Firefly luciferase gene was inserted into the pNL4-3 nef gene. Two frameshifts (5Env and Vpr aa 26) render this clone Env- and Vpr-. Competent for a single round of replication. Requires cotransfection with env expression vector to produce infectious virus. Virus can be produced by transfecting 2x10^6 293 or 293T cells with 20 ug of the NL4-3 DNA, or with 10 ug NL4-3 DNA and 10 ug env expression vector DNA. Transfections can be performed in a 10 cm^2 tissue culture dish using standard calcium phosphate protocols. Virus is typically harvested 48 hours post-transfection. Infections should be performed in a total volume of 0.5 ml. Amphotropic pseudotypes generally have much higher infectivity than those bearing HIV-1 env. Cultures infected with the luciferase viruses can be lysed 2-5 days post-infection and assayed using commercial lysis buffer and luciferase reagents
| 载体基本信息 | |||
| 载体名称 | pNL4-3.Luc.R-E- | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 16390 bp | 载体类型 | Viral Expression & Packaging Vectors |
| 载体来源 | Dr. Nathaniel Landau, Aaron Diamond AIDS Research Center, The Rockefeller University. | 拷贝数 | High copy number |
| 表达方法 | Constiutive, Stable | ||
pNL4-3.Luc.R-E- 载体载体图谱
pNL4-3.Luc.R-E- 载体载体序列
LOCUS Exported 16390 bp ds-DNA circular SYN 08-OCT-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pnl4-3-luc-r-e-
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 16390)
AUTHORS Triple Threat
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..16390
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 669..773
/gene="bla"
/note="AmpR promoter"
CDS 774..1634
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 1805..2393
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
LTR 4204..4837
/note="3 LTR"
/note="3 long terminal repeat (LTR) from HIV-1"
CDS complement(5027..6673)
/codon_start=1
/gene="luc"
/product="firefly luciferase"
/note="luciferase"
/translation="EDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAH
IEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPA
NDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSM
YTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHAR
DPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKI
QSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYG
LTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSG
YVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILL
QHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVF
VDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
CDS complement(6937..6963)
/codon_start=1
/product="nuclear export signal from the HIV Rev protein
(Fischer et al., 1995)"
/note="NES"
/translation="LPPLERLTL"
misc_feature 7486..7719
/note="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS complement(10382..13393)
/codon_start=1
/gene="pol"
/product="pol protein from human immunodeficiency virus 1"
/note="HIV-1 pol"
/translation="FFREDLAFPQGKAREFSSEQTRANSPTRRELQVWGRDNNSLSEAG
ADRQGTVSFSFPQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIG
GIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNFPISPIETV
PVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPVFAIKKKD
STKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKQKKSVTVLDVGDAYFSVPLDKDFRK
YTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQCSMTKILEPFRKQNPDIVIYQYMD
DLYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPI
VLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVRQLCKLLRGTKALTEVVPLTEEAELEL
AENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMKGAHT
NDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPP
LVKLWYQLEKEPIIGAETFYVDGAANRETKLGKAGYVTDRGRQKVVPLTDTTNQKTELQ
AIHLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAH
KGIGGNEQVDKLVSAGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVA
SCDKCQLKGEAMHGQVDCSPGIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQET
AYFLLKLAGRWPVKTVHTDNGSNFTSTTVKAACWWAGIKQEFGIPYNPQSQGVIESMNK
ELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQK
QITKIQNFRVYYRDSRDPVWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQ
MAGDDCVASRQDED"
misc_feature 10580..10697
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
CDS complement(13186..14688)
/codon_start=1
/gene="gag"
/product="gag protein from human immunodeficiency virus 1"
/note="HIV-1 gag"
/translation="MGARASVLSGGELDKWEKIRLRPGGKKQYKLKHIVWASRELERFA
VNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTIAVLYCVHQRIDVKDTKEALDKI
EEEQNKSKKKAQQAAADTGNNSQVSQNYPIVQNLQGQMVHQAISPRTLNAWVKVVEEKA
FSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRLHPVHAGP
IAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVRMYSPTS
ILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPG
ATLEEMMTACQGVGGPGHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEG
HIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAP
PEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQ"
LTR 14844..15101
/note="5 LTR (truncated)"
/note="truncated 5 long terminal repeat (LTR) from HIV-1"
