Mouse Cd19 ORF clone (NM_009844) (Cat. No.: V021584)
- Name:
- pLVX-Puro-Cd19(mouse)-mCherry-luciferase
- Accession ID:
- NM_009844.3
- Antibiotic Resistance:
- Ampicillin
- Length:
- 12143 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- Mammalian cells, Lentivirus
- Selection Marker:
- Puro;mCherry;luciferase
- Promoter:
- CMV
- Fusion Tag:
- mCherry-luciferase
- Expression Method:
- Constiutive, Stable / Transient
- Transcript Definition:
- Mus musculus CD19 antigen (Cd19), transcript variant 1, mRNA
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.
Mouse Cd19 ORF clone (NM_009844) (Cat. No.: V021584) Sequence
LOCUS V021584 12143 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION V021584
VERSION V021584
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"
enhancer 2201..2504
/note="human cytomegalovirus immediate early enhancer"
/label="CMV enhancer"
promoter 2505..2708
/note="human cytomegalovirus (CMV) immediate early
promoter"
/label="CMV promoter"
CDS 2826..4466
/label="Cd19(NM_009844)"
/note="Cd19(NM_009844)"
/gene="Cd19"
CDS 4482..5189
/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
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEGYTIVEQYERA
EGRHSTGGMDELYK"
/label="mCherry"
CDS 5196..5249
/codon_start=1
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/product="2A peptide from Thosea asigna virus capsid
protein"
/protein_id=""
/transl_table=1
/translation="EGRGSLLTCGDVEENPGP"
/label="T2A"
CDS 5253..6902
/EC_number=""
/codon_start=1
/gene="luc+"
/note="enhanced luc+ version of the luciferase gene"
/product="firefly luciferase"
/protein_id=""
/transl_table=1
/translation="EDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAH
IEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPA
NDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSM
YTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHAR
DPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKI
QSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYG
LTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSG
YVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILL
QHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVF
VDEVPKGLTGKLDARKIREILIKAKKGGKIAV*"
/label="luciferase"
promoter 6931..7430
/note="mouse phosphoglycerate kinase 1 promoter"
/label="PGK promoter"
CDS 7451..8050
/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 8064..8652
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
/label="WPRE"
LTR 8859..9492
/note="3' long terminal repeat (LTR) from HIV-1"
/label="3' LTR"
primer_bind complement(9621..9637)
/note="common sequencing primer, one of multiple similar
variants"
/label="M13 rev"
protein_bind 9645..9661
/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(9669..9699)
/note="promoter for the E. coli lac operon"
/label="lac promoter"
protein_bind 9714..9735
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
/label="CAP binding site"
rep_origin complement(10023..10611)
/direction=LEFT
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
/label="ori"
CDS complement(10782..11642)
/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(11643..11747)
/gene="bla"
/label="AmpR promoter"
polyA_signal 11795..11929
/note="SV40 polyadenylation signal"
/label="SV40 poly(A) signal"