LOCUS Exported 12448 bp ds-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 pLenti-HF1RA-P2A-GFP-PGK-Puro
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)
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."
FEATURES Location/Qualifiers
source 1..12448
/organism="synthetic DNA construct"
/mol_type="other DNA"
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
/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 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
/regulatory_class="other"
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
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
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/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"
primer_bind complement(7195..7218)
/label=MSCV-rev
/note="Murine stem cell virus, reverse primer"
primer_bind 7592..7611
/label=mPGK-F
/note="Mouse PGK promoter, forward primer"
CDS 7680..8279
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind complement(7680..7699)
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
primer_bind 8176..8196
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
misc_feature 8295..8883
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(8348..8368)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(8766..8777)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 8955..9188
/label=3 LTR (Delta-U3)
/note="self-inactivating 3 long terminal repeat (LTR) from
HIV-1"
polyA_signal 9266..9387
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(9303..9322)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 9357..9376
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin 9427..9562
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 9489..9508
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
primer_bind complement(9582..9601)
/label=T7
/note="T7 promoter, forward primer"
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
/gene="bla"
/label=AmpR promoter
CDS 10355..11215
/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 complement(10573..10592)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 11386..11974
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 11875..11894
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 12128..12145
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 12262..12283
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/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
/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 12341..12363
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 12360..12376
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 12360..12376
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 12395..12415
/label=T3
/note="T3 promoter, forward primer"
promoter 12397..12415
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter join(12443..12448,1..221)
/label=RSV promoter
/note="Rous sarcoma virus enhancer/promoter"
