| 质粒名称: | BPK2139,pBPK2139-SaCas9 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 65776 |
| 存储实验室: | Keith Joung |
|
载体骨架基本信息 |
|
| 载体名称: | pCAG-CFP |
| 载体抗性: | 氨苄青霉素 |
| 载体类型: | 哺乳细胞表达,CRISPR |
| 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: | mammalian codon-optimized Staphylococcus aureus Cas9-NLS-3xFlag |
| 启动子 | CAG |
| 标签/融合蛋白: |
NLS（插接件上的C端子）
3x FLAG（插接件上的C端子）
|
| 克隆方法: | Gibson克隆 |
| 5' 测序引物 | oBK280 (5'-CCTACAGCTCCTGGGCAACG-3') |
|
文献引用方法 |
|
| 材料和方法部分: | BPK2139 was a gift from Keith Joung (Addgene plasmid # 65776 ; http://n2t.net/addgene:65776 ; RRID:Addgene_65776) |
| 参考文献部分: | Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, Gonzales AP, Li Z, Peterson RT, Yeh JJ, Aryee MJ, Joung JK. Nature. 2015 Jun 22. doi: 10.1038/nature14592. 10.1038/nature14592 PubMed 26098369 |
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 BPK2139 8100 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Human expression vector for S .aureus Cas9:
CAG-humanSaCas9-NLS-3xFLAG.
ACCESSION .
VERSION .
KEYWORDS BPK2139
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8100)
AUTHORS Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z,
Gonzales AP, Li Z, Peterson RT, Yeh JJ, Aryee MJ, Joung JK
TITLE Engineered CRISPR-Cas9 nucleases with altered PAM specificities.
JOURNAL Nature. 2015 Jun 22. doi: 10.1038/nature14592.
PUBMED 26098369
REFERENCE 2 (bases 1 to 8100)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8100
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 4..383
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 386..661
/label=chicken beta-actin promoter
intron 662..1673
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind 1597..1619
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 1681..1700
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
regulatory 1766..1775
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1772..4933
/codon_start=1
/product="Cas9 endonuclease from the Staphylococcus aureus
Type II CRISPR/Cas system"
/label=SaCas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="MGKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENN
EGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEE
EFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDG
EVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPF
GWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEK
FQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARK
EIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLK
AINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQS
IKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKEN
AKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLV
KQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINR
FSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKE
RNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKE
IFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDK
DNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSK
KDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVK
NLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDL
LNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHP
QIIKKG"
CDS 4940..4960
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4967..5032
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
primer_bind complement(5122..5141)
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
polyA_site 5187..5242
/label=-beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal"
primer_bind complement(5241..5260)
/label=rbglobpA-R
/note="Rabbit beta-globin polyA, reverse primer. Also
called rb-glob-pA-term-R"
primer_bind complement(5603..5619)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5603..5619)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(5616..5638)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 5627..5643
/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(5651..5681)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5696..5717
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 5776..5971
/label=SV40 promoter
/note="SV40 early promoter"
rep_origin 5822..5957
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 5884..5903
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
polyA_signal 5977..6111
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(6014..6033)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 6068..6087
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(6179..6196)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(6350..6938)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(6430..6449)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(7109..7969)
/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 7732..7751
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(7970..8074)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
1381 gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct
1441 ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc
1501 ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg
1561 gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg
1621 gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg
1681 gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattctgc agtcgacggt
1741 accgcgggcc cgggatccac cggtcgccac catggggaaa cggaactaca tcctggggct
1801 tgacattggg ataaccagcg ttggctacgg aattattgat tatgagacac gcgatgtgat
1861 tgacgccggg gttaggctgt tcaaagaggc caacgttgaa aacaacgagg gaagacggag
1921 taagcgcgga gcaagaagac tcaagcgcag acggagacat cggattcaga gggtgaaaaa
1981 gctgctcttc gattacaatc tcctgaccga tcatagtgag ctgagcggaa tcaaccccta
2041 cgaggcgcga gtgaaagggc tttcccagaa gctgtccgaa gaggagttct ccgccgcgtt
2101 gctgcacctg gccaaacgga ggggggttca caatgtaaac gaagtggagg aggacacggg
2161 caatgaactt agtacgaaag aacagatcag taggaactct aaggctctcg aagagaaata
2221 cgtcgctgag ttgcagcttg agagactgaa aaaagacggc gaagtacgcg gatctattaa
2281 taggttcaag acttcagatt acgtaaagga agccaagcag ctcctgaaag tacagaaagc
2341 gtaccatcag ctcgatcaga gcttcatcga tacctacata gatttgctgg agacacggag
2401 gacatactac gagggcccag gggaaggatc tccttttggg tggaaggaca tcaaggaatg
2461 gtacgagatg cttatgggac attgtacata ttttccggag gagctcagga gcgtcaagta
