cat Vectors for Promoter Analysis pKK232-8 (27-4925-01) Cloning: Multiple cloning site contains four unique restriction sites. Promoter analysis: Fragments containing a promoter inserted into the multiple cloning site serve as promoter for the promoterless CAT gene. Promoter strength is measured by transcription of CAT. Levels are dependent on the strength of the inserted promoter. Host(s): E. coli. Selectable marker(s): Plasmid confers resistance to 100 ug/ml ampicillin. Amplification: Recommended. pCM7 (27-4931-01) Promoter analysis: See gene cartridge below. Gene cartridge: The CAT cartridge can be excised from the parent plasmid and inserted behind an upstream promoter sequence to give rise to chloramphenicol-resistant transformants. Level of resistance is relative to promoter strength. Host(s): E. coli. Selectable marker(s): Plasmid confers resistance to 100 ug/ml ampicillin. Amplification: Recommended. cDNA Cloning Vectors pExCell Description: pExCell is the phagemid which is released from the Lambda ExCell Cloning Vectors (27-5011-01, 27-5013-01). pExCell contains an MCS with seven unique sites: Xho I, Sfi I, EcoR I, BamH I, Not I, Mlu I, Hind III. pExCell is not available separately. It is only obtained after in vivo release from one of the Lambda ExCell Cloning Vectors. Transcription: Opposed T7 and SP6 promoters allow in vitro transcription of both strands. Sequencing: M13 Universal Sequencing Primer and M13 Reverse Sequence Primer allow sequencing of both strands. Note: M13 Reverse Sequence Primer from Pharmacia will work in this capacity, but reverse primers from other suppliers may not. Prokaryotic expression: In-frame inserts give beta-galactosidase fusion proteins. Expression can be induced with 1-5 mM IPTG. Single-strand DNA: The (+) strand is produced upon infection with M13KO7 helper phage. Host: E. coli NM522. Selectable marker: Plasmid confers resistance to 100 mg/ml ampicillin. Phagemid Directional Cloning Vector (27-4987-01) Directional cloning: Plasmid is provided EcoR I and Not I cut and dephosphorylated for the cloning of directional cDNA produced with TimeSaver cDNA Synthesis Kit and the Directional Cloning Toolbox. Transcription: Opposed T3 and T7 promoters allow in vitro transcription of both strands. Sequencing: M13 Universal Sequencing Primer and M13 Reverse Sequence Primer allow sequencing of both strands. Note: Pharmacia P-L M13 Reverse Sequence Primer will work in this capacity, but reverse primers from other suppliers may not. Prokaryotic expression: Inserts cloned in frame are expressed as fusions with beta galactosidase. Induce the lac promoter with 1-5 mM IPTG. Single-stranded DNA: The (+) strand is produced when host cell is infected with M13KO7 helper phage. A protocol for production of single- stranded DNA is provided with the vector. Host(s): lac Iq strains. NM522 has consistently produced high levels of single-stranded DNA. Selectable marker(s): Plasmid confers resistance to 100 ug/ml ampicillin. Amplification: Recommended. pT7T3 18U EcoR I/BAP (27-4982-01) Transcription: Opposed T3 and T7 promoters allow in vitro transcription of both strands. Sequencing: M13 Universal Sequencing Primer and M13 Reverse Sequence Primer allow sequencing of both strands. Note: Pharmacia P-L M13 Reverse Sequence Primer will work in this capacity, but reverse primers from other suppliers may not. Prokaryotic expression: Inserts cloned in frame are expressed as fusions with beta galactosidase induce the lac promoter with 1-5 mM IPTG. Cloning: Plasmid is provided EcoR I cut and dephosphorylated. Recombinants appear as white colonies when grown on media containing X-gal. Single-stranded DNA: The (+) strand is produced when host cell is infected with M13KO7 helper phage. A protocol for production of single- stranded DNA is provided with the vector. Host(s): lac Iq strains. NM522 has consistently produced high levels of single-stranded DNA. Selectable marker(s): Plasmid confers resistance to 100 ug/ml ampicillin. Amplification: Recommended. Eukaryotic