pLVX-EF1α-mCherry-C1 is a lentiviral expression vector that can be used to generate high-titer lentivirus for transducing virtually any dividing or nondividing mammalian cell type, including primary and stem cells. The vector allows a gene-ofinterest to be fused to the C-terminus of the red fluorescent protein mCherry. Expression of the fusion is driven by the human elongation factor 1 alpha (EF1α) promoter, which continues to be constitutively active even after stable integration of the vector into the host cell genome. Stable expression of the fusion allows the monitoring of a variety of cellular processes (such as differentiation in primary or stem cells) without the transgene silencing associated with CMV promoters. In addition, the vector allows efficient flow cytometric detection of stably or transiently transfected mammalian cells expressing mCherry fusions, without time-consuming drug and clonal selection.
| 载体基本信息 | |||
| 载体名称 | pLVX-EF1α-mCherry-C1 | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 9510 bp | 载体类型 | Viral Expression & Packaging Vectors |
| 载体来源 | Clontech | 筛选标记 | Puromycin |
| 拷贝数 | High copy number | 启动子 | EF1α/ EF1a |
| 克隆方法 | Enzyme Cut | 载体标签 | mCherry |
| 表达方法 | Constiutive, Stable | ||
pLVX-EF1α-mCherry-C1 载体载体图谱
pLVX-EF1α-mCherry-C1 载体载体序列
LOCUS Exported 9510 bp ds-DNA circular SYN 08-APR-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pLVX-EF1-alpha-mCherry-C1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9510)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..9510
/organism="synthetic DNA construct"
/mol_type="other DNA"
LTR 1..634
/note="3 LTR"
/note="3 long terminal repeat (LTR) from HIV-1"
misc_feature 681..806
/note="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1303..1536
/note="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 2028..2143
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1 (lacking the first T)"
promoter 2338..3519
/note="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 2568..3510
/note="EF-1-alpha intron A"
/note="intron upstream of the start codon of human
EF-1-alpha"
CDS 3539..4246
/codon_start=1
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/note="mCherry"
/note="mammalian codon-optimized"
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK"
misc_feature 4247..4312
/note="MCS"
/note="multiple cloning site"
promoter 4345..4844
/note="PGK promoter"
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 4865..5464
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/note="PuroR"
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
misc_feature 5478..6066
/note="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
CDS complement(5949..5960)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/note="Factor Xa site"
/translation="IEGR"
LTR 6273..6906
/note="3 LTR"
/note="3 long terminal repeat (LTR) from HIV-1"
primer_bind complement(7035..7051)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 7059..7075
/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(7083..7113)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 7128..7149
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(7437..8025)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8196..9056)
/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(9057..9161)
/gene="bla"
/note="AmpR promoter"
polyA_signal 9209..9343
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
