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).
| 载体基本信息 | |||
| 载体名称 | pET-32 Ek/LIC | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 5917 bp | 载体类型 | pET & Duet Vectors (Novagen) |
| 载体来源 | Novagen (EMD Millipore) | 拷贝数 | High copy number |
| 克隆方法 | Enzyme digestion and ligation | 5引物 | T7: 5-TAATACGACTCACTATAGGG-3; Trx-F: 5 TTCCTCGACGCTAACCTG 3 |
| 载体标签 | thioredoxin; 6xHis, N-EK | ||
pET-32 Ek/LIC 载体载体图谱
pET-32 Ek/LIC 载体载体序列
LOCUS Exported 5917 bp ds-DNA circular SYN 02-12-2013
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
COMMENT For LIC, linearize with BseRI and treat with T4 DNA polymerase plus
dTTP.
FEATURES Location/Qualifiers
source 1..5917
/organism="synthetic DNA construct"
/lab_host="Escherichia coli"
/mol_type="other DNA"
terminator 26..73
/note="T7 terminator"
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(140..157)
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
misc_feature 158..216
/note="MCS"
/note="multiple cloning site"
CDS complement(236..250)
/codon_start=1
/product="enterokinase recognition and cleavage site"
/note="enterokinase site"
/translation="DDDDK"
CDS complement(266..310)
/codon_start=1
/product="affinity and epitope tag derived from pancreatic
ribonuclease A"
/note="S-Tag"
/translation="KETAAAKFERQHMDS"
CDS complement(317..334)
/codon_start=1
/product="thrombin recognition and cleavage site"
/note="thrombin site"
/translation="LVPRGS"
CDS complement(344..361)
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
CDS complement(383..709)
/codon_start=1
/gene="trxA"
/product="E. coli thioredoxin"
/note="TrxA"
/translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
DANLA"
RBS 718..723
/note="ribosome binding site"
protein_bind 754..778
/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(779..797)
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 1110..1187
/gene="lacI"
/note="lacI promoter"
/note="
"
CDS 1188..2270
/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 3079..3270
/codon_start=1
/gene="rop"
/product="Rop protein"
/note="rop"
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
rep_origin complement(3700..4288)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4459..5319)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(5320..5424)
/gene="bla"
/note="AmpR promoter"
rep_origin complement(5451..5906)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
