251 X15 phage display library 15 5.7e8 2.5e11 Ivone M. Takenaka Completely random NNM Linear The 15-mer phage display random peptide library was constructed using the fUSE-5 vector, kindly provided by G. Smith (University of Missouri). FUSE-5 replicative Form DNA was purified from K802 Escherichia coli and linearized by SfiI digestion, and the resulting vector DNA was ligated to synthetic DNA fragments containing complementary BglI termini. These duplex oligonucleotides contained (NNM)15 triplets in the coding strand, where N corresponds to equivalent amounts of A, T, G, and C and where M corresponds to equivalent amounts of G and T (minimizes occurrence of translational stop codons). The noncoding strand consisted of 18 or 45 residue stretches of the inosine nucleotide analog. Recombinant DNA was electroporated into MC1061 E. coli electrocompetent cells, spread on 94 plates (24 x 24 cm) with NZamine-yeast extract (NZY) agar plus 40 μg/ml tetracycline (tet) and incubated overnight at 37 °C. Phage were collected by polyethylene glycol precipitation and purified on cesium chloride gradients. The number of transducing units (TU) was determined by infecting K91Kan-resistant cells with an aliquot from each library and plating on tet plates. FUSE5-vector DNA containing an insert restores the reading frame of the pIII gene product, enabling the phage to infect K91Kan-resistant cells and produce colonies on tet plates. 252 XC(X)10CX phage display library 14 J. K. Scott Semi-random NNK Circular The library was constructed in the vector f88.4. 253 FMC12C phage display library 12 Completely random NNK Circular The library was constructed in a modified fd-tet vector. 254 X9 T7 phage display library 9 2e10 Lars Hellman Completely random NNK Linear The general structure of the aa sequence in the phage clone is PGG(X)9(H)6. 255 pComb M13 phage display library 6 5.0e6 6.4e7 Roger Y. Tsien Completely random Linear Amino acid sequences of the format Met-(His6)-(E)9-(X)6-(R)9 (where X 6 represents 6 randomized amino acids) were fused to the N terminus (0-4 copies per phage) of a truncated form of the M13 phage gIII coat protein. A hexahistidine motif was added at the N terminus to allow separation by immobilized metal affinity chromatography of unmodified phage from phage whose peptides had been cleaved releasing the hexahistidine tag. 256 CX10C T7 phage display library 10 Maria A. S. Pinhal Completely random NNK Circular 257 TN phage display library pool 12-18 Dyax Corp Semi-random Three disulfide-constrained cyclic peptide phage display libraries (TN6-6, TN10-9, and TN12-1) were pooled in approximately equal amounts. All of the cyclic peptide libraries (TN6-6, TN10-9, and TN12-1) were constructed in same manner, i.e. in a derivative of M13mp18, MANP, having the following modifications: bla (AmpR) gene, modified junction between signal of iii and coding region for mature III, and removal of LacZ complementation system. MANP comprises 8164 bases. The bla gene was from pGEM3Zf1(+), was bound by BamHI-HindIII sites at the 5' end and HindIII-SalI sites at the 3' end, replaced bases 6001 through 6432 of M13mp18, and was in the same orientation as the phage genes. The junction between the III signal sequence and mature III was modified with an NcoI restriction site embedded in the last three codons of the signal sequence. After the III signal, MANP contained the sequence of BPTI with restriction sites for the enzymes StyI, XhoI, PflMI, ApaI, Bsp120I, EcoO109I, PspOMI, BssHII, StuI, BstZ17I, BlpI, EagI, and SphI, which were all unique within MANP. Following BPTI, a unique PstI site and a Factor Xa cleavage site were inserted in a short linker region before mature III. The DNA encoding the library was synthesized with constant DNA on either side so that the DNA could be PCR amplified using TAQ DNA polymerase, cleaved with NcoI and PstI, and ligated to similarly cleaved vector. The variegated parts were synthesized with TRIM technology, which incorporates trinucleotides and allows mixtures of any set of amino acid types in any desired proportions. The flanking and varied DNA sequence positions of TN10-9 phage library is depicted as "AEGTGS-X1X1X2 CX3X3X3X3X3X3X3X3C X2X1X1-APGPTDS", where X1 belongs to any residue of DFHLNPRSWY, X2 denotes any residue of ADFGHLNPQRSVWY, and X3 is any natural amino acids except cysteine. More information on TN6-6 and TN12-1 phage library, check the library with ID 340 and 341 respectively. 258 Byungheon Lee (Department of Biochemistry and Cell Biology, School Medicine, Kyungpook National University, Republic of Korea) 7 5.0e8 In-San Kim, Byung-Heon Lee Completely random NNK Circular The phage display library is based on T7 415-1b phage vector. 259 T7 phage display library (X2PX4) 7 Shunsuke Kamijo Semi-random Linear The phage display library is based on T7 415-1b phage vector. 260 CX7C fUSE5 phage display library 7 Erkkl Ruoslahti Completely random NNK Circular 261 fUSE5-based X15 phage display library 15 2.5e8 Toshinori Sato Completely random NNK Linear 262 fUSE5-based X10 phage display library 10 1.4e9 R. Mandeville (Institute Armand-Frappier, University of Quebec, Laval, Canada) Completely random NNK Linear 263 X18 T7 phage display library 18 Thomas D. Wang Completely random NNK Linear The T7 library was constructed with the T7Select 10-3b vector as reported in the T7Select System Manual (Novagen, Gibbstown, NJ). 264 X12 M13 phage display library 12 Angela M. Belcher Completely random Linear 265 Phage display library ON159.3 (X12) 12 Stephen Albert Johnston Completely random NNK Linear The library was constructed in the vector fd-tet. 266 ON543 phage display library (X20) 20 Stephen Albert Johnston (Affymax, Palo Alto, CA) Completely random NNK Linear The library was constructed in the vector fd-tet. 267 X9 phage display library 9 R. Cortese (IRBM, Pomezia, Italy) Completely random Linear 268 JCFN-RGD phage display library (XRGDXXXX) 8 1.5e9 Semi-random NNK Linear The phage-display vector for FNfn10, JCFN, was constructed by cloning the FNfn10 gene7 in the modified M13 vector, JC-M13-88. A FNfn10 library, JCFN-RGD, was constructed in such a way that the FG loop has XRGDXXXX sequence where X stands for any amino acid (residues 77–84). Mutagenesis on the JCFN template was performed using an oligonucleotide JCFNFGRGD (GTTAATCGAGATTGGCTTGGAMNNMNNMNNMNNATCGCCGCGMNNAGTAACAGCGTATAC, where N is a mixture of A, G, C and T, and M is a mixture of A and C). 269 X15 T7 phage display library 15 1.7e10 Susumu Kobayashi, Fumio Sugawara, Kengo Sakaguchi Completely random NNK Linear The T7 library was constructed with the T7Select 10-3b vector. 270 X12 phage display library 12 Yasser Perera Completely random Linear 271 XCX(3)SDLX(3)CI phage display library 13 J. K. Scott Semi-random NNK Circular 272 X(7)SDLX(3)CI phage display library 15 J. K. Scott Semi-random NNK Circular 273 X12 T7 phage display library 12 7.0e8 Toshiyuki Mori Completely random NNK Linear The T7 library was constructed with the T7Select 10-3b vector. 274 CX5C T7 phage display library 5 1.2e9 Toshiyuki Mori Completely random NNK Circular The T7 library was constructed with the T7Select 10-3b vector. 275 CX7C T7 phage display library 7 1.4e9 Toshiyuki Mori Completely random NNK Circular The T7 library was constructed with the T7Select 10-3b vector.