Expression Vectors pSVK3 (27-4511-01) Expression: Expression is controlled by the uninducible SV40 early promoter. Expression is transient, but can be stable if the gene inserted is selectable. Sequencing: Both double-stranded and single-stranded [(+) strand] sequencing are possible. A protocol for production of single-stranded DNA is provided with the vector. In vitro transcription: A T7 RNA polymerase promoter allows for in vitro transcription of cloned inserts. Host(s): E. coli and mammalian cells (including CV-1, cos and HeLa cells). Selectable marker(s): Plasmid confers resistance to 100 ug/ml ampicillin in E. coli. Relevant Control Regions SV40 origin and early promoter region Minimal region for replication: 239-314; TATA box: 252-258; 21-bp repeats: 170-232; 72-bp repeats: 23-166; Transcription start region: 279-286 MCS: 377-435 SV40 early splice region Small T antigen intron: 630-695 SV40 polyadenylation region PolyA signal sequence: 1331-1336; PolyA site: 1352 T7 promoter: 352-374 beta-lactamase gene region: Promoter region: -10: 2017-2022; -35: 1994-1999; Start codon (ATG): 2064; Stop codon (TTA): 2922 Plasmid replication region: origin of replication: 3683-3685; Region necessary for replication: 2989-3685 f1 region: 1478-1932; Origin of f1 replication: 1770 pBPV (27-4930-01) Expression: Stable expression in eukaryotes is under the control of an expression unit consisting of an enhancer from the long terminal repeat of the Moloney murine sarcoma virus and the promoter from the mouse metallothionein I gene. This promoter is not inducible. Episomal maintenance: pBPV is maintained episomally in eukaryotic cells at a copy number of 20-150 molecules per cell. Host(s): E. coli recA- and mammalian cells [including C127 and 3T3 mouse cells, and Fisher rat fibroblasts (FR3T3). C127 cells produce the most easily identified transformed foci]. Selectable marker(s): The plasmid confers resistance to 100 ug/ml ampicillin in E. coli. Transformed eukaryotic cells form easily identifiable foci. pMSG (27-4506-01) Induction: Multiple cloning site is located downstream of the MMTV promoter, which is inducible with 0.1 uM dexamethasone. Genes inserted into the multiple cloning site are translated from the first ATG of the insert. Selection in eukaryotes: The E. coli gpt gene is under control of the SV40 early promoter. Cells with a functional gpt gene are resistant to mycophenolic acid. Stable expression: pMSG will integrate randomly into the host genome under selective pressure of the mycophenolic acid. Transient expression: After transfection, transient expression is inducible with dexamethasone. Host(s): E. coli recA-; pMSG contains regions of internal homology. Use with some bacterial strains may lead to deletions within the vector. We recommend E. coli HB101. Mammalian cells, including mouse 3T3, CHO, BHK and HeLa cells. For induction, mammalian cells must be responsive to glucocorticoid compounds. Selectable marker(s): Plasmid confers resistance to 100 ug/ml ampicillin in E. coli. Stably transformed mammalian cells are resistant to mycophenolic acid. Amplification: Recommended. Relevant Control Regions MCS: 1-23 MMTV LTR region LTR: 6178-7492; Hormone response element: 7122-7306; Approximate site for transcription initiation: 7358 SV40 early splice region Small T antigen intron: 101-165 SV40 polyadenylation region: PolyA signal sequence: 801-806 PolyA site: 822 SV40 origin and early promoter region Minimal region for replication: 1116-1191; TATA box: 1129-1135; 21-bp repeats: 1047-1109; 72-bp repeats: 900-1043; Transcription initiation region: 1156-1163 gpt gene region gpt gene: 1218-2279; Start codon (ATG): 1416; Stop codon (TAA): 1872 SV40 early splice region Small T antigen intron: 2351-2415 SV40 polyadenylation region PolyA signal sequence: 3051-3056; PolyA site: 3072 beta-lactamase gene region Promoter region: -10: 4036-4041; -35: 4013-4018; Start codon (ATG): 4083; Stop codon (TAA): 4941 Plasmid replication region: Site of replication initiation: 5702-5704; Region