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    V011004

    pET-28b(+) vector (V011004)

    pET-28b(+) vector (V011004)

    Basic Vector Information

     

        • Vector Name:
        • pET-28b(+)
        • Antibiotic Resistance:
        • Kanamycin
        • Length:
        • 5368 bp
        • Type:
        • pET & Duet Vectors (Novagen)
        • Source/Author:
        • Novagen (EMD Millipore)
        • Copy Number:
        • High copy number

    pET-28b(+) vector Vector Map

     
    pET-28b(+)5368 bp6001200180024003000360042004800T7 terminator6xHisMCST7 tagthrombin site6xHisATGRBSlac operatorT7 promoterlacI promoterlacIroporiKanRf1 ori

    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 ℃.


    pET-28b(+) vector Sequence

    LOCUS       Exported                5368 bp ds-DNA    circular SYN 02-12-2013
DEFINITION  Bacterial vector for expression of N-terminally 6xHis-tagged 
            proteins with a thrombin site. For other reading frames, use 
            pET-28a(+) or pET-28c(+).
ACCESSION   .
VERSION     .
KEYWORDS    pET-28b(+)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5368)
  AUTHORS   Novagen (EMD Millipore)
  TITLE     Direct Submission
COMMENT     
FEATURES             Location/Qualifiers
     source          1..5368
                     /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"
     misc_feature    158..203
                     /note="MCS"
                     /note="multiple cloning site"
     CDS             complement(206..238)
                     /codon_start=1
                     /product="epitope tag from a T7 major capsid protein"
                     /note="T7 tag"
                     /translation="MASMTGGQQMG"
     CDS             complement(242..259)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /note="thrombin site"
                     /translation="LVPRGS"
     CDS             complement(269..286)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             complement(296..298)
                     /codon_start=1
                     /product="start codon"
                     /note="ATG"
                     /translation="M"
     RBS             306..311
                     /note="ribosome binding site"
     protein_bind    342..366
                     /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(367..385)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        694..771
                     /gene="lacI"
                     /note="lacI promoter"
                     /note="
                     "
     CDS             772..1854
                     /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             2663..2854
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     rep_origin      complement(3284..3872)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             3994..4809
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /note="KanR"
                     /note="confers resistance to kanamycin"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      complement(4902..5357)
                     /direction=LEFT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
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