The dual-purpose pIEx/Bac vectors are designed for cloning and high-level expression of proteins by transiently transfecting Spodoptera-derived insect cells or by generating baculovirus recombinants. Transient transfection and early baculovirus expression is driven by a promoter/enhancer combination that recruits endogenous insect cell transcription machinery, the AcNPV derived hr5 enhancer and ie1 promoter. Late/very late expression in the baculovirus mode is driven by the strong p10 promoter. The pIEx/Bac-1 vector carries an N-terminal Strep•Tag II coding sequence (1) followed by a recognition site for enterokinase.The multiple cloning region is followed by an optional C-terminal His•Tag coding sequence. The presence of two gentle elution tags at both the N- and C-terminus is ideal for dual purification strategies designed to isolate full-length fusion proteins (2). Unique restriction sites are shown on the circle map.
| 载体基本信息 | |||
| 载体名称 | pIEx/Bac-1 | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 6796 bp | 载体类型 | Insect Cell Vectors |
| 载体来源 | Novagen (EMD Millipore) | 拷贝数 | High copy number |
| 启动子 | IE1, p10 | 克隆方法 | Enzyme digestion and ligation |
| 载体标签 | C-His,N-Strep | ||
pIEx/Bac-1 载体载体图谱
pIEx/Bac-1 载体载体序列
LOCUS Exported 6796 bp ds-DNA circular SYN 28-4-2014
DEFINITION Insect cell and baculovirus vector encoding a cleavable N-terminal
Strep-Tag(R) II and a C-terminal 10xHis tag.
ACCESSION .
VERSION .
KEYWORDS pIEx Bac-1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6796)
AUTHORS Novagen (EMD Millipore)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..6796
/organism="synthetic DNA construct"
/lab_host="Insect Cells"
/mol_type="other DNA"
misc_recomb 329..1156
/note="baculovirus recombination region (lef2/ORF603)"
/note="contains ORF603 and part of lef2"
CDS 329..453
/codon_start=3
/product="baculovirus late expression factor 2"
/note="lef2"
/translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
CDS complement(491..1096)
/codon_start=1
/product="baculovirus ORF603 protein"
/note="ORF603"
/translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
enhancer 1196..1678
/gene="Autographa californica hr5"
/note="hr5 enhancer"
/note="baculovirus early transcription enhancer"
promoter 1682..2273
/note="IE1 promoter"
/note="promoter of the ie1 gene from the baculovirus
Autographa californica"
promoter 2283..2392
/note="p10 promoter"
/note="baculovirus promoter for expression in insect cells"
CDS 2394..2396
/codon_start=1
/product="start codon"
/note="ATG"
/translation="M"
CDS 2403..2426
/codon_start=1
/product="peptide that binds Strep-Tactin(R), an engineered
form of streptavidin"
/note="Strep-Tag II"
/translation="WSHPQFEK"
CDS 2433..2447
/codon_start=1
/product="enterokinase recognition and cleavage site"
/note="enterokinase site"
/translation="DDDDK"
misc_feature 2447..2506
/note="MCS"
/note="multiple cloning site"
CDS 2517..2546
/codon_start=1
/product="10xHis affinity tag"
/note="10xHis"
/translation="HHHHHHHHHH"
terminator 2565..2871
/note="IE1 terminator"
/note="terminator of the ie1 gene from the baculovirus
Autographa californica"
misc_recomb 2900..4257
/note="baculovirus recombination region (ORF1629)"
/note="contains part of ORF1629"
CDS complement(2916..4257)
/codon_start=2
/product="baculovirus capsid-associated protein"
/note="ORF1629"
/note="required for viral replication"
/translation="PSLRQSLYNTIAYIERLLNIGTVNDSEITMLIADFYDLYSNYNIE
LPPPQALPRSRRPSVVQPAAPAPVPTIVREQTKPEQIIPAAPPPPPSPVPNIPAPPPPP
PPSMSELPPAPPMPTEPQPAAPLDDRQQLLEAIRNEKNRTRLRPVKPKTAPETSTIVEV
PTVLPKETFEPKPPSASPPPPPPPPPPPAPPAPPPMVDLSSAPPPPPLVDLPSEMLPPP
APSLSNVLSELKSGTVRLKPAQKRPQSEIIPKSSTTNLIADVLADTINRRRVAMAKSSS
EATSNDEGWDDDDNRPNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVE
PLLQNKTQTNIDKARLLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDL
IFKFRYKDAENHLIFALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
rep_origin complement(4520..5108)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5279..6139)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(6140..6244)
/gene="bla"
/note="AmpR promoter"
rep_origin 6271..6726
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
