Basic Vector Information
The pET-38b(+) vector (Cat. No. 70137-3) is designed for expression of C-terminal CBD cex fusion proteins. Target sequences inserted between the N-terminal signal sequence and C-terminal CBD cex •Tag and His•Tag sequences allow for potential export of the expressed protein. Sequences inserted at the Nde I site will be expressed in the cytoplasm. Note that inserts must allow read-through in the proper reading frame (shown below) to produce the desired polypeptide fusions. A variety of cloning sites and strategies are available. 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-38b(+)
- Antibiotic Resistance:
- Kanamycin
- Length:
- 5821 bp
- Type:
- E. coli Expression Vectors
- Replication origin:
- ori
- Copy Number:
- High copy number
- Cloning Method:
- Enzyme digestion and ligation
- 5' Primer:
- 5'-TAATACGACTCACTATAGGG-3'
- Fusion Tag:
- His,C-CBD
pET-38b(+) vector Vector Map
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-38b(+) vector Sequence
LOCUS 40924_17926 5821 bp DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5821)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 5821)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..5821
/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(150..173)
/codon_start=1
/label=8xHis
/note="8xHis affinity tag"
/translation="HHHHHHHH"
CDS complement(528..545)
/codon_start=1
/label=thrombin site
/note="thrombin recognition and cleavage site"
/translation="LVPRGS"
RBS complement(754..776)
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind complement(791..815)
/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(816..834)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 1147..1224
/label=lacI promoter
CDS 1225..2304
/codon_start=1
/label=lacI
/note="lac repressor"
/translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
protein_bind 2320..2341
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS 3116..3304
/codon_start=1
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
/translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
misc_feature 3409..3551
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(3737..4325)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS 4447..5259
/codon_start=1
/label=KanR
/note="aminoglycoside phosphotransferase"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
rep_origin complement(5355..5810)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
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