pLVTHM vector (V012792)

MSDS

Basic Vector Information

pLVTHM allows for direct cloning of shRNAs into the lentiviral vectors and replaces the old pLVTH system from Trono lab. pLVTHM is similar to pLVTH, but contains a 3bp substitution that generates a unique MluI site for direct cloning of an shRNA into MluI-ClaI. Please note that there is an additional ClaI site in this vector that is blocked by Dam methylation (this site does not appear in the depositor's full sequence). The plasmid needs to be grown in a Dam+ bacterial strain in order to use ClaI for cloning. Note that ClaI has lower salt concentration requirements than MluI. One way to handle this is to digest with ClaI for 45 minutes, followed by addition of diluted MluI buffer and MluI, then incubation for 90 more minutes. Be sure not to use a large amount of vector (1.7 ug to 2.5 ug is known to work). Also, if your shRNA is already in pSUPER (or another plasmid under the control of the PolIII promoter) you may use the EcoRI-ClaI sites to replace the H1 promoter in pLVTHM with the H1-shRNA cassette from pSUPER (or another plasmid).

Vector Name:: pLVTHM

Antibiotic Resistance:: Ampicillin

Length:: 11085 bp

Type:: RNAi

Replication origin:: ori

Source/Author:: Didier Trono

Copy Number:: High copy number

Promoter:: EF-1α

pLVTHM 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 ℃.

pLVTHM vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       pLVTHM.       11085 bp DNA     circular SYN 13-DEC-2021
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    pLVTHM.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11085)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 11085)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..11085
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             2..635
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    682..807
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    1300..1533
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1718..1762
                     /label=gp41 peptide
                     /note="antigenic peptide corresponding to amino acids 655
                     to 669 of the HIV envelope protein gp41 (Lutje Hulsik et 
                     al., 2013)"
     CDS             1911..1952
                     /note="Protein Tat from Human immunodeficiency virus type 1
                     group M subtype B (isolate WMJ22). Accession#: P12509"
     promoter        2122..3299
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     misc_feature    3347..3464
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     CDS             3554..4270
                     /label=EGFP
                     /note="enhanced GFP"
     misc_feature    4343..4931
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     protein_bind    5099..5369
                     /label=tetracycline response element
                     /note="contains seven copies of the tetracycline operator
                     tetO"
     promoter        5398..5612
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     protein_bind    complement(5647..5680)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     LTR             5700..5880
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(5905..5923)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     promoter        6711..6815
                     /label=AmpR promoter
     CDS             6816..7673
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      7847..8435
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        8681..9010
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     intron          10144..10209
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             10339..10359
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     polyA_signal    10784..10918
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"

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