FUW-tetO-EGFP vector (V000223)

Basic Vector Information

      • Vector Name:
      • FUW-tetO-EGFP
      • Antibiotic Resistance:
      • Ampicillin
      • Length:
      • 10457 bp
      • Type:
      • Mammalian Expression, Lentiviral
      • Copy Number:
      • High Copy
      • Promoter:
      • TRE-CMV
      • Cloning Method:
      • Restriction Enzyme
      • 5' Primer:
      • LNCX
      • 3' Primer:
      • pBluescriptKS

FUW-tetO-EGFP vector Vector Map

FUW-tetO-EGFP10457 bp50010001500200025003000350040004500500055006000650070007500800085009000950010000CMV enhancerCMV promoter5' LTR (truncated)HIV-1 PsiRREgp41 peptidecPPT/CTStetracycline response elementminimal CMV promoterKozak sequenceIRES2PuroRWPREKS primer5' LTR (truncated)BGH-revf1 oripBABE 3'EM7 promoterBleoRSV40 poly(A) signalM13 revlac operatorlac promoterCAP binding siteL4440oriAmpRAmpR promoterpRS-marker

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 ℃.

FUW-tetO-EGFP vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       Exported               10457 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  3rd generation lentiviral transfer plasmid. Expresses EGFP and 
            puromycin resistance in mammalian cells under the control of a 
            doxycycline-inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    FUW-tetO-EGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10457)
  AUTHORS   Panciera T, Azzolin L, Fujimura A, Di Biagio D, Frasson C, Bresolin 
            S, Soligo S, Basso G, Bicciato S, Rosato A, Cordenonsi M, Piccolo S
  TITLE     Induction of Expandable Tissue-Specific Stem/Progenitor Cells 
            through Transient Expression of YAP/TAZ.
  JOURNAL   Cell Stem Cell. 2016 Sep 9. pii: S1934-5909(16)30256-9. doi: 
            10.1016/j.stem.2016.08.009.
  PUBMED    27641305
REFERENCE   2  (bases 1 to 10457)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Stem 
            Cell. 2016 Sep 9. pii: S1934-5909(16)30256-9. doi: 
            10.1016/j.stem.2016.08.009."
FEATURES             Location/Qualifiers
     source          1..10457
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        27..406
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        408..606
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     561..581
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             624..804
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    851..976
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1469..1702
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1887..1931
                     /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"
     misc_feature    2229..2346
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    2399..2669
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        2702..2740
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2737..2761
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2895..2904
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2901..3620
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2901..3617
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2946..2967)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3207..3226)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3554..3575
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3660..4246
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3827..3844)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4054..4073
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4247..4846
                     /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(4247..4266)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4743..4763
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4938..5526
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4991..5011)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5409..5420)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5529..5545)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5530..5546)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6051..6231
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6257..6274)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6263..6487
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6533..6961
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6620..6639)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6830..6851
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6970..6990)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6975..7304
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7155..7290
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7217..7236
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7352..7399
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7418..7792
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    7922..8043
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7959..7978)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8013..8032
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8092..8108)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8092..8108)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8105..8127)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8116..8132
                     /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)."
     promoter        complement(8140..8170)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8185..8206
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8323..8340)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8494..9082)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8574..8593)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(9253..10113)
                     /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     9876..9895
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10114..10218)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10293..10312)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"

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