| 质粒名称: | BPK2101,pBPK2101-saCas9-gRNA |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 65770 |
| 存储实验室: | Keith Joung |
|
载体骨架基本信息 |
|
| 载体名称: | pACYCDuet-1 |
| 载体出品公司: | EMD Millipore |
| 载体抗性: | 氯霉素 |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 细菌表达,CRISPR |
| 筛选标记: | -- |
| 高拷贝/低拷贝: | 低拷贝 |
| 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, and SaCas9 gRNA |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | T7 (x2) |
| 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' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | ACYC-DuetUP1 |
| 3' 测序引物 | oBK390 (5'-GCAAATTCGACCCGGTCGTCG-3') |
|
文献引用方法 |
|
| 材料和方法部分: | BPK2101 was a gift from Keith Joung (Addgene plasmid # 65770 ; http://n2t.net/addgene:65770 ; RRID:Addgene_65770) |
| 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 BPK2101 7157 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Bacterial expression plasmid for S. aureus Cas9.
ACCESSION .
VERSION .
KEYWORDS BPK2101
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7157)
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 7157)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7157
/organism="synthetic DNA construct"
/mol_type="other DNA"
protein_bind 3..27
/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)."
CDS 71..3232
/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 3239..3259
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3266..3331
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
promoter 3401..3419
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(3596..3614)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 3610..3657
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(3878..4537)
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
primer_bind 4452..4471
/label=CAT-R
/note="Chloramphenicol resistance gene, reverse primer"
promoter complement(4538..4640)
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
primer_bind complement(5032..5049)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5166..5710)
/direction=LEFT
/label=p15A ori
/note="Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin.
"
primer_bind complement(5246..5265)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(5935..7017)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/label=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)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
primer_bind 6979..6998
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(7018..7095)
/gene="lacI"
/label=lacI promoter
promoter 7141..2
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
61 gagatatacc atgggcaaac ggaactacat cctggggctt gacattggga taaccagcgt
121 tggctacgga attattgatt atgagacacg cgatgtgatt gacgccgggg ttaggctgtt
181 caaagaggcc aacgttgaaa acaacgaggg aagacggagt aagcgcggag caagaagact
241 caagcgcaga cggagacatc ggattcagag ggtgaaaaag ctgctcttcg attacaatct
301 cctgaccgat catagtgagc tgagcggaat caacccctac gaggcgcgag tgaaagggct
361 ttcccagaag ctgtccgaag aggagttctc cgccgcgttg ctgcacctgg ccaaacggag
421 gggggttcac aatgtaaacg aagtggagga ggacacgggc aatgaactta gtacgaaaga
481 acagatcagt aggaactcta aggctctcga agagaaatac gtcgctgagt tgcagcttga
541 gagactgaaa aaagacggcg aagtacgcgg atctattaat aggttcaaga cttcagatta
601 cgtaaaggaa gccaagcagc tcctgaaagt acagaaagcg taccatcagc tcgatcagag
661 cttcatcgat acctacatag atttgctgga gacacggagg acatactacg agggcccagg
721 ggaaggatct ccttttgggt ggaaggacat caaggaatgg tacgagatgc ttatgggaca
781 ttgtacatat tttccggagg agctcaggag cgtcaagtac gcctacaatg ccgacctgta
841 caatgccctc aatgacctca ataacctcgt gattaccagg gacgagaacg agaagctgga
