| 质粒名称: | pcDNA3.1-Cry2FL-VP64 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 60554 |
| 存储实验室: | Charles Gersbach |
|
载体骨架基本信息 |
|
| 载体名称: | pcDNA3.1 |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 5333 bp |
| 完整质粒大小: | 7541 bp |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | 新霉素（G418） |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | Cry2FL-VP64 |
| 物种来源: | A. thaliana (mustard weed) |
| 插入大小: | 2181 bp |
| 启动子 | CMV |
| 标签/融合蛋白: |
FLAG（插入N端子）
SV40 NLS（插入件上的N端子）
SV40 NLS（插接件上的C端子）
HA tag （插接件上的C端子）
|
| 克隆方法: | Gibson克隆 |
| 5' 测序引物 | ATTGACGCAAATGGGCGGTAGGCGTGT |
| 3' 测序引物 | GCCTGCTATTGTCTTCCCAAT |
|
文献引用方法 |
|
| 材料和方法部分: | pcDNA3.1-Cry2FL-VP64 was a gift from Charles Gersbach (Addgene plasmid # 60554 ; http://n2t.net/addgene:60554 ; RRID:Addgene_60554) |
| 参考文献部分: | A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Polstein LR, Gersbach CA. Nat Chem Biol. 2015 Feb 9. doi: 10.1038/nchembio.1753. 10.1038/nchembio.1753 PubMed 25664691 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pcDNA3.1-Cry2FL-VP64 7541 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses Cry2FL-VP64 in mammalian cells.
ACCESSION .
VERSION .
KEYWORDS pcDNA3.1-Cry2FL-VP64
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7541)
AUTHORS Polstein LR, Gersbach CA
TITLE A light-inducible CRISPR-Cas9 system for control of endogenous gene
activation.
JOURNAL Nat Chem Biol. 2015 Feb 9. doi: 10.1038/nchembio.1753.
PUBMED 25664691
REFERENCE 2 (bases 1 to 7541)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7541
/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"
CDS 907..972
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 979..999
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1006..2841
/codon_start=1
/product="Arabidopsis thaliana cryptochrome 2"
/label=CRY2
/note="binds to CIB1 upon exposure to blue light (Kennedy
et al., 2010)"
/translation="MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQ
FYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLY
DPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSI
ESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLN
EFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGE
ESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPL
VDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQY
ISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKA
SGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAAPDEIVADSFEALGANTIKE
PGLCPSVSSNDQQVPSAVRYNGSKRVKPEEEEERDMKKSRGFDERELFSTAESSSSSSV
FFVSQSCSLASEGKNLEGIQDSSDQITTSLGKNGCK"
CDS 2866..2886
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 2896..3045
/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 3052..3078
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
primer_bind complement(3135..3152)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 3141..3365
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 3411..3839
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3498..3517)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 3708..3729
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(3848..3868)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 3853..4182
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 4033..4168
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 4095..4114
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 4249..5043
/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(4303..4322)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 4913..4932
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 5217..5338
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(5254..5273)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 5308..5327
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(5387..5403)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5387..5403)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(5400..5422)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 5411..5427
/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(5435..5465)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5480..5501
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(5618..5635)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5789..6374)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(5869..5888)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6545..7405)
/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 7168..7187
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(7406..7510)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
901 gccatggact acaaagacca tgacggtgat tataaagatc atgacatcga ttacaaggat
961 gacgatgaca agatggcccc caagaagaag aggaaggtgg gccgcatgaa gatggacaaa
1021 aagactatag tttggtttag aagagaccta aggattgagg ataatcctgc attagcagca
1081 gctgctcacg aaggatctgt ttttcctgtc ttcatttggt gtcctgaaga agaaggacag
1141 ttttatcctg gaagagcttc aagatggtgg atgaaacaat cacttgctca cttatctcaa
1201 tccttgaagg ctcttggatc tgacctcact ttaatcaaaa cccacaacac gatttcagcg
1261 atcttggatt gtatccgcgt taccggtgct acaaaagtcg tctttaacca cctctatgat
1321 cctgtttcgt tagttcggga ccataccgta aaggagaagc tggtggaacg tgggatctct
1381 gtgcaaagct acaatggaga tctattgtat gaaccgtggg agatatactg cgaaaagggc
1441 aaacctttta cgagtttcaa ttcttactgg aagaaatgct tagatatgtc gattgaatcc
1501 gttatgcttc ctcctccttg gcggttgatg ccaataactg cagcggctga agcgatttgg
1561 gcgtgttcga ttgaagaact agggctggag aatgaggccg agaaaccgag caatgcgttg
1621 ttaactagag cttggtctcc aggatggagc aatgctgata agttactaaa tgagttcatc
1681 gagaagcagt tgatagatta tgcaaagaac agcaagaaag ttgttgggaa ttctacttca
1741 ctactttctc cgtatctcca tttcggggaa ataagcgtca gacacgtttt ccagtgtgcc
1801 cggatgaaac aaattatatg ggcaagagat aagaacagtg aaggagaaga aagtgcagat
1861 ctttttctta ggggaatcgg tttaagagag tattctcggt atatatgttt caacttcccg
1921 tttactcacg agcaatcgtt gttgagtcat cttcggtttt tcccttggga tgctgatgtt
