pGFPuv原核荧光表达载体 BioVector NTCC中国质粒载体菌种细胞基因保藏中心

pGFPuv原核荧光表达载体 BioVector NTCC中国质粒载体菌种细胞基因保藏中心 pGFPuv原核荧光表达载体，lac启动子，GFPuv荧光基因编码cycle 3型GFP绿色荧光蛋白，Amp抗性。 pGFPuv carries the “cycle 3” variant of GFP described by Crameri et al. (1). This gene was cloned between the two MCSs of the pUC19 derivative pPD16.43 (2). The GFPuv gene can be easily excised from pGFPuv. Alternatively, the GFPuv coding sequence can be amplified by PCR. The GFPuv gene was inserted in frame with the lacZ initiation codon from pUC19 so that a β-galactosidase-GFPuv fusion protein is expressed from the lac promoter in E. coli . Note, however, that if you excise the GFPuv coding sequence using a restriction site in the 5' MCS, the resulting fragment will encode the native (i.e., non-fusion) GFPuv protein. The pUC backbone of pGFPuv provides a high copy number origin of replication and ampicillin resistance gene for propagation in E. coli. Location of features: • lac promoter: 95–178 CAP binding site: 111–124 –35 region: 143–148; –10 region: 167–172 Transcription start point: 179 lac operator: 179–199 • lacZ-GFPuv fusion protein expressed in E. coli Ribosome binding site: 206–209 Start codon (ATG): 217–219; Stop codon: 1003–1005 • 5' MCS: 234–281 • GFPuv gene Start codon (ATG): 289–291; Stop codon: 1003–1005 GFP chromophore: 481–489 wt GFP cDNA sequences (3): 289–454 Synthetic GFP gene with "cycle 3" mutations from pBAD-GFPuv (1): 455–1007 Cycle 3 mutation F99S (T→C): 584 Cycle 3 mutation M153T (T→C): 7Cycle 3 Cycle 3 mutation V163A (T→C): 776 Cycle 3 silent mutation in L137 (T→C): 699 Cycle 3 silent mutation in T225 (A→T): 963 Q80R mutation (A→G) (4): 527 Arg codons optimized for E. coli: R73 (AgA→CgT): 505–507, R96 (AgA→CgC): 574–576, R12(AgA→CgT): 652–654, R168 (AgA→CgC): 790–792, R1215 (AgA→CgT): 931–933 Silent mutations (CccA→TccG) creating BspE I site: 510 & 513 Silent mutation (A→G) creating Mlu I site: 612 Silent mutations (TtGgaA→CtCgaG) creating Xho I site: 709, 711 & 714 Silent mutations (AG→TC) creating BamH I site: 811–812 Silent mutation (C→G) creating Sal I site: 894 Silent mutations (ActA→GctC) creating Sac I site: 993 & 996 Silent mutation in S72 (A→C): 504 • 3' MCS: 107–1091 • Ampicilin resistance gene Promoter: –35 region: 1467–1472; –10 region: 1490–1495 Transcription start point: 1502 Ribosome binding site: 1525–1529 β-lactamase coding sequences: Start codon (ATG): 1537–1539; Stop codon: 2395–2397 β-lactamase signal peptide: 1537–1605 β-lactamase mature protein: 1606–2394 • pUC plasmid replication origin: 2545–3188 Primer Location: • GFP-N Sequencing Primer: 331–352 (Note: The GFP-C Sequencing Primer cannot be used with pGFPuv.) Propagation in E. coli: • Recommended host strain: JM109 or DH5α • Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) on E. coli hosts. • E. coli replication origin: pUC • Copy number: ≈500 • Plasmid incompatibility group: pMB1/ColE1 References: 1. Crameri, A., et al. (1996) Nature Biotechnol. 14:315–319. 2. Fire, A., et al. (1990) Gene 93:189–198. 3. Prasher, D. C., et al. (1992) Gene 111:229–233. 4. Chalfie, M., et al. (1994) Science 263:802–805. BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心www.biovector.net [Supplier来源] http://www.biovector.net