LOCUS Exported 15072 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Variant of lentiCRISPRv2 that confers G418 resistance.
ACCESSION .
VERSION .
KEYWORDS lentiCRISPRv2 neo
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 15072)
AUTHORS Stringer BW, Day BW, DSouza RCJ, Jamieson PR, Ensbey KS, Bruce ZC,
Lim YC, Goasdoue K, Offenhauser C, Akgul S, Allan S, Robertson T,
Lucas P, Tollesson G, Campbell S, Winter C, Do H, Dobrovic A, Inglis
PL, Jeffree RL, Johns TG, Boyd AW
TITLE A reference collection of patient-derived cell line and xenograft
models of proneural, classical and mesenchymal glioblastoma.
JOURNAL Sci Rep. 2019 Mar 20;9(1):4902. doi: 10.1038/s41598-019-41277-z.
PUBMED 30894629
REFERENCE 2 (bases 1 to 15072)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep.";
date: "2019-03-20"; volume: "9(1):4902. doi"; pages: "
10.1038/s41598-019-41277-z"
FEATURES Location/Qualifiers
source 1..15072
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(47..66)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 242..621
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 623..821
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 776..796
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
LTR 839..1019
/label=5 LTR (truncated)
/note="truncated 5 long terminal repeat (LTR) from HIV-1"
misc_feature 1066..1191
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1684..1917
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 2102..2146
/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 2444..2561
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2612..2852
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 2612..2632
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 2783..2802
/label=LKO.1 5
/note="Human U6 promoter, forward primer"
misc_RNA 4742..4817
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 4879..5090
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
regulatory 5109..5118
/regulatory_class="other"
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 5115..9218
/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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 9219..9266
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 9267..9290
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 9300..9356
/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 9357..10148
/codon_start=1
/gene="aph(3)-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="IEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPV
LFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLSS
HLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQG
LAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIAL
ATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(9408..9427)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 10018..10037
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
misc_feature 10164..10752
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(10217..10237)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(10635..10646)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 10824..11057
/label=3 LTR (Delta-U3)
/note="self-inactivating 3 long terminal repeat (LTR) from
HIV-1"
primer_bind complement(11083..11100)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 11089..11313
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 11359..11787
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(11446..11465)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 11656..11677
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(11796..11816)
/label=pBABE 3
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 11801..12130
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 11981..12116
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 12043..12062
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 12178..12225
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 12244..12618
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/label=BleoR
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
polyA_signal 12748..12869
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(12785..12804)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 12839..12858
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(12918..12934)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(12918..12934)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(12931..12953)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 12942..12958
/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(12966..12996)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 13011..13032
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(13149..13166)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(13320..13908)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(13400..13419)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(14079..14939)
/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 14702..14721
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(14940..15044)
/gene="bla"
/label=AmpR promoter