1981 gataagttca aggcctggag acaaggcagg accggttatc cgttggtgga tgccggaatg
2041 agagagcttt gggctaccgg atggatgcat aacagaataa gagtgattgt ttcaagcttt
2101 gctgtgaagt ttcttctcct tccatggaaa tggggaatga agtatttctg ggatacactt
2161 ttggatgctg atttggaatg tgacatcctt ggctggcagt atatctctgg gagtatcccc
2221 gatggccacg agcttgatcg cttggacaat cccgcgttac aaggcgccaa atatgaccca
2281 gaaggtgagt acataaggca atggcttccc gagcttgcga gattgccaac tgaatggatc
2341 catcatccat gggacgctcc tttaaccgta ctcaaagctt ctggtgtgga actcggaaca
2401 aactatgcga aacccattgt agacatcgac acagctcgtg agctactagc taaagctatt
2461 tcaagaaccc gtgaagcaca gatcatgatc ggagcagcac ctgatgagat tgtagcagat
2521 agcttcgagg ccttaggggc taataccatt aaagaacctg gtctttgccc atctgtgtct
2581 tctaatgacc aacaagtacc ttcggctgtt cgttacaacg ggtcaaagag agtgaaacct
2641 gaggaagaag aagagagaga catgaagaaa tctaggggat tcgatgaaag ggagttgttt
2701 tcgactgctg aatcttcttc ttcttcgagt gtgtttttcg tttcgcagtc ttgctcgttg
2761 gcatcagaag ggaagaatct ggaaggtatt caagattcat ctgatcagat tactacaagt
2821 ttgggaaaaa atggttgcaa accaattgcc ggatccaagg ctagcccgaa aaagaaacgc
2881 aaagttgggc gcgccgacgc gctggacgat ttcgatctcg acatgctggg ttctgatgcc
2941 ctcgatgact ttgacctgga tatgttggga agcgacgcat tggatgactt tgatctggac
3001 atgctcggct ccgatgctct ggacgatttc gatctcgata tgttaattaa ctacccgtac
3061 gacgttccgg actacgcttc ttgaatcggt aggaattcgc ggccgtctag acttaagttt
3121 aaaccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
3181 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
3241 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
3301 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
3361 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
3421 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
3481 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
3541 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
3601 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
3661 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
3721 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
3781 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
3841 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
3901 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
3961 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
4021 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
4081 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
4141 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
4201 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
4261 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
4321 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
4381 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
4441 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
4501 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
4561 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
4621 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
4681 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
4741 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
4801 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
4861 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
4921 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
4981 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
5041 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
5101 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
5161 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
5221 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
5281 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
5341 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
5401 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
5461 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
5521 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
5581 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
5641 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
5701 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
5761 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
5821 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
5881 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
5941 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
6001 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
6061 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
6121 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
6181 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
6241 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
6301 gcaaacaaac caccgctggt agcggttttt ttgtttgcaa gcagcagatt acgcgcagaa
6361 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
6421 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
6481 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
6541 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
6601 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
6661 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
6721 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
6781 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
6841 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
6901 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
6961 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
7021 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
7081 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
7141 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
7201 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
7261 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
7321 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
7381 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
7441 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
7501 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
