Tet-on circRNA Expression Vector vector (V000415)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V000415	Tet-on circRNA Expression Vector	In stock, instant shipping	Buy one, get one free!

Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

Low Ampicillin. The vector can efficiently produce a circRNA (circmCherry) from a split EGFP gene after Dox treatment.

Vector Name:: Tet-on circRNA Expression Vector

Antibiotic Resistance:: Ampicillin

Length:: 10054 bp

Type:: Mammalian Expression, Lentiviral

Replication origin:: ori

Copy Number:: High Copy

Promoter:: tight TRE

Cloning Method:: Restriction Enzyme

5' Primer:: none specified

Tet-on circRNA Expression Vector vector Map

Copy Sequence View Map

Plasmid 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 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

References

Zhang Y, Xue W, Li X, et al. The Biogenesis of Nascent Circular RNAs. Cell Rep. 2016;15(3):611-624. doi:10.1016/j.celrep.2016.03.058

Tet-on circRNA Expression Vector vector Sequence

LOCUS       V000415                10054 bp    DNA     circular SYN 13-MAY-2021
DEFINITION  Exported.
ACCESSION   V000415
VERSION     V000415
KEYWORDS    Tet-on circRNA Expression Vector
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 10054)
  AUTHORS   Zhang Y, Xue W, Li X, Zhang J, Chen S, Zhang JL, Yang L, Chen LL
  TITLE     The Biogenesis of Nascent Circular RNAs.
  JOURNAL   Cell Rep. 2016 Apr 19;15(3):611-24. doi:
            10.1016/j.celrep.2016.03.058. Epub 2016 Apr 7.
   PUBMED   27068474
REFERENCE   2  (bases 1 to 10054)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 10054)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi:
            "10.1016/j.celrep.2016.03.058"; journalName: "Cell Rep"; date:
            "2016-04-19- 19"; volume: "15"; issue: "3"; pages: "611-24"
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..10054
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label="3' LTR"
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label="HIV-1 Psi"
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    1303..1536
                     /label="RRE"
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to the
                     cytoplasm."
     CDS             1721..1765
                     /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             1914..1955
                     /note="Protein Tat from Human immunodeficiency virus type 1
                     group M subtype B (isolate WMJ22). Accession#: P12509"
                     /label="Protein Tat"
     misc_feature    2058..2175
                     /label="cPPT/CTS"
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     promoter        2227..2541
                     /label="tight TRE promoter"
                     /note="Tet-responsive promoter PTight, consisting of seven
                     tet operator sequences followed by the minimal CMV
                     promoter"
     regulatory      2569..2578
                     /label="Kozak sequence"
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2620..2641)
                     /label="EGFP-N"
                     /note="EGFP, reverse primer"
     primer_bind     3942..3961
                     /label="mCherry-F"
                     /note="mCherry, forward primer"
     misc_feature    4284..4870
                     /label="IRES2"
                     /note="internal ribosome entry site (IRES) of the
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5890..5909)
                     /label="EXFP-R"
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6237..6258
                     /label="EGFP-C"
                     /note="EGFP, forward primer"
     misc_feature    6444..7032
                     /label="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             7107..7340
                     /label="3' LTR (Delta-U3)"
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(7459..7477)
                     /label="pBRforEco"
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        7545..7649
                     /label="AmpR promoter"
     CDS             7650..8507
                     /label="AmpR"
                     /note="beta-lactamase"
     rep_origin      8681..9269
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     primer_bind     9423..9440
                     /label="L4440"
                     /note="L4440 vector, forward primer"
     primer_bind     9532..9551
                     /label="SV40pro-F"
                     /note="SV40 promoter/origin, forward primer"
     protein_bind    9680..9701
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        9716..9746
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    9754..9770
                     /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     9759..9781
                     /label="M13/pUC Reverse"
                     /note="In lacZ gene"
     primer_bind     9778..9794
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     primer_bind     9778..9794
                     /label="M13 Reverse"
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     9844..9863
                     /label="EBV-rev"
                     /note="SV40 polyA terminator, reverse primer"