psPAX2-D64V vector (Cat. No.: V001086)

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Basic Information

Note: psPAX2-D64V is derived from psPAX2 plasmid. It is a packaging plasmid for making Integrase deficient lentiviral vectors since has a point mutation in the integrase gene.

Name:: psPAX2-D64V

Antibiotic Resistance:: Ampicillin

Length:: 10711 bp

Type:: integrase deficient lentiviral packaging vector

Replication origin:: ori

Growth Strain(s):: stbl3

Growth Temperature:: 37℃

$ 198.8

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Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Resources

Plasmid 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 ℃.

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.

References

Certo MT, Ryu BY, Annis JE, Garibov M, Jarjour J, Rawlings DJ, Scharenberg AM. Tracking genome engineering outcome at individual DNA breakpoints. Nat Methods. 2011 Jul 10;8(8):671-6. doi: 10.1038/nmeth.1648.

psPAX2-D64V vector (Cat. No.: V001086) Sequence

LOCUS       psPAX2-D64V            10711 bp    DNA     circular SYN 26-DEC-2025
DEFINITION  psPAX2-D64V is derived from psPAX2 plasmid. It is a packaging 
            plasmid for making Integrase deficient lentiviral vectors since has 
            a point mutation in the integrase gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10711)
  AUTHORS   Certo MT, Ryu BY, Annis JE, Garibov M, Jarjour J, Rawlings DJ, 
            Scharenberg AM
  TITLE     Tracking genome engineering outcome at individual DNA breakpoints.
  JOURNAL   Nat Methods. 2011 Jul 10;8(8):671-6. doi: 10.1038/nmeth.1648.
  PUBMED    21743461
REFERENCE   2  (bases 1 to 10711)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 10711)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nmeth"; 
            journalName: "Nat Methods"; date: "2011-07-10- 10"; volume: "8"; 
            issue: "8"; pages: "671-6"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..10711
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          2807..2873
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..196
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      47..182
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     109..128
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    202..336
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(239..258)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     293..312
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(404..421)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(575..1163)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(655..674)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1334..2194)
                     /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     1957..1976
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2195..2299)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        2330..2709
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2711..2988
                     /label=chicken beta-actin promoter
     intron          2989..4005
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3929..3951
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4013..4032
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids,
                     forward primer"
     CDS             4111..5613
                     /codon_start=1
                     /gene="gag"
                     /product="gag protein from human immunodeficiency virus 1"
                     /label=HIV-1 gag
                     /translation="MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFA
                     VNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALDKI
                     EEEQNKSKKKAQQAAADTGHSNQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKA
                     FSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGP
                     IAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVRMYSPTS
                     ILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPG
                     ATLEEMMTACQGVGGPGHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEG
                     HIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAP
                     PEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQ"
     CDS             5406..8417
                     /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
                     SCDKCQLKGEAMHGQVDCSPGIWQLVCTHLEGKVILVAVHVASGYIEAEVIPAETGQET
                     AYFLLKLAGRWPVKTVHTDNGSNFTSTTVKAACWWAGIKQEFGIPYNPQSQGVIESMNK
                     ELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQK
                     QITKIQNFRVYYRDSRDPVWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQ
                     MAGDDCVASRQDED"
     misc_feature    8102..8219
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     misc_feature    8991..9224
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             9409..9453
                     /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             9747..9773
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     primer_bind     complement(10058..10077)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    10123..10178
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(10177..10196)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also
                     called rb-glob-pA-term-R"
     primer_bind     complement(10539..10555)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10539..10555)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10552..10574)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10563..10579
                     /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(10587..10617)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10632..10653
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."