Basic Vector Information
- Vector Name:
- pMCSG25
- Antibiotic Resistance:
- Ampicillin
- Length:
- 5664 bp
- Type:
- Structural Genomics Vectors
- Replication origin:
- ori
- Source/Author:
- Osipiuk J, Mulligan R, Bargassa M, Hamilton JE, Cunningham MA,
- Copy Number:
- High copy number
pMCSG25 vector Vector Map
Plasmid Resuspension Protocol:
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5.Store the plasmid at -20 ℃.
pMCSG25 vector Sequence
LOCUS pMCSG25. 5664 bp DNA circular SYN 01-JAN-1980 DEFINITION Bacterial expression vector encoding an aspartic endopeptidase fused to a 6xHis-TEV leader. ACCESSION . VERSION . KEYWORDS pMCSG25. SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5664) AUTHORS Osipiuk J, Mulligan R, Bargassa M, Hamilton JE, Cunningham MA, Joachimiak A. TITLE Characterization of member of DUF1888 protein family, self-cleaving and self-assembling endopeptidase. JOURNAL J. Biol. Chem. 2012;287:19452-61. PUBMED 22493430 REFERENCE 2 (bases 1 to 5664) AUTHORS Midwest Center for Structural Genomics TITLE Direct Submission REFERENCE 3 (bases 1 to 5664) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J. Biol. Chem."; date: "2012"; volume: "287"; pages: "19452-61" COMMENT SGRef: number: 2; type: "Journal Article" COMMENT For ligation-independent cloning (LIC), linearize with SspI and treat with T4 DNA polymerase plus dGTP. FEATURES Location/Qualifiers source 1..5664 /mol_type="other DNA" /organism="synthetic DNA construct" terminator complement(26..73) /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" CDS complement(140..157) /label=6xHis /note="6xHis affinity tag" CDS complement(223..243) /label=TEV site /note="tobacco etch virus (TEV) protease recognition and cleavage site" CDS complement(268..285) /label=6xHis /note="6xHis affinity tag" CDS complement(292..666) /codon_start=1 /product="self-cleaving aspartic endopeptidase SO1698 from Shewanella oneidensis" /note="aspartic endopeptidase" /translation="MFAPQGLAQFIKVNVTLENGEPVFIYTDANGQVCQGDITVTQAGT ITYLLNDQTLKGLKFVGVGFVTPFDGIIDAVTISSDGMLVQLVDLDKTPGTTKFQFVLS NTANTLLVLSPDPQIINRPQN" RBS complement(674..696) /label=RBS /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" protein_bind complement(711..735) /label=lac operator /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(736..754) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 1067..1144 /label=lacI promoter CDS 1145..2224 /label=lacI /note="lac repressor" protein_bind 2240..2261 /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." CDS 3036..3224 /label=rop /note="Rop protein, which maintains plasmids at low copy number" misc_feature 3329..3471 /label=bom /note="basis of mobility region from pBR322" rep_origin complement(3657..4245) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(4419..5276) /label=AmpR /note="beta-lactamase" promoter complement(5277..5380) /label=AmpR promoter
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