pET- 35b

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  • Model: PVT0093
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pET-35b

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pET-35b,Plasmid pET-35b,pET-35b vector

Bacterial Resistance:Ampicillin
Growth Strain:DH5α
Expression: Bacterial
Use:pET Plasmid

 

Promoter: T7/lac
Replicator: ColE1, ori, F1, ori
Terminator: T7 Terminator
Plasmid classification: Escherichia coli vector, PET series of expressed plasmids
Plasmid size: 5927bp
Plasmid Tags: N-CBDclos, C-6 * His
Prokaryotic resistance: kanamycin Kan
Clone strain: DH5 alpha
Culture conditions: 37 DEG C, aerobic, LB
Expression host: BL21 (DE3)
Culture conditions: 37 DEG C, aerobic, LB
Induction: IPTG or lactose and its analogues
Primers for 5'sequencing: T7:TAATACGACTCACTATAGGG
Primers for 3'sequencing: T7-ter:TGCTAGTTATTGCTCAGCGG

Description

 The pET-35b(+) vector is designed for expression of CBDclos fusion proteins.A variety of cloning sites and strategies are available. Rapid directional cloning of PCR-amplified DNA is available with the pET-35 Xa/LIC Vector Kit, which contains linearized pET-35b(+) ready for annealing with appropriately prepared inserts. When cloned with the Xa/LIC method, resulting CBDclos•Tag™ fusion proteins can be cleaved precisely at the vector-encoded junction using Factor Xa. Unique sites are shown on the circle map. Note that the sequence is num-bered 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.

Sequencing

LOCUS       Exported                5927 bp ds-DNA     circular SYN 07-8-2015
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    Untitled
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5927)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported 2015-8-7  from MLCC
            
FEATURES             Location/Qualifiers
     source          1..5927
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          860..882
                     /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(249..260)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /note="Factor Xa site"
                     /translation="IEGR"
     CDS             complement(291..335)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic
                     ribonuclease A"
                     /note="S-Tag"
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(351..368)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /note="thrombin site"
                     /translation="LVPRGS"
     RBS             860..882
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    897..921
                     /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(922..940)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1253..1330
                     /gene="lacI"
                     /note="lacI promoter"
     CDS             1331..2413
                     /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             3222..3413
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     misc_feature    3515..3657
                     /note="bom"
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3843..4431)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             4553..5368
                     /codon_start=1
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