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pLKO.1-TRC vector (V012791#)

Basic Vector Information

This is the recommended vector for cloning and expressing new shRNA sequences. This plasmid is being used by The RNAi Consortium to produce their shRNA library http://www.broad.mit.edu/genome_bio/trc/ The 1.9kb stuffer can be released with AgeI and EcoRI and replaced with your shRNA sequence of choice.

Basic Vector Information
Vector Name pLKO.1-TRC Antibiotic Resistance Ampicillin
Length 8901 bp Type RNAi
Source David Root Copy Number High copy number
Promoter U6 Cloning Method Enzyme Cut
5' Primer LKO.15' :GACTATCATATGCTTACCGT Expression Method Constiutive, Stable / Transient

pLKO.1-TRC vector Vector Map

PuroR 3' LTR (Delta-U3) SV40 poly(A) signal SV40 ori T7 promoter M13 fwd f1 ori bla bla ori CAP binding site lac promoter lac operator M13 rev T3 promoter RSV promoter 5' LTR (truncated) HIV-1 Psi RRE U6 promoter stuffer cPPT/CTS

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 ℃.

pLKO.1-TRC vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported                8901 bp ds-DNA     circular SYN 13-DEC-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pLKO.1-TRC
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8901)
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8901
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             529..1128
                     /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             1256..1489
                     /note="3' LTR (Delta-U3)"
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1561..1682
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      1722..1857
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     promoter        complement(1878..1896)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1906..1922)
                     /note="M13 fwd"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      2064..2519
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        2545..2649
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             2650..3510
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3681..4269
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    4557..4578
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding site"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4593..4623
                     /note="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    4631..4647
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     4655..4671
                     /note="M13 rev"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        4692..4710
                     /note="T3 promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4738..4964
                     /note="RSV promoter"
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             4965..5145
                     /note="5' LTR (truncated)"
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5192..5317
                     /note="HIV-1 Psi"
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5810..6043
                     /note="RRE"
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     promoter        6570..6810
                     /note="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    6822..8690
                     /note="stuffer"
     misc_feature    8732..8849
                     /note="cPPT/CTS"
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        8898..507
                     /note="hPGK promoter"
                     /note="human phosphoglycerate kinase 1 promoter"

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