necessary for replication: 5008-5704 pMSG-CAT (27-4505-01) Similar to pMSG, but includes the CAT gene inserted downstream from the MMTV LTR to provide an easily assayed marker. It is a positive control vector for monitoring expression from the MMTV LTR. Amplification: Recommended. Relevant control regions MMTV LTR region LTR: 6957-8271; Hormone response element: 7901-8085; Approximate site for transcription initiation: 8137 CAT gene region CAT gene: 17-791; Start codon (ATG): 46; Stop codon (TAA): 703 SV40 early splice region Small T antigen intron: 880-944 SV40 polyadenylation region: PolyA signal sequence: 1580-1585; PolyA site: 1601 SV40 origin and early promoter region Minimal region for replication: 1895-1970; TATA box: 1908-1914; 21-bp repeats: 1826-1888; 72-bp repeats: 1659-1822; Transcription initiation region: 1935-1942 gpt gene region gpt gene: 2002-3058; Start codon (ATG): 2195; Stop codon (TAA): 2651 SV40 early splice region: Small T antigen intron: 3130-3194 SV40 polyadenylation region: PolyA signal sequence: 3830-3835; PolyA site: 3851 beta-lactamase gene region: Promoter region: -10: 4815-4820; -35: 4792-4797; Start codon (ATG): 4862; Stop codon (TAA): 5720 Plasmid replication region: Site of replication initiation: 6481-6483; Region necessary for replication: 5787-6483 pSVL (27-4509-01) Expression: Transient expression is under the control of the SV40 late promoter. Genes inserted into the multiple cloning site are expressed from the first ATG. Contains termination, splicing, and polyadenylation sequences. Host(s): E. coli and mammalian cells (the highest level of expression is found in T-antigen producing cells, e.g. COS cells). Selectable marker(s): Plasmid confers resistance to 100 ug/ml ampicillin in E. coli. Can isolate stable transformants in eukaryotes if the inserted gene is selectable. Amplification: Recommended. Relevant Control Regions SV40 origin and late promoter region: Minimal region for replication: 4824-34; 21-bp repeats: 41-103; 72-bp repeats: 107-250; Major transcription start site: 325 VP1 region: VP1 leader sequence: 323-525; VP1 intron: 526-1493 MCS: 1506-1535 SV40 polyadenylation region: PolyA signal sequence: 1654-1659; PolyA site: 1671 beta-lactamase gene region: Promoter region: -35: 4230-4225; -10: 4207-4202; Start codon (ATG): 4160; Stop codon (TAA): 3302 Plasmid replication region: Site of replication initiation: 2541-2539; Region necessary for replication: 3235-2539 pCH110 (27-4508-01) Expression: Transient expression of b-galactosidase is readily assayable in eukaryotic cells and provides an internal marker for monitoring expression. Expression in eukaryotes is from the SV40 early promoter, and from the E. coli gpt promoter in prokaryotes. Promoter screening: Insertion of a promoter into the Hind III site allows monitoring of beta galactosidase expression. Host(s): E. coli and mammalian cells (including COS monkey cells, and mouse Ltk- cells). Selectable marker(s): Plasmid confers resistance to 100 ug/ml ampicillin in E. coli. Amplification: Recommended. Relevant control regions SV40 origin and early promoter region: Minimal region for replication: 7098-7023; TATA box: 7036-7042; 21-bp repeats: 6954-7017; 72-bp repeats: 6807-6950; Transcription start region: 7063-7070 gpt gene region: gpt gene: 7-208; TATAA: 13-17; Start codon (ATG): 85 trpS gene region: 209-292 lacZ gene region: lacZ gene: 295-3605; Stop codon (TAA): 3340 SV40 polyadenylation region: PolyA signal sequence: 3666-3671; PolyA site: 3687 beta-lactamase gene region: Promoter region: -10: 4651-4656; -35: 4628-4633; Start codon (ATG): 4698; Stop codon (TAA): 5556 Plasmid replication region: Site of replication initiation: 6317-6319; Region necessary for replication: 5623-6319 General Cloning Vectors M13 Series Cloning: DNA inserted into the multiple cloning site inactivates the lacZ' gene resulting in white plaques when infected cells are grown on media containing X-gal and induced with IPTG (1-5 mM). Double-stranded sequencing: Inserts can be sequenced from both