Basic Vector Information
Translation reporter: Encodes Histone H2B (125 aa) fused to smFLAG (Spaghetti Monster FLAG: 10 copies of an epitope tag FLAG).
pUB_smFLAG_H2B_MS2 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 ℃.
pUB_smFLAG_H2B_MS2 vector Sequence
LOCUS Exported 9371 bp DNA circular SYN 06-NOV-2023
DEFINITION Translation reporter: Encodes Histone H2B (125 aa) fused to smFLAG
(Spaghetti Monster FLAG: 10 copies of an epitope tag FLAG)..
ACCESSION .
VERSION .
KEYWORDS pUB_smFLAG_H2B_MS2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9371)
AUTHORS Morisaki T, Lyon K, DeLuca KF, DeLuca JG, English BP, Zhang Z, Lavis
LD, Grimm JB, Viswanathan S, Looger LL, Lionnet T, Stasevich TJ
TITLE Real-time quantification of single RNA translation dynamics in
living cells.
JOURNAL Science. 2016 Jun 17;352(6292):1425-9. doi: 10.1126/science.aaf0899.
Epub 2016 May 5.
PUBMED 27313040
REFERENCE 2 (bases 1 to 9371)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1126/science.aaf0899"; journalName: "Science"; date:
"2016-06-17- 17"; volume: "352"; issue: "6292"; pages: "1425-9"
FEATURES Location/Qualifiers
source 1..9371
/mol_type="other DNA"
/organism="synthetic DNA construct"
LTR 1..634
/label=3' LTR
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 681..806
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1303..1536
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1721..1765
/codon_start=1
/product="antigenic peptide corresponding to amino acids
655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
et al., 2013)"
/label=gp41 peptide
/note="recognized by the 2H10 single-chain llama nanobody"
/translation="KNEQELLELDKWASL"
misc_feature 2058..2175
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2218..2616
/label=UbC promoter
/note="human ubiquitin C promoter"
regulatory 2622..2631
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 2631..2654
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 2658..2681
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 2685..2708
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 3213..3236
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 3243..3266
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 3279..3302
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 3309..3332
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 3525..3548
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 3552..3575
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 3579..3602
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 3639..4013
/codon_start=1
/gene="HIST1H2BJ"
/product="human histone H2B"
/label=H2B
/translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
ELAKHAVSEGTKAITKYTSAK"
misc_feature 5761..6349
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(5814..5834)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(6232..6243)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 6424..6657
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
primer_bind complement(6776..6794)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 6862..6966
/gene="bla"
/label=AmpR promoter
CDS 6967..7827
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(7185..7204)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 7998..8586
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 8487..8506
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 8740..8757
/label=L4440
/note="L4440 vector, forward primer"
primer_bind 8849..8868
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
protein_bind 8997..9018
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 9033..9063
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 9071..9087
/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)."
primer_bind 9076..9098
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 9095..9111
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 9095..9111
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 9161..9180
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
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