Basic Vector Information
- Vector Name:
- pMCSG19C
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7468 bp
- Type:
- Structural Genomics Vectors
- Replication origin:
- ori
- Source/Author:
- Donnelly MI, Zhou M, Millard CS, Clancy S, Stols L, Eschenfeldt WH,
- Copy Number:
- High copy number
pMCSG19C 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 ℃.
pMCSG19C vector Sequence
LOCUS 40924_30096 7468 bp DNA circular SYN 01-JAN-1980 DEFINITION Bacterial expression vector with an MBP-TVMV-6xHis-TEV leader and inducible TVMV protease. See also pMCSG19B. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7468) AUTHORS Donnelly MI, Zhou M, Millard CS, Clancy S, Stols L, Eschenfeldt WH, Collart FR, Joachimiak A. TITLE An expression vector tailored for large-scale, high-throughput purification of recombinant proteins. JOURNAL Protein Expr. Purif. 2006;47:446-54. PUBMED 16497515 REFERENCE 2 (bases 1 to 7468) AUTHORS Midwest Center for Structural Genomics TITLE Direct Submission REFERENCE 3 (bases 1 to 7468) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Protein Expr. Purif."; date: "2006"; volume: "47"; pages: "446-54" 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..7468 /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(223..243) /codon_start=1 /label=TEV site /note="tobacco etch virus (TEV) protease recognition and cleavage site" /translation="ENLYFQS" CDS complement(268..285) /codon_start=1 /label=6xHis /note="6xHis affinity tag" /translation="HHHHHH" CDS complement(286..306) /codon_start=1 /label=TVMV site /note="tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site" /translation="ETVRFQS" protein_bind complement(309..333) /label=attB1 /note="recombination site for the Gateway(R) BP reaction" CDS complement(343..1443) /codon_start=1 /label=MBP /note="maltose binding protein from E. coli" /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQT" RBS complement(1451..1473) /label=RBS /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" protein_bind complement(1488..1512) /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(1513..1531) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 1830..1903 /label=PLtetO-1 promoter /note="modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)" CDS 1927..2634 /codon_start=1 /label=TVMV protease /note="tobacco vein mottling virus NIa protease (Nallamsetty et al., 2004)" /translation="MSKALLKGVRDFNPISACVCLLENSSDGHSERLFGIGFGPYIIAN QHLFRRNNGELTIKTMHGEFKVKNSTQLQMKPVEGRDIIVIKMAKDFPPFPQKLKFRQP TIKDRVCMVSTNFQQKSVSSLVSESSHIVHKEDTSFWQHWITTKDGQCGSPLVSIIDGN ILGIHSLTHTTNGSNYFVEFPEKFVATYLDAADGWCKNWKFNADKISWGSFTLVEDAPE DDFMAKKTVAAIMD" terminator 2670..2756 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" promoter 2871..2948 /label=lacI promoter CDS 2949..4028 /codon_start=1 /label=lacI /note="lac repressor" /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" protein_bind 4044..4065 /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." CDS 4840..5028 /codon_start=1 /label=rop /note="Rop protein, which maintains plasmids at low copy number" /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" misc_feature 5133..5275 /label=bom /note="basis of mobility region from pBR322" rep_origin complement(5461..6049) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(6223..7080) /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(7081..7184) /label=AmpR promoter
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