Basic Vector Information
- Vector Name:
- pMCSG27
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6362 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
pMCSG27 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 ℃.
pMCSG27 vector Sequence
LOCUS 40924_30136 6362 bp DNA circular SYN 01-JAN-1980
DEFINITION Bacterial vector encoding a C-terminal 10xHis-TVMV-MBP cassette, for
high-throughput purification of recombinant proteins.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6362)
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 6362)
AUTHORS Midwest Center for Structural Genomics
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6362)
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..6362
/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(213..1310)
/codon_start=1
/label=MBP
/note="maltose binding protein from E. coli"
/translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
YAVRTAVINAASGRQTVDEALKDAQT"
CDS complement(1311..1331)
/codon_start=1
/label=TVMV site
/note="tobacco vein mottling virus (TVMV) NIa protease
recognition and cleavage site"
/translation="ETVRFQS"
CDS complement(1335..1361)
/codon_start=1
/label=9xHis
/note="9xHis affinity tag"
/translation="HHHHHHHHH"
RBS complement(1373..1393)
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind complement(1409..1433)
/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(1434..1452)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 1765..1842
/label=lacI promoter
CDS 1843..2922
/codon_start=1
/label=lacI
/note="lac repressor"
/translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
protein_bind 2938..2959
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS 3734..3922
/codon_start=1
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
/translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
misc_feature 4027..4169
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(4355..4943)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5117..5974)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(5975..6078)
/label=AmpR promoter
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