| 质粒名称: | pAAV-CMV-Cas9C-VPR |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 80933 |
| 存储实验室: | George Church |
|
载体骨架基本信息 |
|
| 载体名称: | pZac2.1 |
| 载体出品公司: | University of Pennsylvannia |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 3000 bp |
| 完整质粒大小: | 7637 bp |
| 载体修饰: | -- |
| 载体类型: | 哺乳动物表达，AAV，CRISPR，合成生物学 |
| 筛选标记: | -- |
| 高拷贝/低拷贝: | -- |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | Cas9C-VPR,Split-Cas9 C-terminal half with VPR transcriptional activator |
| 物种来源: | Synthetic |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | -- |
| 标签/融合蛋白: | -- |
| 克隆方法: | -- |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | -- |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pAAV-CMV-Cas9C-VPR was a gift from George Church (Addgene plasmid # 80933 ; http://n2t.net/addgene:80933 ; RRID:Addgene_80933) |
| 参考文献部分: | A multifunctional AAV-CRISPR-Cas9 and its host response. Chew WL, Tabebordbar M, Cheng JK, Mali P, Wu EY, Ng AH, Zhu K, Wagers AJ, Church GM. Nat Methods. 2016 Sep 5. doi: 10.1038/nmeth.3993. 10.1038/nmeth.3993 PubMed 27595405 |
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 pAAV-CMV-Cas9C-VPR 7637 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses Cas9C-VPR in mammalian cells..
ACCESSION .
VERSION .
KEYWORDS pAAV-CMV-Cas9C-VPR
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7637)
AUTHORS Chew WL, Tabebordbar M, Cheng JK, Mali P, Wu EY, Ng AH, Zhu K,
Wagers AJ, Church GM
TITLE A multifunctional AAV-CRISPR-Cas9 and its host response.
JOURNAL Nat Methods. 2016 Sep 5. doi: 10.1038/nmeth.3993.
PUBMED 27595405
REFERENCE 2 (bases 1 to 7637)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7637
/organism="synthetic DNA construct"
/mol_type="other DNA"
repeat_region 1..130
/label=AAV2 ITR
promoter 311..514
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 465..485
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
intron 606..738
/label=chimeric intron
/note="chimera between introns from human beta-globin and
immunoglobulin heavy chain genes"
regulatory 768..777
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
regulatory 772..781
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1378..1389
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
CDS 2907..2927
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
protein_bind 2964..2988
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 3018..3167
/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 3192..3212
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3642..3998
/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
EDFSSIADMDFSALL"
CDS 4017..4586
/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"
repeat_region 4686..4815
/label=AAV2 ITR
primer_bind complement(4845..4862)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(4845..4861)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4854..4876)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 5003..5458
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(5090..5109)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 5300..5321
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 5485..5589
/gene="bla"
/label=AmpR promoter
CDS 5590..6450
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVVMVTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(5808..5827)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6621..7209
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7110..7129
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 7363..7380
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 7497..7518
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 7533..7563
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 7571..7587
/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)."
primer_bind 7576..7598
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 7595..7611
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 7595..7611
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
ORIGIN
1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacccg ctagcaggca
181 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
241 ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat tagtcatcgc
301 tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc ggtttgactc
361 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
421 tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag
481 gcgtgtacgg tgggaggtct atataagcag aggtcgttta gtgaaccgtc agatcactag
541 tagctttatt gcggtagttt atcacagtta aattgctaac gcagtcagtg ctcgactgat
601 cacaggtaag tatcaaggtt acaagacagg tttaaggagg ccaatagaaa ctgggcttgt
661 cgagacagag aagattcttg cgtttctgat aggcacctat tggtcttact gacatccact
721 ttgcctttct ctccacaggg gtaccgaagc cgctagcgct accggtcgcc accatggcgg
781 cggcgtgccc ggaactgcgt cagctggcgc agagcgatgt gtattgggat ccgattgtga
841 gcattgaacc ggatggcgtg gaagaagtgt ttgatctgac cgtgccgggc ccgcataact
901 ttgtggcgaa cgatattatt gcgcataact ctggccaggg ggacagtctt cacgagcaca
961 tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc gttaaggtcg
1021 tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt atcgagatgg
1081 cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg atgaagagga
1141 ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca gttgaaaaca
1201 cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg gacatgtacg
1261 tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat atcgtgcccc
1321 agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc gataaaaata
1381 gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa aattattggc
1441 ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg actaaggctg
1501 aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag cttgttgaga
1561 cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac accaagtacg
1621 atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct aagctggtct
1681 cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat taccaccatg
1741 cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa tatcccaagc
1801 ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa atgatcgcaa
1861 agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc aatattatga
1921 attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga ccacttatcg
1981 aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc gcgacagtcc
