RNAi-Ready pSIREN-RetroQ vector (V012742)

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
      • Source/Author:
      • Clontech
      • Copy Number:
      • Low copy number
      • Promoter:
      • U6
      • Expression Method:
      • Transient

RNAi-Ready pSIREN-RetroQ vector Vector Map

RNAi-Ready pSIREN-RetroQ6455 bp300600900120015001800210024002700300033003600390042004500480051005400570060006300CMV promoter5' LTR (truncated)MMLV Psigag (truncated)U6 promoterPGK promoterPuroR3' LTR (Delta-U3)SV40 promoteroriAmpRAmpR promoter

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

Copy Sequence

Download GeneBank File(.gb)

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"

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