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pRE112自杀质粒细菌同源置换载体 BioVector NTCC质粒载体菌种细胞基因保藏中心

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  • 货  号:pRE112自杀质粒细菌同源置换载体
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pRE112自杀质粒细菌同源置换载体

BioVector NTCC质粒载体菌种细胞基因保藏中心                


    Vector backbone pRE107 (modified from pGP704)
(Search Vector Database)
Backbone manufacturer Addgene plasmid 43829
Backbone size (bp) 5173
Modifications to backbone From pGP704: SacB1 from pUC58-sacB1 cloned into the EcoRI site. BamHI site in oriV converted to a ClaI site by partial digestion and Klenow treatment. From pRE107: Ampicillin resistance replaced with Chloramphenicol resistance (from pACYC184) by blunted-BamHI digestion.
Vector type Bacterial Expression ; Bacterial allelic exchange vector with sacB1
Selectable markers SacB (sucrose sensitivity)
GROWTH IN BACTERIA

Bacterial Resistance(s) Chloramphenicol
Growth Temperature 37°C
Growth Strain(s) SM10 λpir
Growth instructions Contains lambda pir-dependent R6K replication origin; requires lambda pir-containing bacteria strain
Copy number Low Copy
CLONING INFORMATION

Cloning method Restriction Enzyme
5′ sequencing primer CAT-F
(Common Sequencing Primers)
RESOURCE INFORMATION

Supplemental Documents
pRE112 annotated Genbank file
A portion of this plasmid was derived from a plasmid made by SM10 λpir bacterial strain and pGP704 plasmid backbone from John Mekalanos, Harvard Medical School, Boston, MA. SacB1 gene from plasmid pUC58-sacBI from Dennis Ohman, VCU Medical Center, Richmond, VA. CmR gene from pACYC184 (standard cloning vector - commercially available)
Terms and Licenses
UBMTA
DEPOSITOR COMMENTS

The plasmids deposited here comprise a set of SacB1-dependent allelic exchange vectors improved from a previously described suicide vector, pGP704 (Miller and Mekalanos, 1988), by including a system to select for plasmid loss by recombination.

Plasmid pRE107 (Addgene plasmid #43829) was constructed by cloning the appropriate EcoRI fragment from pUC58-sacB1 (McIver et al., 1995) into pGP704 (see associated schematic image). The sacB1 allele is a modified variation of sacB, where unique restriction sites were removed by site directed mutagenesis (McIver et al., 1995). Additionally, the BamHI site in the R6K ori of the resulting plasmid was removed by partial BamHI digestion, blunting the resulting overhangs with PolIk (resulting in the formation of a ClaI site) and screening for its loss by restriction analysis.

To use this plasmid with bla fusions and increase the functionality of this system, the depositing laboratory replaced the ApR gene as follows.

The ApR gene flanked by the remaining BamHI sites was removed and replaced with either CmR, TcR or KmR resistance markers (see associated schematic image).

The CmR gene was PCR-amplified from pACYC184 using two primers

5′-CATGGTACCCGGGCCCTAAATACCTGTGACGGAAGAT-3′
and
5′-AACTGCAGACCCGGGCCCTATCACTTATTCAGGCGTAGC-3′

and standard conditions to produce a 958-bp fragment with overlapping SmaI/ApaI sites. This fragment was cloned into the blunted BamHI sites to produce plasmid pRE112.

The resulting plasmid together with others in this deposited series contain the conditional R6K ori, the origin of transfer (oriT) which allows conjugative transfer from permissive hosts, the sacB1 gene to provide negative selection and a MCS, along with a range of different AbR markers.
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