pMCSG25 载体 (V010695#)

载体基本信息
载体名称 pMCSG25 载体抗性 Ampicillin
载体长度 5664 bp 载体类型 Structural Genomics Vectors
载体来源 Osipiuk J, Mulligan R, Bargassa M, Hamilton JE, Cunningham MA, 拷贝数 High copy number

pMCSG25 载体载体图谱

pMCSG255664 bp60012001800240030003600420048005400T7 terminator6xHisTEV site6xHisaspartic endopeptidaseRBSlac operatorT7 promoterlacI promoterlacIroporiAmpRAmpR promoter

pMCSG25 载体载体序列

LOCUS       Exported                5664 bp ds-DNA    circular SYN 10-2-2014
DEFINITION  Bacterial expression vector encoding an aspartic endopeptidase fused
            to a 6xHis-TEV leader.
ACCESSION   .
VERSION     .
KEYWORDS    pMCSG25
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5664)
  AUTHORS   Osipiuk J, Mulligan R, Bargassa M, Hamilton JE, Cunningham MA, 
            Joachimiak A.
  TITLE     Characterization of member of DUF1888 protein family, self-cleaving 
            and self-assembling endopeptidase.
  JOURNAL   J. Biol. Chem. 2012;287:19452-61.
  PUBMED    22493430
REFERENCE   2  (bases 1 to 5664)
  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..5664
                     /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(223..243)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /note="TEV site"
                     /translation="ENLYFQS"
     CDS             complement(268..285)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             complement(292..666)
                     /codon_start=1
                     /locus_tag="
                     "
                     /product="self-cleaving aspartic endopeptidase SO1698 from 
                     Shewanella oneidensis"
                     /note="aspartic endopeptidase"
                     /protein_id="
                     "
                     /translation="MFAPQGLAQFIKVNVTLENGEPVFIYTDANGQVCQGDITVTQAGT
                     ITYLLNDQTLKGLKFVGVGFVTPFDGIIDAVTISSDGMLVQLVDLDKTPGTTKFQFVLS
                     NTANTLLVLSPDPQIINRPQN"
     RBS             675..680
                     /note="ribosome binding site"
     protein_bind    711..735
                     /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(736..754)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1067..1144
                     /gene="lacI"
                     /note="lacI promoter"
                     /note="
                     "
     CDS             1145..2227
                     /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             3036..3227
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     rep_origin      complement(3657..4245)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4416..5276)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5277..5380)
                     /gene="bla"
                     /note="AmpR promoter"

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