directions using the M13 Universal Sequencing Primer and M13 Reverse Sequence Primer, and from one direction using the M13 Universal Sequencing Primer with single-stranded DNA. Single-stranded sequencing: The M13 Universal Sequencing Primer can be used with single-stranded DNA. A protocol for production of single- stranded DNA is provided with the vector. Host(s): A lac Iq host is recommended. Infection requires a host with an F' factor, such as JM105 or NM522. Selectable marker(s): None pUC Plasmids Cloning: DNA inserted into the multiple cloning site inactivates the lacZ' gene resulting in white colonies when transformed cells are grown on media containing X-gal. Expression: DNA inserted in frame with the lacZ' gene will be expressed as a fusion protein under the control of the lac promoter. The lac promoter is inducible with 1-5 mM IPTG, and contains both an initiation codon and ribosome-binding site. Sequencing: Inserts can be sequenced from both directions using the M13 Universal Sequencing Primer and M13 Reverse Sequence Primer. Host(s): A lac Iq host is required for blue/white selection. Selectable marker(s): Plasmid confers resistance to 100 ug/ml ampicillin. pBR 322 (27-4902-01, 27-4902-02) Cloning: More than 30 unique sites exist in the plasmid. Negative selection is possible by cloning into a site that inactivates either of the selectable markers. Sequencing: Double-stranded sequencing. Host(s): E. coli. Selectable marker(s): Plasmid confers resistance to 100 ug/ml ampicillin and to 15 ug/ml tetracycline. Amplification: Recommended. Multifunctional Phagemids and Transcription Vectors pSL1180/1190 Superlinker Phagemids (pSL1180 27-4384-01, pSL1190 27-4386-01) Cloning: Blue/white selection is not possible. Sequencing: The M13 Universal Sequencing Primer can be used depending on cloning site chosen in the superlinker. If the cloning site is too far from the primer site, sequencing results will not be satisfactory. See Brosius, J., DNA 8, 759 (1989) for useful primer sequences. Double- stranded sequencing is possible using the M13 -40 Sequencing Primer and M13 Reverse Sequence Primer. Note: The M13 Reverse Sequence Primer from Pharmacia P-L will work in this application, but primers from other suppliers may not, due to a single-base mutation present in the pSL series. Single-stranded DNA: The (+) strand is produced when the host cell is infected with M13KO7 helper phage. A protocol for production of single- stranded DNA is provided with the vector. Host(s): E. coli. F' factor is required for infection with M13KO7 helper phage. Selectable marker(s): Plasmid confers resistance to 100 ug/ml ampicillin. Useful applications: Useful as a source of restriction sites for vector construction, and also for procedures requiring digestion with successive enzymes (e.g. Maxam and Gilbert sequencing). pTZ18R/19R (pTZ18R 27-4984-01, pTZ19R 27-4986-01) Cloning: Recombinants appear as white colonies when grown on media containing X-gal. Transcription: T7 promoter allows transcription with T7 RNA polymerase. Sequencing: Sequencing of the reverse strand is possible with the M13 Reverse Sequence Primer. Expression: Inserts cloned in frame are expressed as fusions with beta galactosidase. The lac promoter is inducible with 1-5 mM IPTG. Single-stranded DNA: The reverse strand is produced when host cell is infected with M13KO7 helper phage. A protocol for production of single-stranded DNA is provided with the vector. Host(s): lac Iq strains. NM522 has consistently produced high levels of single-stranded DNA. Selectable marker(s): Plasmid confers resistance to 100 ug/ml ampicillin. Amplification: Recommended. Cloning: Recombinants appear as white colonies when grown on media containing X-gal. pT7T3 18U/19U (pT3T318U 27-3512-01, pT7T319U 27-3513-01) Transcription: Opposed T3 and T7 promoters allow in vitro transcription of both strands. Sequencing: M13 Universal Sequencing Primer and M13 Reverse Sequence Primer allow sequencing of both strands. Note: Pharmacia P-L M13 Reverse Sequence