Basic Vector Information
| Basic Vector Information | |||
|---|---|---|---|
| Vector Name | NL4-3 mCherry Luciferase | Antibiotic Resistance | Ampicillin |
| Length | 17833 bp | Type | Lentiviral |
| Selection Marker | mCherry, Luciferase | Promoter | EF-1 alpha– driven mCherry:Puromycin |
| Cloning Method | Restriction Enzyme | 5' Primer | from pNLENG1 5' |
| 3' Primer | from pNLENG1 3' | ||
NL4-3 mCherry Luciferase 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 ℃.
NL4-3 mCherry Luciferase vector Sequence
LOCUS Exported 17833 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION HIV dual reporter vector expressing mCh.
ACCESSION .
VERSION .
KEYWORDS NL4-3 mCherry Luciferase
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 17833)
AUTHORS Lassen KG, Hebbeler AM, Bhattacharyya D, Lobritz MA, Greene WC
TITLE A flexible model of HIV-1 latency permitting evaluation of many
primary CD4 T-cell reservoirs.
JOURNAL PLoS One. 2012;7(1):e30176. doi: 10.1371/journal.pone.0030176. Epub
2012 Jan 24.
PUBMED 22291913
REFERENCE 2 (bases 1 to 17833)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
date: "2012"; volume: "7(1):e30176. doi"; pages: "
10.1371/journal.pone.0030176. Epub 2012 Jan 24"
FEATURES Location/Qualifiers
source 1..17833
/organism="synthetic DNA construct"
/mol_type="other DNA"
LTR 377..634
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
CDS 790..2292
/codon_start=1
/gene="gag"
/product="gag protein from human immunodeficiency virus 1"
/label=HIV-1 gag
/translation="MGARASVLSGGELDKWEKIRLRPGGKKQYKLKHIVWASRELERFA
VNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTIAVLYCVHQRIDVKDTKEALDKI
EEEQNKSKKKAQQAAADTGNNSQVSQNYPIVQNLQGQMVHQAISPRTLNAWVKVVEEKA
FSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRLHPVHAGP
IAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVRMYSPTS
ILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPG
ATLEEMMTACQGVGGPGHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEG
HIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAP
PEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQ"
CDS 2085..5096
/codon_start=1
/gene="pol"
/product="pol protein from human immunodeficiency virus 1"
/label=HIV-1 pol
/translation="FFREDLAFPQGKAREFSSEQTRANSPTRRELQVWGRDNNSLSEAG
ADRQGTVSFSFPQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIG
GIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNFPISPIETV
PVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPVFAIKKKD
STKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKQKKSVTVLDVGDAYFSVPLDKDFRK
YTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQCSMTKILEPFRKQNPDIVIYQYMD
DLYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPI
VLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVRQLCKLLRGTKALTEVVPLTEEAELEL
AENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMKGAHT
NDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPP
LVKLWYQLEKEPIIGAETFYVDGAANRETKLGKAGYVTDRGRQKVVPLTDTTNQKTELQ
AIHLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAH
KGIGGNEQVDKLVSAGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVA
SCDKCQLKGEAMHGQVDCSPGIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQET
AYFLLKLAGRWPVKTVHTDNGSNFTSTTVKAACWWAGIKQEFGIPYNPQSQGVIESMNK
ELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQK
QITKIQNFRVYYRDSRDPVWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQ
MAGDDCVASRQDED"
misc_feature 4781..4898
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
misc_feature 7759..7992
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 8177..8221
/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"
CDS 8515..8541
/codon_start=1
/product="nuclear export signal from the HIV Rev protein
(Fischer et al., 1995)"
/label=NES
/translation="LPPLERLTL"
CDS 8787..9494
/codon_start=1
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/label=mCherry
/note="mammalian codon-optimized"
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEGYTIVEQYERA
EGRHSTGGMDELYK"
primer_bind complement(8932..8950)
/label=mCherry-R
/note="mCherry, reverse primer"
primer_bind 9200..9219
/label=mCherry-F
/note="mCherry, forward primer"
CDS 9501..9554
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 9558..11207
/codon_start=1
/gene="luc+"
/product="firefly luciferase"
/label=luciferase
/note="enhanced luc+ version of the luciferase gene"
/translation="EDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAH
IEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPA
NDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSM
YTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHAR
DPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKI
QSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYG
LTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSG
YVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILL
QHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVF
VDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
primer_bind complement(9621..9639)
/label=LucNrev
/note="Firefly luciferase, reverse primer"
misc_feature 11212..11794
/label=IRES2
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
primer_bind complement(11378..11395)
/label=IRES reverse
/note="IRES internal ribosome entry site, reverse primer.
Also called pCDH-rev"
primer_bind 11605..11624
/label=IRES-F
/note="IRES internal ribosome entry site, forward primer"
LTR 12084..12717
/label=3' LTR
/note="3' long terminal repeat (LTR) from HIV-1"
primer_bind complement(14357..14374)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(14528..15116)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(14608..14627)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(15287..16147)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind 15910..15929
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(16148..16252)
/gene="bla"
/label=AmpR promoter
primer_bind 16320..16338
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
primer_bind complement(16376..16398)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind 16498..16517
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
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