NLSx3-CRY2PHR-p65 载体 (V000077#)

载体基本信息
载体名称 NLSx3-CRY2PHR-p65 载体抗性 Ampicillin
载体长度 8026 bp 载体类型 Mammalian Expression
筛选标记 Neomycin (select with G418) 拷贝数 High Copy
启动子 CMV 克隆方法 Restriction Enzyme
5'引物 CMV-F 3'引物 BGH Reverse

NLSx3-CRY2PHR-p65 载体载体图谱

NLSx3-CRY2PHR-p658026 bp400800120016002000240028003200360040004400480052005600600064006800720076008000pRS-markerCMV enhancerCMV promoterT7Kozak sequenceSV40 NLSSV40 NLSCRY2PHRHARelA (p65) ADV5 tag6xHisBGH-revf1 oripBABE 3'NeoR/KanRSV40 poly(A) signalM13 revlac operatorlac promoterCAP binding siteL4440oriAmpRAmpR promoter

NLSx3-CRY2PHR-p65 载体载体序列

LOCUS       Exported                8026 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Photoactivatable transcription system. Expression of activator 
            probe, which includes the photolyase homology region of CRY2 
            (CRY2PHR) and the transcriptional activator domain..
ACCESSION   .
VERSION     .
KEYWORDS    NLSx3-CRY2PHR-p65
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8026)
  AUTHORS   Nihongaki Y, Yamamoto S, Kawano F, Suzuki H, Sato M
  TITLE     CRISPR-Cas9-based Photoactivatable Transcription System.
  JOURNAL   Chem Biol. 2015 Feb 19;22(2):169-74. doi: 
            10.1016/j.chembiol.2014.12.011. Epub 2015 Jan 22.
  PUBMED    25619936
REFERENCE   2  (bases 1 to 8026)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.chembiol.2014.12.011"; journalName: "Chem Biol"; date: 
            "2015-02-19- 19"; volume: "22"; issue: "2"; pages: "169-74"
FEATURES             Location/Qualifiers
     source          1..8026
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      908..917
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             923..943
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             959..979
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1085..2578
                     /codon_start=1
                     /product="N-terminal photolyase homology region of 
                     Arabidopsis thaliana cryptochrome 2"
                     /label=CRY2PHR
                     /note="binds to CIB1 upon exposure to blue light (Kennedy 
                     et al., 2010)"
                     /translation="MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQ
                     FYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLY
                     DPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSI
                     ESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLN
                     EFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGE
                     ESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPL
                     VDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQY
                     ISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKA
                     SGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAA"
     CDS             2636..2662
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3092..3463
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALLSQISS"
     CDS             3524..3565
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3575..3592
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3615..3632)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3621..3845
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3891..4319
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3978..3997)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4188..4209
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4328..4348)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4333..4662
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4513..4648
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4575..4594
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4729..5523
                     /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4783..4802)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5393..5412
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5699..5820
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5736..5755)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5790..5809
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5869..5885)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5869..5885)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5882..5904)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5893..5909
                     /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(5917..5947)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5962..5983
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6100..6117)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6271..6859)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6351..6370)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7030..7890)
                     /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     7653..7672
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7891..7995)
                     /gene="bla"
                     /label=AmpR promoter

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