2041 ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta cagaccggag
2101 gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc gcacgcaaaa
2161 aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct tacagtgtac
2221 tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc aaggaactgc
2281 tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac tttctcgagg
2341 cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag tactctctct
2401 ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg cagaaaggta
2461 acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc cactatgaaa
2521 agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa caacacaaac
2581 actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg atcctcgccg
2641 acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag cccatcaggg
2701 agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg cctgcagcct
2761 tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag gaggtcctgg
2821 acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc gacctctctc
2881 agctcggtgg agacagcagg gctgacccca agaagaagag gaaggtgtcg ccagggatcc
2941 gtcgacttga cgcgttgata tcaacaagtt tgtacaaaaa agcaggctac aaagaggcca
3001 gcggttccgg acgggctgac gcattggacg attttgatct ggatatgctg ggaagtgacg
3061 ccctcgatga ttttgacctt gacatgcttg gttcggatgc ccttgatgac tttgacctcg
3121 acatgctcgg cagtgacgcc cttgatgatt tcgacctgga catgctgatt aactctagaa
3181 gttccggatc tccgaaaaag aaacgcaaag ttggtagcca gtacctgccc gacaccgacg
3241 accggcaccg gatcgaggaa aagcggaagc ggacctacga gacattcaag agcatcatga
3301 agaagtcccc cttcagcggc cccaccgacc ctagacctcc acctagaaga atcgccgtgc
3361 ccagcagatc cagcgccagc gtgccaaaac ctgcccccca gccttacccc ttcaccagca
3421 gcctgagcac catcaactac gacgagttcc ctaccatggt gttccccagc ggccagatct
3481 ctcaggcctc tgctctggct ccagcccctc ctcaggtgct gcctcaggct cctgctcctg
3541 caccagctcc agccatggtg tctgcactgg ctcaggcacc agcacccgtg cctgtgctgg
3601 ctcctggacc tccacaggct gtggctccac cagcccctaa acctacacag gccggcgagg
3661 gcacactgtc tgaagctctg ctgcagctgc agttcgacga cgaggatctg ggagccctgc
3721 tgggaaacag caccgatcct gccgtgttca ccgacctggc cagcgtggac aacagcgagt
3781 tccagcagct gctgaaccag ggcatccctg tggcccctca caccaccgag cccatgctga
3841 tggaataccc cgaggccatc acccggctcg tgacaggcgc tcagaggcct cctgatccag
3901 ctcctgcccc tctgggagca ccaggcctgc ctaatggact gctgtctggc gacgaggact
3961 tcagctctat cgccgatatg gatttctcag ccttgctggg ctctggcagc ggcagccggg
4021 attccaggga agggatgttt ttgccgaagc ctgaggccgg ctccgctatt agtgacgtgt
4081 ttgagggccg cgaggtgtgc cagccaaaac gaatccggcc atttcatcct ccaggaagtc
4141 catgggccaa ccgcccactc cccgccagcc tcgcaccaac accaaccggt ccagtacatg
4201 agccagtcgg gtcactgacc ccggcaccag tccctcagcc actggatcca gcgcccgcag
4261 tgactcccga ggccagtcac ctgttggagg atcccgatga agagacgagc caggctgtca
4321 aagcccttcg ggagatggcc gatactgtga ttccccagaa ggaagaggct gcaatctgtg
4381 gccaaatgga cctttcccat ccgcccccaa ggggccatct ggatgagctg acaaccacac
4441 ttgagtccat gaccgaggat ctgaacctgg actcacccct gaccccggaa ttgaacgaga
4501 ttctggatac cttcctgaac gacgagtgcc tcttgcatgc catgcatatc agcacaggac
4561 tgtccatctt cgacacatct ctgttttgac aataaaatat ctttattttc attacatctg
4621 tgtgttggtt ttttgtgtta gctagcgcgt agataagtag catggcgggt taatcattaa
4681 ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
4741 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
4801 cgagcgagcg cgcagcctaa ttaaggcctt aattaaccta attcactggc cgtcgtttta
4861 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
4921 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
4981 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
5041 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
5101 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
5161 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
5221 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
5281 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
5341 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
5401 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tataatttca
5461 ggtggcatct ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
5521 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
5581 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
5641 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
5701 ttgggtgcac gagtgggtta catcgaactg gatctcaata gtggtaagat ccttgagagt
5761 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
5821 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
5881 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
5941 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
6001 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
6061 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
6121 accacgatgc ctgtagtaat ggtaacaacg ttgcgcaaac tattaactgg cgaactactt
6181 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
6241 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
6301 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
6361 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
6421 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
6481 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
6541 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
6601 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
6661 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
6721 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
6781 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
6841 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
6901 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
6961 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
7021 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
7081 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
7141 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
7201 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctgcggtttt
7261 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
7321 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
7381 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
7441 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
7501 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
7561 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
7621 gccagattta attaagg
//
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