Basic Vector Information
- Vector Name:
- Flag-HA-USP53
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9964 bp
- Type:
- Mammalian Expression, Retroviral
- Replication origin:
- ori
- Selection Marker:
- Puromycin
- Copy Number:
- High Copy
- Promoter:
- MSCV
- Cloning Method:
- Gateway Cloning
- 5' Primer:
- CMV-F
- 3' Primer:
- MSCV-rev
Flag-HA-USP53 vector Vector Map
Plasmid Resuspension 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 ℃.
Flag-HA-USP53 vector Sequence
LOCUS 40924_830 9964 bp DNA circular SYN 13-MAY-2021
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9964)
AUTHORS Sowa ME, Bennett EJ, Gygi SP, Harper JW
TITLE Defining the human deubiquitinating enzyme interaction landscape.
JOURNAL Cell. 2009 Jul 23. 138(2):389-403.
PUBMED 19615732
REFERENCE 2 (bases 1 to 9964)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 9964)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2009
Jul 23. 138(2):389-403."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9964
/mol_type="other DNA"
/organism="synthetic DNA construct"
LTR 1..517
/label=5' LTR
/note="5' long terminal repeat from murine embryonic stem
cell virus"
misc_feature 581..922
/label=MESV Psi
/note="packaging signal of murine embryonic stem cell
virus"
CDS 989..1405
/codon_start=1
/label=gag (truncated)
/note="truncated Moloney murine leukemia virus (MMLV) gag
gene lacking the start codon"
/translation="GQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWPTF
NVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVHPKP
PPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGA"
enhancer 1420..1799
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 1800..2003
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
protein_bind 2005..2023
/label=tet operator
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 2026..2044
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
primer_bind 2054..2078
/label=LNCX
/note="Human CMV promoter, forward primer"
regulatory 2151..2160
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 2160..2183
/codon_start=1
/label=FLAG
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/translation="DYKDDDDK"
CDS 2196..2222
/codon_start=1
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
/translation="YPYDVPDYA"
protein_bind 2238..2262
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
protein_bind complement(5290..5310)
/label=attB2
/note="core recombination site for the Gateway(R) BP
reaction"
promoter 5330..5829
/label=PGK promoter
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 5850..6446
/codon_start=1
/label=PuroR
/note="puromycin N-acetyltransferase"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
protein_bind 7035..7051
/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(7059..7089)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(7104..7125)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(7242..7259)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(7413..8001)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8175..9032)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(9033..9137)
/label=AmpR promoter
primer_bind 9205..9223
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
primer_bind complement(9261..9283)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind 9383..9402
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 9596..9618
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 9746..9765
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
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