Primer will work in this capacity, but reverse primers from other suppliers may not. Expression: Inserts cloned in frame are expressed as fusions with beta galactosidase. The lac promoter is inducible with 1-5 mM IPTG. Single-stranded DNA: The (+) strand is produced when host cell is infected with M13KO7 helper phage. A protocol for production of single- stranded DNA is provided with the vector. Host(s): lac Iq strains. NM522 has consistently produced high levels of single-stranded DNA. Selectable marker(s): Plasmid confers resistance to 100 ug/ml ampicillin. Amplification: Recommended. Selection in prokaryotes: Plasmid confers resistance to 100 ug/ml ampicillin. Prokaryotic Expression Vectors pTrc 99A (21-5007-01) Induction: The trc promoter is inducible with 1-5 mM IPTG. The trc promoter contains the trp -35 region and lac UV5 -10 region spaced apart by 17 base pairs. This promoter is very strong; uninduced cells may show a low level of expression. Expression: Nco I linker supplies an ATG start codon in all three reading frames for expression of insert. Vector provides a ribosome-binding site. Host(s): E. coli. The plasmid provides lac Iq repressor. Selectable marker(s): Plasmid confers resistance to 100 ug/ml ampicillin. Amplification: Recommended. Relative control regions: Expression control region: trc promoter: -10: 216-222; -35: 193-198 Ribosome binding site: 255 MCS: 265-314 rrnB operon region: 5S rRNA region: 409-528; rrnB T1 terminator: 532-575 rrnB T2 terminator: 707-734 beta-lactamase gene region: Promoter: -10: 799-804; -35: 776-781; Start codon (ATG): 846; Stop codon (TAA): 1704 lacIq gene region: Start codon (GTG): 3055; Stop codon (TGA): 4135 Plasmid replication region: Site of replication initiation: 2465-2467; Region necessary for replication: 1771-2467 pKK223-3 (27-4935-01) Induction: The tac promoter is inducible with 1-5 mM IPTG. This promoter is very strong; uninduced cells may show a low level of expression. Expression: Genes containing a ribosome-binding site and ATG can be inserted into any unique site in the MCS for expression. The ribosome-binding site on the plasmid can be utilized if the ATG on the gene is within 5-13 base pairs from the provided ribosome-binding site. Transcription terminators: rrnB transcription terminators stabilize the plasmid, allowing normal replication. Host(s): lac Iq strains. Selectable marker(s): Plasmid confers resistance to 100 ug/ml ampicillin. Amplification: Recommended. Relevant Control Regions: Expression control region: tac promoter: -10: 50-44; -35: 72-67; Ribosome binding site: 11 MCS: 4552-3 rrnB operon region: 5S rRNA region: 4471-4352; rrnB T1 terminator: 4348-4305; rrnB T2 terminator: 4173-4146 beta-lactamase gene region: Promoter: -10: 4082-4077; -35: 4105-4100; Start codon (ATG): 4035; Stop codon (TAA): 3178 Plasmid replication region: Site of replication initiation: 2417-2415; Region necessary for replication: 3111-2415 pPL-Lambda (27-4946-01) Induction: The lambda PL promoter is tightly controlled and induced by shifting the growth temperature from 29C to 42C. Expression: If the insert does not contain its own ribosome-binding site and start codon, a fusion protein is produced. If the insert contains its own ribosome-binding site, start codon and stop codons in all three reading frames upstream of the insert's ribosome-binding site, the product will not be a fusion protein. Note: If the upstream BamH I site is removed with Sma I and the vector religated, it is possible to clone into the downstream BamH I site. In this case a ribosome-binding site and start codon must be included with the insert and a fusion protein will not be produced. Host(s): E. coli N99cI+ with the wild-type lambda repressor is the amplification host. The expression host is E. coli N4830-1 which contains the temperature-sensitive lambda repressor. Note: Expression is also possible in E. coli N99cI+ after induction with nalidixic acid as described in Mott, J., et al., Proc. Natl Acad. Sci. USA 82, 88 (1985). Selectable