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:: 6455 bp

Type:: RNAi

Replication origin:: ori

Source/Author:: Clontech

Copy Number:: Low copy number

Promoter:: mPGK

Expression Method:: Transient

RNAi-Ready pSIREN-RetroQ vector Map

Copy Sequence View Map

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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