LOCUS Exported 15702 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION 3rd generation lenti vector encoding dCas9 (S. pyogenes) fused with
VP64-p65-Rta (VPR) and 2A puromycin resistance marker;
EF1a-dCas9-VPR-P2A-Puro-WPRE).
ACCESSION .
VERSION .
KEYWORDS lenti-EF1a-dCas9-VPR-Puro
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 15702)
AUTHORS Ho SM, Hartley BJ, Flaherty E, Rajarajan P, Abdelaal R, Obiorah I,
Barretto N, Muhammad H, Phatnani HP, Akbarian S, Brennand KJ
TITLE Evaluating Synthetic Activation and Repression of
Neuropsychiatric-Related Genes in hiPSC-Derived NPCs, Neurons, and
Astrocytes.
JOURNAL Stem Cell Reports. 2017 Aug 8;9(2):615-628. doi:
10.1016/j.stemcr.2017.06.012. Epub 2017 Jul 27.
PUBMED 28757163
REFERENCE 2 (bases 1 to 15702)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1016/j.stemcr.2017.06.012"; journalName: "Stem Cell Reports";
date: "2017-08-8- 8"; volume: "9"; issue: "2"; pages: "615-628"
FEATURES Location/Qualifiers
source 1..15702
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 130..1308
/label=EF-1-alpha promoter
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
intron 361..1299
/label=EF-1-alpha intron A
/note="intron upstream of the start codon of human
EF-1-alpha"
primer_bind 1256..1276
/label=EF1a-F
/note="Human elongation factor-1a promoter, forward primer"
regulatory 1333..1342
/regulatory_class="other"
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1339..5442
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/label=Cas9m4
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the mutations D10A, D839A,
H840A, and N863A (Mali et al., 2013)"
/translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 5455..5475
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
protein_bind 5512..5536
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 5566..5715
/codon_start=1
/product="tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al., 1998)"
/label=VP64
/translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
DLDML"
CDS 5740..5760
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 6190..6546
/codon_start=1
/product="transcriptional activation domain of human RelA,
also known as p65 (OShea and Perkins, 2008)"
/label=RelA (p65) AD
/translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
EDFSSIADMDFSALL"
CDS 6565..7134
/codon_start=1
/product="transcriptional activation domain from the human
herpesvirus 4 (Epstein-Barr virus) replication and
transcription activator Rta/BRLF1 (Hardwick et al., 1992;
Chavez et al., 2015)"
/label=Rta AD
/translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
DTFLNDECLLHAMHISTGLSIFDTSLF"
CDS 7150..7206
/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 7219..7812
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="EYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERVT
ELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAAQ
QQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSAP
RNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind 7709..7729
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
misc_feature 7837..8425
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(7890..7910)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(8308..8319)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
primer_bind complement(8428..8444)
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8429..8445)
/label=pBluescriptKS
/note="For pBluescript vector"
LTR 8950..9130
/label=5 LTR (truncated)
/note="truncated 5 long terminal repeat (LTR) from HIV-1"
primer_bind complement(9156..9173)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 9162..9386
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 9432..9860
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(9519..9538)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 9729..9750
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(9869..9889)
/label=pBABE 3
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 9874..10203
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 10054..10189
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 10116..10135
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 10251..10298
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 10317..10691
/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 10821..10942
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(10858..10877)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 10912..10931
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(10991..11007)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(10991..11007)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(11004..11026)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 11015..11031
/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(11039..11069)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 11084..11105
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(11222..11239)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(11393..11981)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(11473..11492)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(12152..13012)
/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 12775..12794
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(13013..13117)
/gene="bla"
/label=AmpR promoter
primer_bind complement(13192..13211)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 13383..13762
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 13764..13962
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 13917..13937
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
LTR 13980..14160
/label=5 LTR (truncated)
/note="truncated 5 long terminal repeat (LTR) from HIV-1"
misc_feature 14207..14332
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 14825..15058
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 15243..15287
/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 15585..15702
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
