACGAC TGTTTGCCCG CCAGTTGTTG 5551 TGCCACGCGG TTGGGAATGT AATTCAGCTC CGCCATCGCC GCTTCCACTT 5601 TTTCCCGCGT TTTCGCAGAA ACGTGGCTGG CCTGGTTCAC CACGCGGGAA 5651 ACGGTCTGAT AAGAGACACC GGCATACTCT GCGACATCGT ATAACGTTAC 5701 TGGTTTCACA TTCACCACCC TGAATTGACT CTCTTCCGGG CGCTATCATG 5751 CCATACCGCG AAAGGTTTTG CGCCATTCGA TGGTGTCCGG GATCTCGACG 5801 CTCTCCCTTA TGCGACTCCT GCATTAGGAA GCAGCCCAGT AGTAGGTTGA 5851 GGCCGTTGAG CACCGCCGCC GCAAGGAATG GTGCATGCAA GGAGATGGCG 5901 CCCAACAGTC CCCCGGCCAC GGGGCCTGCC ACCATACCCA CGCCGAAACA 5951 AGCGCTCATG AGCCCGAAGT GGCGAGCCCG ATCTTCCCCA TCGGTGATGT 6001 CGGCGATATA GGCGCCAGCA ACCGCACCTG TGGCGCCGGT GATGCCGGCC 6051 ACGATGCGTC CGGCGTAGAG GATCGAGATC TCGATCCCGC GAAATTAATA 6101 CGACTCACTA TAGGGGAATT GTGAGCGGAT AACAATTCCC CTCTAGAAAT 6151 AATTTTGTTT AACTTTAAGA AGGAGATATA C