TLCV2 vector (V000856)

Price Information

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V000856 TLCV2 In stock, 1 week for quality controls

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

Basic Vector Information

Vector Name:
TLCV2
Antibiotic Resistance:
Ampicillin
Length:
16733 bp
Type:
Mammalian Expression, Lentiviral, CRISPR ; Doxycyc
Replication origin:
ori
Selection Marker:
Puromycin
Promoter:
U6
5' Primer:
LNCX [5'-AGCTCGTTTAGTGAACCGTCAGATC-3']
3' Primer:
EF1a-R [5'CACGGCGACTACTGCACTTA-3']

TLCV2 vector Map

TLCV216733 bp800160024003200400048005600640072008000880096001040011200120001280013600144001520016000tet operatortet operatortet operatortet operatortet operatortet operatorminimal CMV promoterCas9nucleoplasmin NLSFLAGT2AEGFPEF-1-alpha core promoterPuroRP2ArtTA-AdvancedWPRE3' LTR (Delta-U3)bGH poly(A) signalf1 oriSV40 promoterEM7 promoterBleoRSV40 poly(A) signalIn lacZ genelac promoterCAP binding siteL4440oriAmpRAmpR promoterpRS-markerCMV enhancerCMV promoter5' LTR (truncated)HIV-1 PsiRREgp41 peptideProtein TatcPPT/CTSU6 promotergRNA scaffoldtet operator

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.

TLCV2 vector Sequence

LOCUS       V000856                16733 bp    DNA     circular SYN 13-MAY-2021
DEFINITION  Exported.
ACCESSION   V000856
VERSION     V000856
KEYWORDS    TLCV2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 16733)
  AUTHORS   Barger CJ, Branick C, Chee L, Karpf AR
  TITLE     Pan-Cancer Analyses Reveal Genomic Features of FOXM1 Overexpression
            in Cancer.
  JOURNAL   Cancers (Basel). 2019 Feb 21;11(2). pii: cancers11020251. doi:
            10.3390/cancers11020251.
   PUBMED   30795624
REFERENCE   2  (bases 1 to 16733)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 16733)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cancers
            (Basel)."; date: "2019-02-21"; pages: " 10.3390/cancers11020251"
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..16733
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    14..32
                     /label="tet operator"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    49..67
                     /gene="tetO"
                     /label="tet operator"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    85..103
                     /gene="tetO"
                     /label="tet operator"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    121..139
                     /gene="tetO"
                     /label="tet operator"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    156..174
                     /gene="tetO"
                     /label="tet operator"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    192..210
                     /gene="tetO"
                     /label="tet operator"
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        222..260
                     /label="minimal CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     primer_bind     257..281
                     /label="LNCX"
                     /note="Human CMV promoter, forward primer"
     CDS             316..4419
                     /label="Cas9"
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     CDS             4420..4467
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label="nucleoplasmin NLS"
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4468..4491
                     /label="FLAG"
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             4501..4554
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid
                     protein"
                     /label="T2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding separate
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4555..5268
                     /label="EGFP"
                     /note="enhanced GFP"
     promoter        5282..5493
                     /label="EF-1-alpha core promoter"
                     /note="core promoter for human elongation factor
                     EF-1-alpha"
     CDS             5515..6111
                     /label="PuroR"
                     /note="puromycin N-acetyltransferase"
     CDS             6121..6177
                     /label="P2A"
                     /note="2A peptide from porcine teschovirus-1 polyprotein"
     CDS             6178..6921
                     /label="rtTA-Advanced"
                     /note="improved tetracycline-controlled transactivator"
     misc_feature    6945..7533
                     /label="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             7605..7838
                     /label="3' LTR (Delta-U3)"
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    7870..8094
                     /label="bGH poly(A) signal"
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8140..8568
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        8582..8911
                     /label="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     promoter        8959..9006
                     /label="EM7 promoter"
                     /note="synthetic bacterial promoter"
     CDS             9025..9396
                     /label="BleoR"
                     /note="antibiotic-binding protein"
     polyA_signal    9529..9662
                     /label="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9699..9715)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     primer_bind     complement(9699..9715)
                     /label="M13 Reverse"
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9712..9734)
                     /label="M13/pUC Reverse"
                     /note="In lacZ gene"
     protein_bind    9723..9739
                     /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(9747..9777)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(9792..9813)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(9930..9947)
                     /label="L4440"
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10101..10689)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(10863..11720)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(11721..11825)
                     /label="AmpR promoter"
     primer_bind     complement(11900..11919)
                     /label="pRS-marker"
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     enhancer        12091..12470
                     /label="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        12471..12673
                     /label="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     LTR             12688..12868
                     /label="5' LTR (truncated)"
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    12915..13040
                     /label="HIV-1 Psi"
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    13533..13766
                     /label="RRE"
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to the
                     cytoplasm."
     CDS             13951..13995
                     /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             14144..14185
                     /note="Protein Tat from Human immunodeficiency virus type 1
                     group M subtype B (isolate WMJ22). Accession#: P12509"
                     /label="Protein Tat"
     misc_feature    14293..14410
                     /label="cPPT/CTS"
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     promoter        14461..14701
                     /label="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        16591..16666
                     /label="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes
                     CRISPR/Cas9 system"
     protein_bind    16711..16729
                     /label="tet operator"
                     /note="bacterial operator O2 for the tetR and tetA genes"