2521 cgcctacaat gccgacctgt acaatgccct caatgacctc aataacctcg tgattaccag
2581 ggacgagaac gagaagctgg agtactatga aaagttccag attatcgaga atgtgtttaa
2641 gcagaagaag aagccgacac ttaagcagat tgcaaaggaa atcctcgtga atgaggaaga
2701 tatcaaggga tacagagtga caagtacagg caagcccgag ttcacaaatc tgaaggtgta
2761 ccacgatatt aaggacataa ccgcacgaaa ggagataatc gaaaacgctg agctcctcga
2821 tcagatcgca aaaattctta ccatctacca gtctagtgag gacattcagg aggaactgac
2881 taatctgaac agtgagctca cccaagagga aattgagcag atttcaaacc tgaaaggcta
2941 caccgggacg cacaatctga gcctcaaagc aatcaacctc attctggatg aactttggca
3001 cacaaatgac aaccaaattg ccatattcaa ccgcctgaaa ctggtgccaa aaaaagtgga
3061 tctgtcacag caaaaggaaa tccctacaac cttggttgac gattttattc tgtcccccgt
3121 tgtcaagcgg agcttcatcc agtcaatcaa ggtgatcaat gccatcatta aaaaatacgg
3181 attgccaaac gatataatta tcgagcttgc acgagagaag aactcaaagg acgcccagaa
3241 gatgattaac gaaatgcaga agcgcaaccg ccagacaaac gaacgcatag aggaaattat
3301 aagaacaacc ggcaaagaga atgccaagta tctgatcgag aaaatcaagc tgcacgacat
3361 gcaagaaggc aagtgcctgt actctctgga agctatccca ctcgaagatc tgctgaataa
3421 tccattcaat tacgaggtgg accacatcat ccctagatcc gtaagctttg acaattcctt
3481 caataacaaa gttctggtta aacaggagga aaattctaaa aaagggaacc ggaccccgtt
3541 ccagtacctg agctccagtg acagcaagat tagctacgag acttttaaga aacatattct
3601 gaatctggcc aaaggcaaag gcaggatcag caagaccaag aaggagtacc tcctcgaaga
3661 acgcgacatt aacagattta gtgtgcagaa agatttcatc aaccgaaacc ttgtcgatac
3721 tcggtacgcc acgagaggcc tgatgaatct cctcaggagc tacttccgcg tcaataatct
3781 ggacgttaaa gtcaagagca taaatggggg attcaccagc tttctgagga gaaagtggaa
3841 gtttaagaag gaacgaaaca aaggatacaa gcaccatgct gaggatgctt tgatcatcgc
3901 taacgcggac tttatcttta aggaatggaa aaagctggat aaggcaaaga aagtgatgga
3961 aaaccagatg ttcgaggaga agcaggcaga gtcaatgcct gagatcgaga cagagcagga
4021 atacaaggaa attttcatca cccctcatca gattaaacac ataaaggact tcaaagacta
4081 taaatactct catagggtgg acaaaaaacc caatcgcgag ctcattaatg acaccctgta
4141 ctcaacacgg aaggatgata aaggtaatac cttgattgtg aataatctta atggattgta
4201 tgacaaagat aacgacaagc tcaagaagct gatcaacaag tctccagaga agctccttat
4261 gtatcaccac gacccacaga cttatcagaa attgaaactg atcatggagc aatacgggga
4321 tgagaagaac ccactctaca aatattatga ggaaacaggt aattacctga ccaagtactc
4381 caagaaggat aacggaccag tgatcaaaaa gataaagtac tatggcaaca aacttaatgc
4441 gcatttggac ataactgacg attaccccaa ttctcgaaac aaggttgtga agctctccct
4501 gaagccttat agatttgacg tgtacctgga taatggggtt tataaattcg tcaccgtgaa
4561 aaatctggac gtgatcaaaa aggagaacta ttatgaagta aactcaaagt gctatgagga
4621 ggcgaagaag ctgaagaaga tctccaatca ggccgagttc atcgcttcct tctataataa
4681 cgatctcatc aagatcaatg gagagcttta tcgcgtcatt ggtgtgaaca atgacttgct
4741 gaacaggatc gaagtcaata tgatagacat tacctaccgg gagtatctcg aaaacatgaa
4801 tgataaacgg ccgcctcgca tcatcaagac aatcgcatct aaaactcagt caataaaaaa
4861 gtactctacc gatatcctgg ggaatctcta tgaagtgaag tcaaagaagc acccacaaat
4921 cattaaaaaa ggtggatccc ccaagaagaa gaggaaagtc tcgagcgact acaaagacca
4981 tgacggtgat tataaagatc atgacatcga ttacaaggat gacgatgaca agtaaagcgg
5041 ccgcactcct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct
5101 ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg acatcatgaa
5161 gccccttgag catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg
5221 ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca
5281 tcagaatgag tatttggttt agagtttggc aacatatgcc catatgctgg ctgccatgaa
5341 caaaggttgg ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt
5401 attccataga aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt
5461 tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct
5521 ctcctgacta ctcccagtca tagctgtccc tcttctctta tggagatccc tcgacctgca
5581 gcccaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
5641 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
5701 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
5761 cgtgccagcg gatccgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
5821 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
5881 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
5941 aggctttttt ggaggcctag gcttttgcaa aaagctaact tgtttattgc agcttataat
6001 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
6061 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat ccgctgcatt
6121 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
6181 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
6241 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
6301 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
6361 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
6421 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
6481 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
6541 ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
6601 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
6661 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
6721 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
6781 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
6841 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
6901 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
6961 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
7021 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
7081 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
7141 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
7201 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
7261 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
7321 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
7381 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
7441 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
7501 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
7561 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
7621 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
7681 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
7741 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
7801 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
7861 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
7921 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
7981 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
8041 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
//
