Basic Vector Information
- Vector Name:
- pMCSG33
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6457 bp
- Type:
- Structural Genomics Vectors
- Replication origin:
- ori
- Source/Author:
- Eschenfeldt WH, Maltseva N, Stols L, Donnelly MI, Gu M, Nocek B, Tan
- Copy Number:
- High copy number
pMCSG33 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 ℃.
pMCSG33 vector Sequence
LOCUS 40924_30166 6457 bp DNA circular SYN 01-JAN-1980 DEFINITION Bacterial vector encoding N-terminal 10xHis-MBP-TEV and C-terminal TEV-6xHis, for high-throughput purification of recombinant proteins. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6457) AUTHORS Eschenfeldt WH, Maltseva N, Stols L, Donnelly MI, Gu M, Nocek B, Tan K, Kim Y, Joachimiak A. TITLE Cleavable C-terminal His-tag vectors for structure determination. JOURNAL J. Struct. Funct. Genomics 2010;11:31-9. PUBMED 20213425 REFERENCE 2 (bases 1 to 6457) AUTHORS Midwest Center for Structural Genomics TITLE Direct Submission REFERENCE 3 (bases 1 to 6457) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J. Struct. Funct. Genomics"; date: "2010"; volume: "11"; pages: "31-9" COMMENT SGRef: number: 2; type: "Journal Article" COMMENT For ligation-independent cloning (LIC), linearize with SmaI and treat with T4 DNA polymerase plus dATP. FEATURES Location/Qualifiers source 1..6457 /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) /codon_start=1 /label=6xHis /note="6xHis affinity tag" /translation="HHHHHH" CDS complement(208..225) /codon_start=1 /label=6xHis /note="6xHis affinity tag" /translation="HHHHHH" CDS complement(232..252) /codon_start=1 /label=TEV site /note="tobacco etch virus (TEV) protease recognition and cleavage site" /translation="ENLYFQS" CDS complement(263..283) /codon_start=1 /label=TEV site /note="tobacco etch virus (TEV) protease recognition and cleavage site" /translation="ENLYFQS" CDS complement(305..1402) /codon_start=1 /label=MBP /note="maltose binding protein from E. coli" /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW YAVRTAVINAASGRQTVDEALKDAQT" CDS complement(1430..1456) /codon_start=1 /label=9xHis /note="9xHis affinity tag" /translation="HHHHHHHHH" RBS complement(1467..1489) /label=RBS /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" protein_bind complement(1504..1528) /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(1529..1547) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 1860..1937 /label=lacI promoter CDS 1938..3017 /codon_start=1 /label=lacI /note="lac repressor" /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" protein_bind 3033..3054 /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." CDS 3829..4017 /codon_start=1 /label=rop /note="Rop protein, which maintains plasmids at low copy number" /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" misc_feature 4122..4264 /label=bom /note="basis of mobility region from pBR322" rep_origin complement(4450..5038) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(5212..6069) /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(6070..6173) /label=AmpR promoter
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