marker(s): Plasmid confers resistance to 100 ug/ml ampicillin. pDR540 (27-4932-01) Induction: The tac promoter is inducible with 1-5 mM IPTG. Expression: Proteins are expressed as fusions with gal K protein. Expression is from the first ATG of the insert. Host(s): lac Iq strains. Selectable marker(s): Plasmid confers resistance to 100 ug/ml ampicillin. Amplification: Recommended. Prokaryotic Gene Fusion Vectors GST Gene Fusion System Induction: tac promoter inducible with 1-5 mM IPTG. Expression: Proteins are expressed as fusion proteins with the 26-kDa glutathione S-transferase (GST). The GST gene contains an ATG and ribosome- binding site, and is under control of the tac promoter. A translation terminator is provided in each reading frame. The resulting fusion protein may be purified using the GST Purification Module (27-4570-01, -02). Enzymatic cleavage with thrombin: pGEX-1lT, pGEX-2T, pGEX-2TK, pGEX-4T-1, -2, -3 allow for removal of the GST carrier protein from the fusion protein by enzymatic cleavage with thrombin. Enzymatic cleavage with factor Xa: pGEX-3X, pGEX-5X-1, -2, -3 allow for removal of the GST carrier protein from the fusion protein by enzymatic cleavage with factor Xa. Direct labelling in vitro: pGEX-2TK allows for direct labelling of fusion proteins in vitro with 32P using the catalytic subunit of cAMP-dependent protein kinase. Reading frame: pGEX-1 lambda T: GGA TCC CCG GAA TTC ATC GTG ACT GAC TGA pGEX-2T: GGA TCC CCG GGA ATT CAT CGT GAC TGA pGEX-2TK: GGA TCC CCG GGA ATT CAT CGT GAC TGA pGEX-3X: GGG ATC CCC GGG AAT TCA TCG TGA pGEX-4T-1: GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT pGEX-4T-2: GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA pGEX-4T-3: CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC pGEX-5X-1: GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT pGEX-5X-2: GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA pGEX-5X-3: AGG AAT TCC CGG GTC GAC TCG AGC GGC CGC Host(s): E. coli. The plasmid provides lac Iq repressor. Selectable marker(s): Plasmid confers resistance to 100 ug/ml ampicillin. Amplification: Recommended. Relevant Control Regions: All pGEX vectors: Glutathione S-transferase gene region: tac promoter: -10: 205-211; -35: 183-188; lac operator: 217-237; Ribosome binding site for GST: 244; Start codon (ATG) for GST: 258; Primer region for double-stranded sequencing: 874-890 pGEX-1 lambda T (27-4805-01) Coding region for thrombin cleavage: 918-935 MCS: 930-944 beta-lactamase gene region: Promoter: -10: 1308-1313; -35: 1285-1290; Start codon (ATG): 1355; Stop codon (TAA): 2213 lacIq gene region: Start codon (GTG): 3296; Stop codon (TGA): 4376 Plasmid replication region: Site of replication initiation: 2973; Region necessary for replication: 2280-2976 pGEX-2T (27-4801-01) Coding region for thrombin cleavage: 918-935 MCS: 930-945 beta-lactamase gene region: Promoter: -10: 1309-1314; -35: 1286-1291; Start codon (ATG): 1356; Stop codon (TAA): 2214 lacIq gene region: Start codon (GTG): 3297; Stop codon (TGA): 4377 Plasmid replication region: Site of replication initiation: 2974; Region necessary for replication: 2281-2977 pGEX-2TK (27-4587-01) Coding region for thrombin cleavage: 918-935 Coding for kinase recognition site: 936-950 MCS: 951-966 beta-lactamase gene region: Promoter: -10: 1330-1335; -35: 1307-1312; Start codon (ATG): 1377; Stop codon (TAA): 2235 lacIq gene region: Start codon (GTG): 3318; Stop codon (TGA): 4398 Plasmid replication region: Site of replication initiation: 2995; Region necessary for replication: 2302-2998 pGEX-4T-1 (27-4580-01) Coding region for thrombin cleavage: 918-935 MCS: 930-966 beta-lactamase gene region: Promoter: -10: 1330-1335; -35: 1307-1312; Start codon (ATG): 1377; Stop codon (TAA): 2235 lacIq gene region: Start codon (GTG): 3318; Stop codon (TGA): 4398 Plasmid replication region: Site of replication initiation: 2995; Region necessary for replication: 2302-2998 pGEX-4T-2 (27-4581-01) Coding region for thrombin cleavage: 918-935 MCS: 930-967 beta-lactamase gene region: