Basic Vector Information
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.
- Vector Name:
- RNAi-Ready pSIREN-RetroQ
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6543 bp
- Type:
- RNAi
- Replication origin:
- ori
- Source/Author:
- Clontech
- Copy Number:
- Low copy number
- Promoter:
- mPGK
- Expression Method:
- Transient
RNAi-Ready pSIREN-RetroQ vector Vector Map
Plasmid Resuspension Protocol:
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5.Store the plasmid at -20 ℃.
RNAi-Ready pSIREN-RetroQ vector Sequence
LOCUS 40924_48703 6455 bp DNA circular SYN 11-SEP-2021
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6455)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 6455)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6455
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 5..308
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 309..512
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 513..688
/label=5' LTR (truncated)
/note="truncated long terminal repeat from Moloney murine
sarcoma virus"
misc_feature 751..950
/label=MMLV Psi
/note="packaging signal of Moloney murine leukemia virus
(MMLV)"
CDS 1151..1567
/codon_start=1
/label=gag (truncated)
/note="truncated Moloney murine leukemia virus (MMLV) gag
gene lacking the start codon"
/translation="GQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWPTF
NVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVHPKP
PPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGA"
promoter 1591..1831
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
promoter 1861..2360
/label=PGK promoter
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 2381..2977
/codon_start=1
/label=PuroR
/note="puromycin N-acetyltransferase"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
LTR 3161..3586
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
Moloney murine leukemia virus"
promoter 3852..4181
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin complement(4474..5062)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5236..6093)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(6094..6198)
/label=AmpR promoter
enhancer 6384..6455
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
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