901 gtactatgaa aagttccaga ttatcgagaa tgtgtttaag cagaagaaga agccgacact
961 taagcagatt gcaaaggaaa tcctcgtgaa tgaggaagat atcaagggat acagagtgac
1021 aagtacaggc aagcccgagt tcacaaatct gaaggtgtac cacgatatta aggacataac
1081 cgcacgaaag gagataatcg aaaacgctga gctcctcgat cagatcgcaa aaattcttac
1141 catctaccag tctagtgagg acattcagga ggaactgact aatctgaaca gtgagctcac
1201 ccaagaggaa attgagcaga tttcaaacct gaaaggctac accgggacgc acaatctgag
1261 cctcaaagca atcaacctca ttctggatga actttggcac acaaatgaca accaaattgc
1321 catattcaac cgcctgaaac tggtgccaaa aaaagtggat ctgtcacagc aaaaggaaat
1381 ccctacaacc ttggttgacg attttattct gtcccccgtt gtcaagcgga gcttcatcca
1441 gtcaatcaag gtgatcaatg ccatcattaa aaaatacgga ttgccaaacg atataattat
1501 cgagcttgca cgagagaaga actcaaagga cgcccagaag atgattaacg aaatgcagaa
1561 gcgcaaccgc cagacaaacg aacgcataga ggaaattata agaacaaccg gcaaagagaa
1621 tgccaagtat ctgatcgaga aaatcaagct gcacgacatg caagaaggca agtgcctgta
1681 ctctctggaa gctatcccac tcgaagatct gctgaataat ccattcaatt acgaggtgga
1741 ccacatcatc cctagatccg taagctttga caattccttc aataacaaag ttctggttaa
1801 acaggaggaa aattctaaaa aagggaaccg gaccccgttc cagtacctga gctccagtga
1861 cagcaagatt agctacgaga cttttaagaa acatattctg aatctggcca aaggcaaagg
1921 caggatcagc aagaccaaga aggagtacct cctcgaagaa cgcgacatta acagatttag
1981 tgtgcagaaa gatttcatca accgaaacct tgtcgatact cggtacgcca cgagaggcct
2041 gatgaatctc ctcaggagct acttccgcgt caataatctg gacgttaaag tcaagagcat
2101 aaatggggga ttcaccagct ttctgaggag aaagtggaag tttaagaagg aacgaaacaa
2161 aggatacaag caccatgctg aggatgcttt gatcatcgct aacgcggact ttatctttaa
2221 ggaatggaaa aagctggata aggcaaagaa agtgatggaa aaccagatgt tcgaggagaa
2281 gcaggcagag tcaatgcctg agatcgagac agagcaggaa tacaaggaaa ttttcatcac
2341 ccctcatcag attaaacaca taaaggactt caaagactat aaatactctc atagggtgga
2401 caaaaaaccc aatcgcgagc tcattaatga caccctgtac tcaacacgga aggatgataa
2461 aggtaatacc ttgattgtga ataatcttaa tggattgtat gacaaagata acgacaagct
2521 caagaagctg atcaacaagt ctccagagaa gctccttatg tatcaccacg acccacagac
2581 ttatcagaaa ttgaaactga tcatggagca atacggggat gagaagaacc cactctacaa
2641 atattatgag gaaacaggta attacctgac caagtactcc aagaaggata acggaccagt
2701 gatcaaaaag ataaagtact atggcaacaa acttaatgcg catttggaca taactgacga
2761 ttaccccaat tctcgaaaca aggttgtgaa gctctccctg aagccttata gatttgacgt
2821 gtacctggat aatggggttt ataaattcgt caccgtgaaa aatctggacg tgatcaaaaa
2881 ggagaactat tatgaagtaa actcaaagtg ctatgaggag gcgaagaagc tgaagaagat
2941 ctccaatcag gccgagttca tcgcttcctt ctataataac gatctcatca agatcaatgg
3001 agagctttat cgcgtcattg gtgtgaacaa tgacttgctg aacaggatcg aagtcaatat
3061 gatagacatt acctaccggg agtatctcga aaacatgaat gataaacggc cgcctcgcat
3121 catcaagaca atcgcatcta aaactcagtc aataaaaaag tactctaccg atatcctggg
3181 gaatctctat gaagtgaagt caaagaagca cccacaaatc attaaaaaag gtggatcccc
3241 caagaagaag aggaaagtct cgagcgacta caaagaccat gacggtgatt ataaagatca
3301 tgacatcgat tacaaggatg acgatgacaa gtaaagcggc cgcataatgc ttaagtcgaa
3361 cagaaagtaa tcgtattgta cacggccgca taatcgaaat taatacgact cactataggg
3421 agacccatgc catagcgttg ttcggaacag attcaccaac acctagtggt ctccgtttta
3481 gtactctgta attttaggta tgaggtagac gaaaattgta cttataccta aaattacaga
3541 atctactaaa acaaggcaaa atgccgtgtt tatctcgtca acttgttggc gagatccgct
3601 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
3661 aaacctcagg catttgagaa gcacacggtc acactgcttc cggtagtcaa taaaccggta
3721 aaccagcaat agacataagc ggctatttaa cgaccctgcc ctgaaccgac gaccgggtcg
3781 aatttgcttt cgaatttctg ccattcatcc gcttattatc acttattcag gcgtagcaac
3841 caggcgttta agggcaccaa taactgcctt aaaaaaatta cgccccgccc tgccactcat
3901 cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat
