Basic Vector Information
- Vector Name:
- PB-TRE-dCas9-VPR
- Antibiotic Resistance:
- Ampicillin
- Length:
- 13808 bp
- Type:
- Mammalian Expression, CRISPR, Synthetic Biology ;
- Replication origin:
- ori
- Selection Marker:
- Hygromycin
- Copy Number:
- High Copy
- Promoter:
- TRE3G
- Cloning Method:
- Gibson Cloning
- 5' Primer:
- GATTCCCCAGAAGGAAGAGG
PB-TRE-dCas9-VPR 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 ℃.
PB-TRE-dCas9-VPR vector Sequence
LOCUS 40924_5189 13808 bp DNA circular SYN 13-MAY-2021
DEFINITION SP-dCas9-VPR with doxycycline-inducible expression.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13808)
AUTHORS Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, P R Iyer E, Lin
S, Kiani S, Guzman CD, Wiegand DJ, Ter-Ovanesyan D, Braff JL,
Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ,
Church GM
TITLE Highly efficient Cas9-mediated transcriptional programming.
JOURNAL Nat Methods. 2015 Mar 2. doi: 10.1038/nmeth.3312.
PUBMED 25730490
REFERENCE 2 (bases 1 to 13808)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 13808)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Methods. 2015 Mar 2. doi: 10.1038/nmeth.3312."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..13808
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin complement(14..469)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 596..618
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 610..627
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 611..627
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
repeat_region 757..819
/label=piggyBac right (3') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
promoter 1268..1646
/label=TRE3G promoter
/note="3rd-generation Tet-responsive promoter that can be
activated by binding of Tet-On(R) 3G"
CDS 1679..5782
/codon_start=1
/label=Cas9m4
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
/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 5795..5815
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 5840..5989
/codon_start=1
/label=VP64
/note="tetrameric repeat of the minimal activation domain
of herpes simplex virus VP16 (Beerli et al., 1998)"
/translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
DLDML"
CDS 6014..6034
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
CDS 6464..6820
/codon_start=1
/label=RelA (p65) AD
/note="transcriptional activation domain of human RelA,
also known as p65 (O'Shea and Perkins, 2008)"
/translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
EDFSSIADMDFSALL"
CDS 6839..7408
/codon_start=1
/label=Rta AD
/note="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)"
/translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
DTFLNDECLLHAMHISTGLSIFDTSLF"
primer_bind complement(7463..7482)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
polyA_signal 7507..7555
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
promoter 7786..7997
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
LTR 8010..8278
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from human
T-cell leukemia virus (HTLV) type 1"
CDS 8312..9055
/codon_start=1
/label=Tet-On(R) 3G
/note="modified rtTA protein that binds tightly to
promoters containing the tet operator in the presence of
doxycycline"
/translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
DALDDFDLDMLPADALDDFDLDMLPG"
CDS 9056..9109
/codon_start=1
/label=T2A
/note="2A peptide from Thosea asigna virus capsid protein"
/translation="EGRGSLLTCGDVEENPGP"
CDS 9131..10147
/codon_start=1
/label=HygR
/note="aminoglycoside phosphotransferase from E. coli"
/translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
primer_bind complement(10207..10227)
/label=WPRE-R
/note="WPRE, reverse primer"
polyA_signal 10261..10393
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
repeat_region 11145..11179
/label=piggyBac left (5') inverted repeat
/note="piggyBac transposon-specific inverted terminal
repeat sequence (ITR)"
primer_bind complement(11670..11686)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(11670..11686)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(11683..11705)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 11694..11710
/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(11718..11748)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(11763..11784)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(11901..11918)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(12072..12660)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(12834..13691)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(13692..13796)
/label=AmpR promoter
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