Human TSPAN4 ORF clone (NM_001025237) (Cat. No.: V039551)

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Basic Information

Note: It is designed for expression of the human TSPAN4 gene with an mCherry reporter and FLAG tag, driven by the mCMV promoter. The vector contains an SV40 poly(A) signal and a loxP site. It also features Ad5 Ψ, ori, and AmpR for replication and selection.

Name:: pDC316-TSPAN4(human)-mCherry-FLAG

Accession ID:: NM_001025237.2

Antibiotic Resistance:: Ampicillin

Length:: 5380 bp

Type:: Protein expression

Replication origin:: ori

Host:: Mammalian cells, Adenovirus

Selection Marker:: mCherry

Copy Number:: High

Promoter:: mCMV

3' Primer:: Sv40-polyA-R

Fusion Tag:: mCherry-FLAG

Growth Strain(s):: DH10B

Growth Temperature:: 37℃

Expression Method:: Transient

Gene Synonyms:: NAG-2; NAG2; TETRASPAN; TM4SF7; TSPAN-4

Transcript Definition:: Homo sapiens tetraspanin 4 (TSPAN4), transcript variant 1, mRNA

Cat. No.: V039551 pDC316-TSPAN4(human)-mCherry-FLAG

$ 298.4

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Two tubes of transfection grade plasmid, each tube is about 100 µg (lyophilized).

Resources

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.

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.

Human TSPAN4 ORF clone (NM_001025237) (Cat. No.: V039551) Sequence

LOCUS       V039551                 5380 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V039551
VERSION     V039551
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     misc_signal     190..340
                     /note="packaging signal for adenovirus serotype 5"
                     /label="Ad5 Ψ"
     promoter        470..992
                     /label="mCMV promoter"
     CDS             1005..1712
                     /codon_start=1
                     /note="mammalian codon-optimized"
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /transl_table=1
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
                     /label="mCherry"
     CDS             1743..2456
                     /label="TSPAN4(NM_001025237)"
                     /note="TSPAN4(NM_001025237)"
                     /gene="TSPAN4"
     CDS             2457..2480
                     /codon_start=1
                     /product="FLAG® epitope tag, followed by an enterokinase
                     cleavage site"
                     /transl_table=1
                     /translation="DYKDDDDK"
                     /label="FLAG"
     polyA_signal    2503..2637
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     protein_bind    complement(2657..2690)
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core
                     sequence (ATGTATGC) (Shaw et al., 2021)."
                     /label="loxP"
     primer_bind     complement(2712..2728)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    2736..2752
                     /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-β-D-thiogalactopyranoside (IPTG)."
                     /label="lac operator"
     promoter        complement(2760..2790)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    2805..2826
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(3114..3702)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(3873..4733)
                     /codon_start=1
                     /gene="bla"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /product="β-lactamase"
                     /transl_table=1
                     /translation="MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEY
                     SPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR
                     WEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS
                     ALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA
                     SLIKHW*"
                     /label="AmpR"
     promoter        complement(4734..4838)
                     /gene="bla"
                     /label="AmpR promoter"