3961 gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca
4021 tagtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga
4081 aactcaccca gggattggct gagacgaaaa acatattctc aataaaccct ttagggaaat
4141 aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga
4201 aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg
4261 tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgga
4321 actccggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt
4381 gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat
4441 aggtacattg agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata
4501 tatcaacggt ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa
4561 atctcgataa ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg
4621 aacctcttac gtgccgatca acgtctcatt ttcgccaaaa gttggcccag ggcttcccgg
4681 tatcaacagg gacaccagga tttatttatt ctgcgaagtg atcttccgtc acaggtattt
4741 attcggcgca aagtgcgtcg ggtgatgctg ccaacttact gatttagtgt atgatggtgt
4801 ttttgaggtg ctccagtggc ttctgtttct atcagctgtc cctcctgttc agctactgac
4861 ggggtggtgc gtaacggcaa aagcaccgcc ggacatcagc gctagcggag tgtatactgg
4921 cttactatgt tggcactgat gagggtgtca gtgaagtgct tcatgtggca ggagaaaaaa
4981 ggctgcaccg gtgcgtcagc agaatatgtg atacaggata tattccgctt cctcgctcac
5041 tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg aacggggcgg
5101 agatttcctg gaagatgcca ggaagatact taacagggaa gtgagagggc cgcggcaaag
5161 ccgtttttcc ataggctccg cccccctgac aagcatcacg aaatctgacg ctcaaatcag
5221 tggtggcgaa acccgacagg actataaaga taccaggcgt ttcccctggc ggctccctcg
5281 tgcgctctcc tgttcctgcc tttcggttta ccggtgtcat tccgctgtta tggccgcgtt
5341 tgtctcattc cacgcctgac actcagttcc gggtaggcag ttcgctccaa gctggactgt
5401 atgcacgaac cccccgttca gtccgaccgc tgcgccttat ccggtaacta tcgtcttgag
5461 tccaacccgg aaagacatgc aaaagcacca ctggcagcag ccactggtaa ttgatttaga
5521 ggagttagtc ttgaagtcat gcgccggtta aggctaaact gaaaggacaa gttttggtga
5581 ctgcgctcct ccaagccagt tacctcggtt caaagagttg gtagctcaga gaaccttcga
5641 aaaaccgccc tgcaaggcgg ttttttcgtt ttcagagcaa gagattacgc gcagaccaaa
5701 acgatctcaa gaagatcatc ttattaatca gataaaatat ttctagattt cagtgcaatt
5761 tatctcttca aatgtagcac ctgaagtcag ccccatacga tataagttgt aattctcatg
5821 ttagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
5881 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
5941 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
6001 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
6061 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
6121 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
6181 gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga
6241 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
6301 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
6361 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
6421 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
6481 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
6541 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
6601 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
6661 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
6721 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
6781 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
6841 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
6901 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
6961 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
7021 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
7081 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaaat
7141 taatacgact cactata
//
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