Mouse Siglecg ORF clone (NM_172900) (Cat. No.: V034712)

Copy Sequence View Map

Basic Information

Note: PLV-EF1a-IRES-Puro-Siglecg(mouse)-APEX2 is a lentiviral proximity labeling vector expressing mouse Siglecg-APEX2 fusion under EF1a promoter, with IRES-mediated puromycin selection for mammalian proteomics.

Name:: PLV-EF1a-IRES-Puro-Siglecg(mouse)-APEX2

Accession ID:: NM_172900.4

Antibiotic Resistance:: Ampicillin

Length:: 11538 bp

Type:: Protein expression

Replication origin:: ori

Host:: Mammalian cells, Lentivirus

Selection Marker:: Puro

Promoter:: EF1a

5' Primer:: EF1a-F3

Fusion Tag:: APEX2

Growth Strain(s):: Stbl3

Growth Temperature:: 37℃

Expression Method:: Constiutive, Stable / Transient

Gene Synonyms:: 9830164H23; A630096C01Rik; mSiglec-G; Siglec-G; Siglec10

Transcript Definition:: Mus musculus sialic acid binding Ig-like lectin G (Siglecg), transcript variant 1, mRNA

Cat. No.: V034712 PLV-EF1a-IRES-Puro-Siglecg(mouse)-APEX2

$ 298.5

In stock, instant shipping

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

Resources

Transcript ID

NM_172900.4

Fusion Tag

3xFLAG Myc-FLAG APEX2 FLAG Myc

Selection Marker

Neo/G418 Puro Puro;CopGFP

Expression Method

Transient Constiutive, Stable / Transient

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.

References

Martell JD, Deerinck TJ, Lam SS, Ellisman MH, Ting AY. Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells. Nat Protoc. 2017 Sep; 12(9):1792-1816. doi: 10.1038/nprot.2017.065. PMID: 28796234.

Ben-Dor I, Itsykson P, Goldenberg D, Galun E, Reubinoff BE. Lentiviral vectors harboring a dual-gene system allow high and homogeneous transgene expression in selected polyclonal human embryonic stem cells. Mol Ther. 2006 Aug; 14(2):255-67. doi: 10.1016/j.ymthe.2006.02.010. PMID: 16632408.

Angata T, Hingorani R, Varki NM, Varki A. Cloning and characterization of a novel mouse Siglec, mSiglec-F: differential evolution of the mouse and human (CD33) Siglec-3-related gene clusters. J Biol Chem. 2001 Nov 30; 276(48):45128-36. doi: 10.1074/jbc.M108573200. PMID: 11579105.

Mouse Siglecg ORF clone (NM_172900) (Cat. No.: V034712) Sequence

LOCUS       V034712                11538 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V034712
VERSION     V034712
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     LTR             1..634
                     /note="3' long terminal repeat (LTR) from HIV-1"
                     /label="3' LTR"
     misc_feature    681..806
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
                     /label="HIV-1 Ψ"
     misc_feature    1303..1536
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to the
                     cytoplasm."
                     /label="RRE"
     CDS             1721..1765
                     /codon_start=1
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /product="antigenic peptide corresponding to amino acids
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
                     et al., 2013)"
                     /transl_table=1
                     /translation="KNEQELLELDKWASL"
                     /label="gp41 peptide"
     misc_feature    2028..2143
                     /note="central polypurine tract and central termination
                     sequence of HIV-1 (lacking the first T)"
                     /label="cPPT/CTS"
     promoter        2221..2432
                     /note="core promoter for human elongation factor
                     EF-1α"
                     /label="EF-1α core promoter"
     intron          2433..3371
                     /note="intron upstream of the start codon of human EF-1α"
                     /label="EF-1α intron A"
     CDS             3393..5456
                     /label="Siglecg(NM_172900)"
                     /note="Siglecg(NM_172900)"
                     /gene="Siglecg"
     CDS             5466..6215
                     /codon_start=1
                     /note="more active than the original APEX due to the A134P
                     mutation"
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell
                     proteomics (Lam et al., 2015)"
                     /transl_table=1
                     /translation="MGKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAG
                     TFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVA
                     VEVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGH
                     TIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVD
                     KYAADEDAFFADYAEAHQKLSELGFADA"
                     /label="APEX2"
     misc_feature    6332..6881
                     /note="internal ribosome entry site (IRES) of the
                     encephalomyocarditis virus (EMCV)"
                     /label="IRES"
     CDS             6884..7483
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /note="confers resistance to puromycin"
                     /product="puromycin N-acetyltransferase"
                     /transl_table=1
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA*"
                     /label="PuroR"
     misc_feature    7507..8095
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
                     /label="WPRE"
     LTR             8302..8935
                     /note="3' long terminal repeat (LTR) from HIV-1"
                     /label="3' LTR"
     primer_bind     complement(9063..9079)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    9087..9103
                     /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(9111..9141)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    9156..9177
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(9465..10053)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(10224..11084)
                     /codon_start=1
                     /gene="bla"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /product="β-lactamase"
                     /transl_table=1
                     /translation="MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEY
                     SPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR
                     WEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS
                     ALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA
                     SLIKHW*"
                     /label="AmpR"
     promoter        complement(11085..11189)
                     /gene="bla"
                     /label="AmpR promoter"
     polyA_signal    11237..11371
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"