Human MIB1 ORF clone (NM_020774) (Cat. No.: V030145)
- Name:
- pLV-Tet-miniCMV-mCherry-Puro-MIB1(human)-HA-GFP nanobody
- Accession ID:
- NM_020774.4
- Antibiotic Resistance:
- Ampicillin
- Length:
- 14175 bp
- Type:
- Protein expression, Tetracycline inducible
- Replication origin:
- ori
- Host:
- Mammalian cells, Lentivirus
- Selection Marker:
- Puro;mCherry
- Promoter:
- Tet-miniCMV
- 5' Primer:
- miniCMV-F
- Fusion Tag:
- HA-GFP nanobody
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
- Expression Method:
- Tetracycline inducible
- Gene Synonyms:
- DIP-1; DIP1; LVNC7; MIB; ZZANK2; ZZZ6
- Transcript Definition:
- Homo sapiens MIB E3 ubiquitin protein ligase 1 (MIB1), 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.
Human MIB1 ORF clone (NM_020774) (Cat. No.: V030145) Sequence
LOCUS V030145 14175 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION V030145
VERSION V030145
KEYWORDS .
SOURCE .
ORGANISM .
.
FEATURES Location/Qualifiers
misc_feature 25..142
/note="central polypurine tract and central termination
sequence of HIV-1"
/label="cPPT/CTS"
protein_bind 210..228
/bound_moiety="tetracycline repressor TetR"
/gene="tetO"
/note="bacterial operator O2 for the tetR and tetA genes"
/label="tet operator"
protein_bind 245..263
/bound_moiety="tetracycline repressor TetR"
/gene="tetO"
/note="bacterial operator O2 for the tetR and tetA genes"
/label="tet operator"
protein_bind 281..299
/bound_moiety="tetracycline repressor TetR"
/gene="tetO"
/note="bacterial operator O2 for the tetR and tetA genes"
/label="tet operator"
protein_bind 317..335
/bound_moiety="tetracycline repressor TetR"
/gene="tetO"
/note="bacterial operator O2 for the tetR and tetA genes"
/label="tet operator"
protein_bind 352..370
/bound_moiety="tetracycline repressor TetR"
/gene="tetO"
/note="bacterial operator O2 for the tetR and tetA genes"
/label="tet operator"
protein_bind 388..406
/bound_moiety="tetracycline repressor TetR"
/gene="tetO"
/note="bacterial operator O2 for the tetR and tetA genes"
/label="tet operator"
promoter 418..456
/note="human cytomegalovirus (CMV) immediate early
promoter"
/label="minimal CMV promoter"
promoter 524..542
/note="promoter for bacteriophage T7 RNA polymerase"
/label="T7 promoter"
CDS 614..3631
/label="MIB1(NM_020774)"
/note="MIB1(NM_020774)"
/gene="MIB1"
CDS 3632..3658
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/transl_table=1
/translation="YPYDVPDYA"
/label="HA"
CDS 3704..4045
/codon_start=1
/product="GFP-binding fragment of a single-chain camelid
antibody (Rothbauer et al., 2008)"
/transl_table=1
/translation="VQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKERE
WVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYW
GQGTQVTVSS"
/label="GFP nanobody"
promoter 4171..4670
/note="mouse phosphoglycerate kinase 1 promoter"
/label="PGK promoter"
CDS 4691..5398
/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 5399..5455
/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 porcine teschovirus-1
polyprotein"
/transl_table=1
/translation="ATNFSLLKQAGDVEENPGP"
/label="P2A"
CDS 5456..6055
/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 6066..6654
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
/label="WPRE"
LTR 6717..6950
/note="self-inactivating 3' long terminal repeat (LTR)
from HIV-1"
/label="3' LTR (ΔU3)"
promoter complement(6975..6993)
/note="promoter for bacteriophage SP6 RNA polymerase"
/label="SP6 promoter"
promoter 7782..7886
/gene="bla"
/label="AmpR promoter"
CDS 7887..8747
/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"
rep_origin 8918..9506
/direction=RIGHT
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
/label="ori"
promoter 9752..10081
/note="SV40 enhancer and early promoter"
/label="SV40 promoter"
intron 11215..11280
/note="SV40 (simian virus 40) small t antigen intron"
/label="small t intron"
CDS 11410..11430
/codon_start=1
/locus_tag=""
/note=""
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/protein_id=""
/transl_table=1
/translation="PKKKRKV"
/label="SV40 NLS"
polyA_signal 11855..11989
/note="SV40 polyadenylation signal"
/label="SV40 poly(A) signal"
LTR 12158..12791
/note="3' long terminal repeat (LTR) from HIV-1"
/label="3' LTR"
misc_feature 12838..12963
/note="packaging signal of human immunodeficiency virus
type 1"
/label="HIV-1 Ψ"
misc_feature 13455..13688
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
/label="RRE"
CDS 13873..13917
/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"