pET-32 Ek/LIC vector (V010990)

MSDS

Basic Vector Information

The pET-32 Ek/LIC vector is prepared for rapid, directional cloning of PCR-amplified DNA for high-level expression of polypeptides fused with the 109aa Trx•Tag thioredoxin protein. Using specifically designed primers for amplification and the pET-32 Ek/LIC Cloning Kit (Cat. No. 69076-3), inserts can be efficiently cloned without the need for restriction digestion or ligation. Fusion proteins also contain cleavable His•Tag and S•Tag sequences for detection and purification. 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 (Cat. No. 69337-3).

Vector Name:: pET-32 Ek/LIC

Antibiotic Resistance:: Ampicillin

Length:: 5917 bp

Type:: pET & Duet Vectors (Novagen)

Replication origin:: ori

Source/Author:: Novagen (EMD Millipore)

Copy Number:: High copy number

Cloning Method:: Enzyme digestion and ligation

5' Primer:: T7: 5'-TAATACGACTCACTATAGGG-3'; Trx-F: 5' TTCCTCGACGCTAACCTG 3'

Fusion Tag:: thioredoxin; 6xHis, N-EK

pET-32 Ek/LIC vector Vector Map

View Map in NovoBuilder

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-32 Ek/LIC vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       pET-32_Ek_LIC.        5917 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector for ligation-independent cloning (LIC) to express 
            thioredoxin-tagged proteins with an enterokinase site.
ACCESSION   .
VERSION     .
KEYWORDS    pET-32 Ek_LIC.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5917)
  AUTHORS   Novagen (EMD Millipore)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 5917)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     For LIC, linearize with BseRI and treat with T4 DNA polymerase plus 
            dTTP.
FEATURES             Location/Qualifiers
     source          1..5917
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(140..157)
                     /label=6xHis
                     /note="6xHis affinity tag"
     misc_feature    158..216
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     CDS             complement(236..250)
                     /label=enterokinase site
                     /note="enterokinase recognition and cleavage site"
     CDS             complement(266..310)
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
     CDS             complement(317..334)
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
     CDS             complement(344..361)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(383..709)
                     /label=TrxA
                     /note="E. coli thioredoxin"
     RBS             complement(717..739)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(754..778)
                     /label=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(779..797)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1110..1187
                     /label=lacI promoter
     CDS             1188..2267
                     /label=lacI
                     /note="lac repressor"
     protein_bind    2283..2304
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3079..3267
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    3372..3514
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3700..4288)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4462..5319)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5320..5424)
                     /label=AmpR promoter
     rep_origin      complement(5451..5906)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"

This page is informational only.