pET-40b

pET-40b

Search name

pET-40b,Plasmid pET-40b,pET-40b vector

pET-40b Informaiton

Promoter: T7/lac

Copier: ColE1 ori, F1 ori

Terminator: T7 Terminator

Plasmid Classification: E. coli Vector; PET Series Expression Plasmid

Plasmid size: 6190bp

Plasmid tags: N-6 *His, N-DsbC, N-S, N-EK, N-Thrombin, C-6 *His

Prokaryotic resistance: kanamycin Kan

Clonal strain: DH5 alpha

Culture conditions: 37 C, aerobic, LB

Expression host: BL21 (DE3)

Culture conditions: 37 C, aerobic, LB

Induction: IPTG or lactose and its analogues

5'Sequencing Primers: T7:TAATACGACTCACTATAGG

3'Sequencing Primer: T7-ter: TGCTAGTTATTGCTCAGCGG

Note: Expressing DsbC fusion protein

pET-40b Description

The pET-40b(+) vector is designed for expression of DsbC fusion proteins. Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the circle map. The cloning/ expression region of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage will produce virions containing single stranded DNA that corresponds to the coding strand. Therefore, single stranded sequencing should be performed using the T7 terminator primer .

pET-40b Multiple cloning site

pET-40b Sequence

LOCUS       Exported                6190 bp ds-DNA     circular SYN 10-8-2015
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    Untitled 3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6190)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported 2015-8-10   
FEATURES             Location/Qualifiers
     source          1..6190
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          1123..1145
                     /organism="Enterobacteria phage T7"
                     /mol_type="genomic DNA"
                     /db_xref="taxon:10760"
     terminator      26..73
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA
                     polymerase"
     CDS             complement(150..173)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /note="8xHis"
                     /translation="HHHHHHHH"
     CDS             complement(252..266)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /note="enterokinase site"
                     /translation="DDDDK"
     CDS             complement(282..326)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic
                     ribonuclease A"
                     /note="S-Tag"
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(342..359)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /note="thrombin site"
                     /translation="LVPRGS"
     CDS             complement(369..386)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             complement(408..1115)
                     /codon_start=1
                     /gene="dsbC"
                     /product="bacterial periplasmic protein disulfide
                     isomerase"
                     /note="DsbC"
                     /translation="MKKGFMLFTLLAAFSGFAQADDAAIQQTLAKMGIKSSDIQPAPVA
                     GMKTVLTNSGVLYITDDGKHIIQGPMYDVSGTAPVNVTNKMLLKQLNALEKEMIVYKAP
                     QEKHVITVFTDITCGYCHKLHEQMADYNALGITVRYLAFPRQGLDSDAEKEMKAIWCAK
                     DKNKAFDDVMAGKSVAPASCDVDIADHYALGVQLGVSGTPAVVLSNGTLVPGYQPPKEM
                     KEFLDEHQKMTSGK"
     RBS             1123..1145
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1160..1184
                     /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(1185..1203)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1516..1593
                     /gene="lacI"
                     /note="lacI promoter"
     CDS             1594..2676
                     /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             3485..3676
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
              &n  <