pEF4/V5-His A, B, and C are 5.7 kb vectors derived from pEF1/V5-His and designed for overproduction of recombinant proteins in mammalian cell lines. Features of the vectors allow purification and detection of expressed proteins. High-level stable and transient expression can be carried out in most mammalian cells. The vectors contain thefollowing elements:The control plasmid, pEF4/V5-His/lacZ is included for use as a positive control for transfection, expression, purification, and detection in the cell line of choice.
| 载体基本信息 | |||
| 载体名称 | pEF4/V5-His A | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 5769 bp | 载体类型 | Mammalian Expression Vectors |
| 筛选标记 | Zeocin | 拷贝数 | High copy number |
| 启动子 | EF-1a | 克隆方法 | Enzyme digestion and ligation |
| 5引物 | T7 Fwd : 5d[TAATACGACTCACTATAGGG]3 | 载体标签 | 6xHis, V5 |
pEF4/V5-His A 载体载体图谱
pEF4/V5-His A 载体载体序列
LOCUS Exported 5769 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 5769)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..5769
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 475..1653
/note="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 706..1644
/note="EF-1-alpha intron A"
/note="intron upstream of the start codon of human
EF-1-alpha"
promoter 1670..1688
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1807..1848
/codon_start=1
/product="epitope tag from simian virus 5"
/note="V5 tag"
/translation="GKPIPNPLLGLDST"
CDS 1858..1875
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
polyA_signal 1904..2128
/note="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 2174..2602
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2616..2946
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2797..2932
/note="SV40 ori"
/note="SV40 origin of replication"
promoter 2994..3041
/note="EM7 promoter"
/note="synthetic bacterial promoter "
CDS 3060..3434
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/note="BleoR"
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
polyA_signal 3564..3685
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(3734..3750)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 3758..3774
/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(3782..3812)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 3827..3848
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(4136..4724)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4895..5755)
/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(5756..91)
/gene="bla"
/note="AmpR promoter"
