Basic Vector Information
- Vector Name:
- pLenti-HF1RA-P2A-GFP-PGK-Puro
- Antibiotic Resistance:
- Ampicillin
- Length:
- 12448 bp
- Type:
- Lentiviral
- Replication origin:
- ori
- Selection Marker:
- Puromycin ; GFP
- Copy Number:
- Low Copy
- Promoter:
- mPGK
- Cloning Method:
- Gibson Cloning
- 5' Primer:
- TAAGTGCAGTAGTCGCCGTG
- 3' Primer:
- TTAAGAATACCAGTCAATCTTTC
pLenti-HF1RA-P2A-GFP-PGK-Puro 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 ℃.
pLenti-HF1RA-P2A-GFP-PGK-Puro vector Sequence
LOCUS 40924_27742 12448 bp DNA circular SYN 13-MAY-2021 DEFINITION Lentiviral vector for constitutive expression of Cas9-HF1RA-P2A-GFP in mammalian cells (codon optimized). ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 12448) AUTHORS Zafra MP, Schatoff EM, Katti A, Foronda M, Breinig M, Schweitzer AY, Simon A, Han T, Goswami S, Montgomery E, Thibado J, Kastenhuber ER, Sanchez-Rivera FJ, Shi J, Vakoc CR, Lowe SW, Tschaharganeh DF, Dow LE TITLE Optimized base editors enable efficient editing in cells, organoids and mice. JOURNAL Nat Biotechnol. 2018 Jul 3. pii: nbt.4194. doi: 10.1038/nbt.4194. PUBMED 29969439 REFERENCE 2 (bases 1 to 12448) TITLE Direct Submission REFERENCE 3 (bases 1 to 12448) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat Biotechnol. 2018 Jul 3. pii: nbt.4194. doi: 10.1038/nbt.4194." COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..12448 /mol_type="other DNA" /organism="synthetic DNA construct" LTR 222..402 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 449..574 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1067..1300 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1485..1529 /codon_start=1 /label=gp41 peptide /note="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /translation="KNEQELLELDKWASL" misc_feature 1713..1830 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 1899..2110 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" regulatory 2130..2139 /label=Kozak sequence /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" /regulatory_class="other" CDS 2139..2162 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 2169..2189 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 2214..6314 /codon_start=1 /label=SpCas9-HF1 /note="Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system, mutated to improve targeting specificity (Kleinstiver et al., 2016)" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 6315..6362 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 6372..6428 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 6429..7148 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(6474..6495) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(6735..6754) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 7082..7103 /label=EGFP-C /note="EGFP, forward primer" promoter 7160..7659 /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" CDS 7680..8276 /codon_start=1 /label=PuroR /note="puromycin N-acetyltransferase" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" misc_feature 8295..8883 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" LTR 8955..9188 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 9266..9400 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" rep_origin 9427..9562 /label=SV40 ori /note="SV40 origin of replication" promoter complement(9583..9601) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(9611..9628) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(9611..9627) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(9620..9642) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 9769..10224 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(9856..9875) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 10066..10087 /label=F1ori-F /note="F1 origin, forward primer" promoter 10250..10354 /label=AmpR promoter CDS 10355..11212 /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 11386..11974 /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 12128..12145 /label=L4440 /note="L4440 vector, forward primer" protein_bind 12262..12283 /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." promoter 12298..12328 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 12336..12352 /label=lac operator /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 12360..12376 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" promoter 12397..12415 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase"
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