RNAi-Ready pSIREN-RetroQ is a self-inactivating retroviral expression vector designed to express a small hairpin RNA (shRNA) using the human U6 promoter (PU6; RNA Pol III-dependent). RNAi-Ready pSIREN-RetroQ is provided as a linearized vector digested with BamH I and EcoR I. It is used for targeted gene silencing when an oligonuceotide encoding an appropriate shRNA is ligated into the vector. You can transfect your pSIREN-RetroQ construct as a plasmid expression vector, or—upon transfection into a packaging cell line—this vector can transiently express, or integrate and stably express a viral genomic transcript containing the human U6 promoter and the shRNA. The vector contains a puromycin resistance gene for the selection of stable transfectants. This retroviral vector is optimized to eliminate promoter interference through self-inactivation. The hybrid 5' LTR consists of the cytomegalovirus (CMV) type I enhancer and the mouse sarcoma virus (MSV) promoter. This construct drives high levels of transcription in HEK 293-based packaging cell lines due, in part, to the presence of adenoviral E1A (1–4) in these cells. The self-inactivating feature of the vector is provided by a deletion in the 3' LTR enhancer region (U3). During reverse transcription of the retroviral RNA, the inactivated 3' LTR is copied and replaces the 5' LTR, resulting in inactivation of the 5' LTR CMV enhancer sequences. This may reduce the phenomenon known as promoter interference (5) and allow more efficient expression. Also included in the viral genomic transcript are the necessary viral RNA processing elements including the LTRs, packaging signal (Psi+), and tRNA primer binding site. RNAi-Ready pSIREN-RetroQ also contains a bacterial origin of replication and E. coli Ampr gene for propagation and selection in bacteria.
| 载体基本信息 | |||
| 载体名称 | RNAi-Ready pSIREN-RetroQ | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 6543 bp | 载体类型 | RNAi |
| 载体来源 | Clontech | 拷贝数 | Low copy number |
| 启动子 | U6 | 表达方法 | Transient |
RNAi-Ready pSIREN-RetroQ 载体载体图谱
RNAi-Ready pSIREN-RetroQ 载体载体序列
LOCUS Exported 6455 bp ds-DNA circular SYN 11-SEP-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 3
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6455)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..6455
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 309..512
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 513..688
/note="5 LTR (truncated)"
/note="truncated long terminal repeat from Moloney murine
sarcoma virus"
misc_feature 751..950
/note="MMLV Psi"
/note="packaging signal of Moloney murine leukemia virus
(MMLV)"
CDS 1151..1567
/note="gag (truncated)"
/note="truncated Moloney murine leukemia virus (MMLV) gag
gene lacking the start codon"
promoter 1591..1831
/note="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
promoter 1861..2360
/note="PGK promoter"
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 2381..2980
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/note="PuroR"
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
LTR 3161..3586
/note="3 LTR (Delta-U3)"
/note="self-inactivating 3 long terminal repeat (LTR) from
Moloney murine leukemia virus"
promoter 3852..4181
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 4032..4167
/note="SV40 ori"
/note="SV40 origin of replication"
rep_origin complement(4474..5062)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5233..6093)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(6094..6198)
/gene="bla"
/note="AmpR promoter"
enhancer 6384..308
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
