Tet-pLKO-puro-Scrambled vector (V000016)

Basic Vector Information

      • Vector Name:
      • Tet-pLKO-puro-Scrambled
      • Antibiotic Resistance:
      • Ampicillin
      • Length:
      • 8815 bp
      • Type:
      • Mammalian Expression, Lentiviral, RNAi
      • Selection Marker:
      • Puromycin
      • Copy Number:
      • High Copy
      • Promoter:
      • H1
      • Cloning Method:
      • Restriction Enzyme
      • 5' Primer:
      • H1

Tet-pLKO-puro-Scrambled vector Vector Map

Tet-pLKO-puro-Scrambled8815 bp40080012001600200024002800320036004000440048005200560060006400680072007600800084008800Puro-F3' LTR (Delta-U3)SV40 poly(A) signalSV40 oriT7 promoterM13 Forwardf1 oriAmpR promoterAmpRoriL4440CAP binding sitelac promoterlac operatorM13 revT3RSV promoter5' LTR (truncated)HIV-1 PsiRREgp41 peptideH1-2O2 promotercPPT/CTShPGK promoterbeta-globin intronpCAG-FT7 promoterTetRIRESPuroR

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 ℃.

Tet-pLKO-puro-Scrambled vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported                8815 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Tet inducible scrambled shRNA..
ACCESSION   .
VERSION     .
KEYWORDS    Tet-pLKO-puro-Scrambled
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8815)
  AUTHORS   Rudin CM, Durinck S, Stawiski EW, Poirier JT, Modrusan Z, Shames DS,
            Bergbower EA, Guan Y, Shin J, Guillory J, Rivers CS, Foo CK, Bhatt 
            D, Stinson J, Gnad F, Haverty PM, Gentleman R, Chaudhuri S, 
            Janakiraman V, Jaiswal BS, Parikh C, Yuan W, Zhang Z, Koeppen H, Wu 
            TD, Stern HM, Yauch RL, Huffman KE, Paskulin DD, Illei PB, 
            Varella-Garcia M, Gazdar AF, de Sauvage FJ, Bourgon R, Minna JD, 
            Brock MV, Seshagiri S
  TITLE     Comprehensive genomic analysis identifies SOX2 as a frequently 
            amplified gene in small-cell lung cancer.
  JOURNAL   Nat Genet. 2012 Oct;44(10):1111-6. doi: 10.1038/ng.2405. Epub 2012 
            Sep 2.
  PUBMED    22941189
REFERENCE   2  (bases 1 to 8815)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/ng.2405"; 
            journalName: "Nat Genet"; date: "2012-10"; volume: "44"; issue: 
            "10"; pages: "1111-6"
FEATURES             Location/Qualifiers
     source          1..8815
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     258..278
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             489..722
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    794..915
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(831..850)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     885..904
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      955..1090
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1017..1036
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        complement(1111..1129)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1139..1156)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1139..1155)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1148..1170)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1297..1752
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1384..1403)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1594..1615
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1778..1882
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1883..2743
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2101..2120)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2914..3502
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3403..3422
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3656..3673
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3790..3811
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3826..3856
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3864..3880
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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     3869..3891
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3888..3904
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3888..3904
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3923..3943
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3925..3943
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3971..4197
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             4198..4378
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4425..4550
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5043..5276
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5461..5505
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        5794..5888
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     primer_bind     5805..5824
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     protein_bind    5842..5860
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5870..5888
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    5988..6105
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6154..6664
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     6583..6601
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     intron          6673..7245
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     primer_bind     7253..7272
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        7300..7318
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             7329..7946
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG"
     primer_bind     complement(7402..7421)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     misc_feature    7984..8557
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(8151..8168)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     8378..8397
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(8577..8815,1..361)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(8577..8596)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"

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