Basic Vector Information
- Vector Name:
- pMCSG73
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6227 bp
- Type:
- Structural Genomics Vectors
- Replication origin:
- ori
- Source/Author:
- Eschenfeldt WH, Makowska-Grzyska M, Stols L, Donnelly MI,
- Copy Number:
- High copy number
pMCSG73 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 ℃.
pMCSG73 vector Sequence
LOCUS 40924_30361 6227 bp DNA circular SYN 01-JAN-1980 DEFINITION Compact bacterial vector encoding tRNA genes for rare Arg and Ile codons, with a NusA-TVMV-6xHis-Strep-Tag(R) II-TEV leader. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6227) AUTHORS Eschenfeldt WH, Makowska-Grzyska M, Stols L, Donnelly MI, Jedrzejczak R, Joachimiak A. TITLE New LIC vectors for production of proteins from genes containing rare codons. JOURNAL J. Struct. Funct. Genomics 2013;14:135-44. PUBMED 24057978 REFERENCE 2 (bases 1 to 6227) AUTHORS Midwest Center for Structural Genomics TITLE Direct Submission REFERENCE 3 (bases 1 to 6227) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J. Struct. Funct. Genomics"; date: "2013"; volume: "14"; pages: "135-44" 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..6227 /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(155..175) /codon_start=1 /label=TEV site /note="tobacco etch virus (TEV) protease recognition and cleavage site" /translation="ENLYFQS" CDS complement(176..199) /codon_start=1 /label=Strep-Tag II /note="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /translation="WSHPQFEK" CDS complement(200..217) /codon_start=1 /label=6xHis /note="6xHis affinity tag" /translation="HHHHHH" CDS complement(218..238) /codon_start=1 /label=TVMV site /note="tobacco vein mottling virus (TVMV) NIa protease recognition and cleavage site" /translation="ETVRFQS" CDS complement(248..1708) /codon_start=1 /label=NusA /note="transcription elongation protein (N utilization substance protein A)" /translation="MNKEILAVVEAVSNEKALPREKIFEALESALATATKKKYEQEIDV RVQIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAARYEDESLNLGDYVEDQIESVTFDR ITTQTAKQVIVQKVREAERAMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVIL REDMLPRENFRPGDRVRGVLYSVRPEARGAQLFVTRSKPEMLIELFRIEVPEIGEEVIE IKAAARDPGSRAKIAVKTNDKRIDPVGACVGMRGARVQAVSTELGGERIDIVLWDDNPA QFVINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRLASQLSGWELNVMT VDDLQAKHQAEAHAAIDTFTKYLDIDEDFATVLVEEGFSTLEELAYVPMKELLEIEGLD EPTVEALRERAKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLED LAEQGIDDLADIEGLTDEKAGALIMAARN" RBS complement(1716..1738) /label=RBS /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" protein_bind complement(1752..1776) /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(1777..1795) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" tRNA complement(2070..2146) /label=argU tRNA complement(2416..2491) /label=ileX promoter 2631..2708 /label=lacI promoter CDS 2709..3788 /codon_start=1 /label=lacI /note="lac repressor" /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" protein_bind 3804..3825 /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(4220..4808) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(4982..5839) /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(5840..5943) /label=AmpR promoter
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