载体基本信息 | |||
载体名称 | pMCSG18 | 载体抗性 | Ampicillin |
---|---|---|---|
载体长度 | 6009 bp | 载体类型 | Structural Genomics Vectors |
载体来源 | Stols L, Gu M, Dieckman L, Raffen R, Collart FR, Donnelly MI. | 拷贝数 | High copy number |
pMCSG18 载体载体图谱
pMCSG18 载体载体序列
LOCUS Exported 6009 bp ds-DNA circular SYN 05-4-2014 DEFINITION Bacterial vector with a 6xHis-TEV leader for high-throughput purification of C-terminally GFP-tagged proteins. ACCESSION . VERSION . KEYWORDS pMCSG18 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6009) AUTHORS Stols L, Gu M, Dieckman L, Raffen R, Collart FR, Donnelly MI. TITLE A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site. JOURNAL Protein Expr. Purif. 2002;25:8-15. PUBMED 12071693 REFERENCE 2 (bases 1 to 6009) AUTHORS Midwest Center for Structural Genomics TITLE Direct Submission COMMENT For ligation-independent cloning (LIC), linearize with SspI and treat with T4 DNA polymerase plus dGTP. FEATURES Location/Qualifiers source 1..6009 /organism="synthetic DNA construct" /lab_host="Escherichia coli" /mol_type="other DNA" terminator 26..73 /note="T7 terminator" /note="transcription terminator for bacteriophage T7 RNA polymerase" CDS complement(140..157) /codon_start=1 /product="6xHis affinity tag" /note="6xHis" /translation="HHHHHH" CDS complement(208..921) /codon_start=1 /product="green fluorescent protein variant" /note="GFP" /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF ICTTGKLPVPWPTLVTTLCYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN FKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF VTAAGITHGMDELYN" CDS complement(946..966) /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /note="TEV site" /translation="ENLYFQS" CDS complement(991..1008) /codon_start=1 /product="6xHis affinity tag" /note="6xHis" /translation="HHHHHH" CDS complement(1009..1011) /codon_start=1 /product="start codon" /note="ATG" /translation="M" RBS 1020..1025 /note="ribosome binding site" protein_bind 1056..1080 /bound_moiety="lac repressor encoded by lacI" /note="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(1081..1099) /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" promoter 1412..1489 /gene="lacI" /note="lacI promoter" /note=" " CDS 1490..2572 /codon_start=1 /gene="lacI" /product="lac repressor" /note="lacI" /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)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" CDS 3381..3572 /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /note="rop" /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" rep_origin complement(4002..4590) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(4761..5549) /codon_start=1 /gene="bla(M)" /product="beta-lactamase lacking the signal sequence" /note="bla(M)" /note="allows cytosolic expression of beta-lactamase" /translation="PETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMST FKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMS DNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATT LRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAA LGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW" promoter complement(5622..5725) /gene="bla" /note="AmpR promoter"
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