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| 产品名称： | pSIT |
|---|---|
| 商品货号： | TS157033 |
| Designations： | pSIT |
| Depositors： | E Hunter |
| Biosafety Level： | 1 |
| Vector Information： | Size (kb): 6.0999999046325680 Vector: pSIT (phagemid) Promoters: Promoter T7 (phi10) Construction: pSELECT, T7 promoter (pET-11d) Marker(s):tetR Construct size (kb): 6.099999904632568 Features: initiation codon: ATG marker(s): ampS marker(s): tetR operator: lac promoter: T7 (phi10) replicon: f1 replicon: pMB1 repressor gene: lacIq restriction site: BamHI restriction site: EspI restriction site: NcoI ribosome-binding site: T7 (phi10) terminator: T7 (phi10) |
| Applications： | expression vector vector permitting RNA synthesis in vitro vector permitting production of single-stranded DNA vector useful for site-directed mutagenesis |
| Comments： | Restriction digests of the clone give the following sizes (kb): BamHI--6.1; XbaI--6.1; EcoRV--4.35, 1.75. Vector contains the following restriction sites (approximate kb from nt 1): BamHI--0.14; BglI--1.40, 1.63, 4.47, 4.59, 6.04; ClaI--0.49; EcoRV--0.65, 2.40; NcoI--0.11; PvuI--1.86, 4.72, 6.07; PvuII--1.89, 2.16, 2.25; XbaI--0.07. Mutagenesis is achieved through alkali denaturation of the plasmid containing a cloned insert, followed by amplification of the plasmid using an ampicillin repair primer and a mutagenic primer. Single stranded DNA can also be generated by infection with a helper phage such as R408 or M13K07 (ATCC 37468). Following in vitro synthesis of the second DNA strand, the plasmid should be grown in E. coli BMH 71-18 mutS or ES1301 mutS (to avoid repair of the mutation) and can be selected for ampicillin resistance. Clones can then be transformed into a suitable strain for propagation (E. coli JM109 - ATCC 53323) or expression (E. coli BL21 (DE3)). Several rounds of mutation can be achieved by including a tetracycline inactivation primer in the first round, so that tetracycline resistance can be restored and used as a marker for second round mutants. Best mutagenic efficiency was achieved using a 10 fold molar excess of mutagenic primer to ampicillin repair primer. Expression vector allowing site directed mutagenesis of cloned inserts and subsequent selection of mutants for ampicillin resistance. The following oligonucleotides can be used to repair or inactivate the vector markers: ampicillin repair, 5-GTTGCCATTGCTGCAGGCATCGTGGTG-3; ampicillin knockout, 5-GTTGCCATTGCGGCATCGTGGTGTCAC-3; tetracyclin repair, 5-GCCGGGCCTCTTGCGGGATATCGTCCA-3; tetracycline knockout, 5-GCCGGGCCTCTTGCGGGCGTCCATTCC-3. Constructed from pSELECT by replacing the lacZ region with a T7 promoter, under control of the lac repressor, and replacing the rop copy number control sequence with the lacIq repressor gene. |
| Media： | ATCC® Medium 1273: LB medium (ATCC medium 1065) with 20 mcg/ml tetracycline |
| Growth Conditions： | Temperature: 37.0°C |
| References： | Andreansky M, Hunter E. Phagemid pSIT permits efficient in vitro mutagenesis and tightly controlled expression in E. coli. BioTechniques 16: 626-633, 1994. PubMed: 8024782 |
| Shipped： | freeze-dried |