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).
| 载体基本信息 | |||
| 载体名称 | pET-38b(+) | 载体抗性 | Kanamycin |
|---|---|---|---|
| 载体长度 | 5821 bp | 载体类型 | E. coli Expression Vectors |
| 拷贝数 | High copy number | 克隆方法 | Enzyme digestion and ligation |
| 5引物 | 5-TAATACGACTCACTATAGGG-3 | 载体标签 | His,C-CBD |
pET-38b(+) 载体载体图谱
pET-38b(+) 载体载体序列
LOCUS Exported 5821 bp ds-DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5821)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..5821
/organism="synthetic DNA construct"
/mol_type="other DNA"
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(528..545)
/codon_start=1
/product="thrombin recognition and cleavage site"
/note="thrombin site"
/translation="LVPRGS"
RBS 754..776
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind 791..815
/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(816..834)
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 1147..1224
/gene="lacI"
/note="lacI promoter"
CDS 1225..2307
/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 3116..3307
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/note="rop"
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
misc_feature 3409..3551
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(3737..4325)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS 4447..5262
/codon_start=1
/gene="aph(3)-Ia"
/product="aminoglycoside phosphotransferase"
/note="KanR"
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
rep_origin complement(5355..5810)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