Promoter: -10: 1331-1336; -35: 1308-1313; Start codon (ATG): 1378; Stop codon (TAA): 2236 lacIq gene region: Start codon (GTG): 3319; Stop codon (TGA): 4399 Plasmid replication region: Site of replication initiation: 2996; Region necessary for replication: 2303-2999 pGEX-4T-3 (27-4583-01) Coding region for thrombin cleavage: 918-935 MCS: 930-965 beta-lactamase gene region: Promoter: -10: 1329-1334; -35: 1306-1311; Start codon (ATG): 1376; Stop codon (TAA): 2234 lacIq gene region: Start codon (GTG): 3317; Stop codon (TGA): 4397 Plasmid replication region: Site of replication initiation: 2994; Region necessary for replication: 2301-2997 pGEX-3X (27-4803-01) Coding region for Factor Xa cleavage: 921-932; MCS: 934-949 beta-lactamase gene region: Promoter: -10: 1313-1318; -35: 1290-1295; Start codon (ATG): 1360; Stop codon (TAA): 2218 lacIq gene region: Start codon (GTG): 3301; Stop codon (TGA): 4381 Plasmid replication region: Site of replication initiation: 2978; Region necessary for replication: 2285-2981 pGEX-5X-1 (27-4584-01) Coding region for Factor Xa cleavage: 921-932; MCS: 934-969 beta-lactamase gene region: Promoter: -10: 1333-1338; -35: 1310-1315; Start codon (ATG): 1380; Stop codon (TAA): 2238 lacIq gene region: Start codon (GTG): 3321; Stop codon (TGA): 4401 Plasmid replication region: Site of replication initiation: 2998; Region necessary for replication: 2305-3001 pGEX-5X-2 (27-4585-01) Coding region for Factor Xa cleavage: 921-932; MCS: 934-970 beta-lactamase gene region: Promoter: -10: 1334-1339; -35: 1311-1316; Start codon (ATG): 1381; Stop codon (TAA): 2239 lacIq gene region: Start codon (GTG): 3322; Stop codon (TGA): 4402 Plasmid replication region: Site of replication initiation: 2999; Region necessary for replication: 2306-3002 pGEX-5X-3 (27-4586-01) Coding region for Factor Xa cleavage: 921-932; MCS: 934-971 beta-lactamase gene region: Promoter: -10: 1335-1340; -35: 1312-1317; Start codon (ATG): 1382; Stop codon (TAA): 2240 lacIq gene region: Start codon (GTG): 3323; Stop codon (TGA): 4403 Plasmid replication region: Site of replication initiation: 3000; Region necessary for replication: 2307-3003 pEZZ18 (27-4810-01) Expression: Expression is controlled by both the lacUV5 and protein A promoters and is not inducible. Proteins are expressed as fusions with the synthetic ZZ peptide which is based on an IgG binding domain of protein A. The protein A signal sequence is provided so expression in E. coli leads to secretion of fusion proteins into the culture medium. Elements of the protein A gene provide both the ATG and ribosome-binding site. Stop codons must be provided by the insert. Fusion protein may be purified on IgG Sepharose (17-0969-01). The size of the ZZ carrier is about 14 kDa. Sequencing: M13 Universal Sequencing Primer and M13 -40 Sequencing Primer can be used for both double-stranded and single-stranded sequencing. A protocol for production of single-stranded DNA is provided with the vector. Cloning: Inserts containing a stop codon will yield white colonies when grown on media containing X-gal. Host(s): E. coli. Selectable marker(s): Plasmid confers resistance to 70 ug/ml ampicillin. Amplification: Recommended. Relevant Control Regions Protein A region: Protein A promoter: -10: 2390-2400; -35: 2366-2377; lac promoter: -10: 2168-2174; -35: 2144-2149; operator: 2180-2200; Protein A signal sequence: 2432-2539; Partial E domain: 2540-2557; Synthetic ZZ domain: (Z1) 2558-2731; (Z2) 2732-2905 MCS: 2914-2970 beta-galactosidase (lacZ'): Gene region: 2973-3130; Stop codon (TAA): 3155 f1 replication region: f1 region: 3136-4352; Intergenic region: 3441-3948; origin of f1 replication: 3724 beta-lactamase gene region: Promoter: -10: 154-159; -35: 131-136; Start codon (ATG): 201; Stop codon (TAA): 1059 Plasmid replication region: Site of replication initiation: 1820-1822; Region necessary for replication: 1126-1822 pRIT2T (27-4808-01) Induction: The lambda PR promoter is induced by shifting the growth temperature from 30C to 42C for 90 minutes. Expression: Proteins are expressed as fusion proteins with staphylococcal protein A. The lambda cro gene supplies an ATG. No signal sequence is provided; therefore the protein remains intracellular. Transcription and translation termination signals are provided. Fusion protein may be purified on IgG Sepharose (17-0969-01). The size of the protein A carrier protein is about 30 kDa. Host(s): N4830-1/NC99I+. N4830-1 must be used for expression, as it contains the temperature-sensitive cI857 repressor. Selectable marker(s): Plasmid confers resistance to 100 µg/ml ampicillin. pMC1871 (27-4945-01) Expression: The lac gene is promoterless and missing the first eight non- essential amino acids. Inserts cloned into the Sma I site give fusion proteins with beta galactosidase. Insert must contain a promoter, ATG, and ribosome-binding site. Host(s): E. coli strains carrying a lac deletion. Selectable marker(s): Plasmid confers resistance to 15 ug/ml tetracycline. General Note on transformation: For high level transformationof host cells (E. coli), we recommend the "Hanahan" protocol: Hanahan, D., J. Mol. Biol. 166, 557 (1983). General Information on Promoters lac-based promoters: The promoters trc and tac combine the -35 region from the trp promoter with the -10 region from the lacUV5 promoter. The promoters are of the same relative strength (1) and are 11 times more efficient than lacUV5 and three times more efficient than the trp promoters at directing transcription (2). Both promoters are repressed by the lacI gene product and can be induced by IPTG. The only difference between the trc and tac promoters is the spacing between the -10 and -35 region (1). References 1. Brosius, J. et al., J. Biol. Chem. 260, 3539 (1985). 2. deBoer, H. A. et al., Proc. Natl. Acad. Sci. USA 80, 21 (1983). SV40 promoters: The SV40 regulatory region contains several controlling elements. This 300-bp region contains the viral origin of replication (bp 5202-34*), an A-T rich region (bp 14-31), three G-C rich 21-bp direct repeats (bp 41-103), and two 72-bp direct repeats (bp 107-250). Early promoter function is controlled by the A-T rich region, the 21-bp repeats and the 72-bp repeats (1). The A-T region contains the TATA box sequence "TATTTAT" which fixes the early transcription start site within a narrow area. Initiation will occur 27-34 bp downstream from the first "T" of the TATA sequence (2, 3). The TATA box, however, is dispensable and when deleted, transcription occurs, at about the same level, but initiation sites are altered (2, 3). The 21-bp repeats are required for promoter function, and each contains two copies of the G-C hexanucleotide 5'-GGGCGG-3'. Deletion of all three 21-bp repeats drastically reduces promoter activity, but some promoter function is maintained in the presence of only two G-C hexanucleotides (1). The 72-bp repeats function as enhancer elements and can stimulate transcription at promoters located thousands of base pairs away (4, 5). Late promoter function is controlled by two domains, and efficient induction requires the SV40 Large T antigen (6). One domain consists of the 75-bp minimal region required for viral replication. The other includes the second 72-bp repeat and an adjacent downstream sequence. Absence of either domain decreases transcriptional activity by two orders of magnitude. Transcription initiation occurs at several sites, but the major start site occurs 75 bp downstream from the end of the second 72-bp repeat (i.e. bp 325) (6). *Base pairs are numbered according to reference 7. References 1. McKnight, S., and Tijian, R., Cell 46, 795 (1986). 2. Ghosh, P. K. et al., Proc. Natl. Acad. Sci. USA 78, 100 (1981). 3. Benoist, C., and Chambon, P., Nature 290, 305 (1981). 4. Banerji, J. et al., Cell 27, 299 (1981). 5. Moreau, P. et al., Nucl. Acids Res. 9, 6047 (1981). 6. Hartzell, S. W. et al., Proc. Natl. Acad. Sci. USA 81, 6335 (1984). 7. Tooze, J., ed. in DNA Tumor Viruses. Molecular Biology of Tumor Viruses, Vol. 2 Cold Spring Harbor Laboratory, Cold Spring Harbor (1980).