| 质粒名称: | pKMD106e - intein-Cas9(S219) |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 64190 |
| 存储实验室: | David Liu |
|
载体骨架基本信息 |
|
| 载体名称: | pJDS246 |
| 载体抗性: | 氨苄霉素 |
| 空载体大小: | 3399 bp |
| 完整质粒大小: | 8850 bp |
| 载体类型: | 哺乳细胞表达，CRISPR |
| 高拷贝/低拷贝: | 未知 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | mammalian codon-optimized S. pyogenes intein-Cas9(S219) - NLS - 3x FLAG |
| 物种来源: | Streptococcus pyogenes |
| 插入大小: | 5451 bp |
| 基因又名: | intein-Cas9(S219) |
| 启动子 | CMV |
| 标签/融合蛋白: | NLS - 3x FLAG (C terminal on insert) |
| 克隆方法: | 未知 |
| 5' 测序引物 | CMV-F |
| 3' 测序引物 | OK569 |
|
文献引用方法 |
|
| 材料和方法部分: | pKMD106e - intein-Cas9(S219) was a gift from David Liu (爱迪基因 plasmid # 64190) |
| 参考文献部分: | Small molecule-triggered Cas9 protein with improved genome-editing specificity. Davis KM, Pattanayak V, Thompson DB, Zuris JA, Liu DR. Nat Chem Biol. 2015 May;11(5):316-8. doi: 10.1038/nchembio.1793. Epub 2015 Apr 6. 10.1038/nchembio.1793 PubMed 25848930 |
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
质粒图谱.png)
载体简介
载体序列
LOCUS pKMD106e - intein-Cas9(S219) 8850 bp ds-DNA circular SYN 10-JAN-2018
DEFINITION Expresses intein-Cas9(S219) in mammalian cells.
ACCESSION .
VERSION .
KEYWORDS pKMD106e - intein-Cas9(S219)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8850)
AUTHORS Davis KM, Pattanayak V, Thompson DB, Zuris JA, Liu DR
TITLE Small molecule-triggered Cas9 protein with improved genome-editing
specificity.
JOURNAL Nat Chem Biol. 2015 May;11(5):316-8. doi: 10.1038/nchembio.1793.
Epub 2015 Apr 6.
PUBMED 25848930
COMMENT This file is created by Vector NTI
http://www.biofeng.com/
COMMENT ORIGDB|GenBank
COMMENT VNTAUTHORNAME|biofeng.com|
COMMENT VNTNAME|pKMD106e - intein-Cas9(S219)|
FEATURES Location/Qualifiers
source 1..8850
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 132..335
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 286..306
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 377..396
/label=T7
/note="T7 promoter, forward primer"
promoter 377..395
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
regulatory 403..412
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 3364..5748
/codon_start=1
/product="C-terminal portion of Streptococcus pyogenes Cas9
(Zetsche et al., 2015)"
/label=Cas9(C)
/translation="CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED
IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK
TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG
ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQI
LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN
KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK
AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK
ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP
QVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV
EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN
GRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD
EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY
FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
CDS 5755..5775
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 5782..5847
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 5865..5882
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(5905..5922)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 5911..6135
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(6206..6222)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(6206..6222)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(6219..6241)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 6230..6246
/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(6254..6284)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6299..6320
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(6437..6454)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(6608..7196)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(6688..6707)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(7367..8227)
/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 7990..8009
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(8228..8332)
/gene="bla"
/label=AmpR promoter
primer_bind complement(8411..8430)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 8602..131
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
ORIGIN
1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat ggataaaaag
421 tattctattg gtttagacat cggcactaat tccgttggat gggctgtcat aaccgatgaa
481 tacaaagtac cttcaaagaa atttaaggtg ttggggaaca cagaccgtca ttcgattaaa
541 aagaatctta tcggtgccct cctattcgat agtggcgaaa cggcagaggc gactcgcctg
601 aaacgaaccg ctcggagaag gtatacacgt cgcaagaacc gaatatgtta cttacaagaa
661 atttttagca atgagatggc caaagttgac gattctttct ttcaccgttt ggaagagtcc
721 ttccttgtcg aagaggacaa gaaacatgaa cggcacccca tctttggaaa catagtagat
781 gaggtggcat atcatgaaaa gtacccaacg atttatcacc tcagaaaaaa gctagttgac
841 tcaactgata aagcggacct gaggttaatc tacttggctc ttgcccatat gataaagttc
901 cgtgggcact ttctcattga gggtgatcta aatccggaca actcggatgt cgacaaactg
961 ttcatccagt tagtacaaac ctataatcag ttgtttgaag agaaccctat aaatgcaagt
1021 ggcgtggatg cgaaggctat tcttagcgcc cgcctctcta aatgccttgc cgagggtacc
1081 cgaatcttcg atccggtcac tggtacaacg catcgcatcg aggatgttgt cgatgggcgc
1141 aagcctattc atgtcgtggc tgctgccaag gacggaacgc tgctcgcgcg gcccgtggtg
1201 tcctggttcg accagggaac gcgggatgtg atcgggttgc ggatcgccgg tggcgccatc
1261 gtgtgggcga cacccgatca caaggtgctg acagagtacg gctggcgtgc cgccggggaa
1321 ctccgcaagg gagacagggt ggccggaccg ggtggttctg gtaacagcct ggccttgtcc
1381 ctgacggccg accagatggt cagtgccttg ttggatgctg agccccccat actctattcc
1441 gagtatgatc ctaccagtcc cttcagtgaa gcttcgatga tgggcttact gaccaacctg
1501 gcagacaggg agctggttca catgatcaac tgggcgaaga gggtgccagg ctttgtggat
1561 ttgaccctcc atgatcaggc ccaccttcta gaacgtgcct ggctagagat cctgatgatt
1621 ggtctcgtct ggcgctccat ggagcaccca gggaagctac tgtttgctcc taacttgctc
1681 ttggacagga accagggaaa atgtgtagag ggcatggtgg agatcttcga catgctgctg
1741 gctacatcat ctcggttccg catgatgaat ctgcagggag aggagtttgt gtgcctcaaa
1801 tctattattt tgcttaattc tggagtgtac acatttctgt ccagcaccct gaagtctctg
1861 gaagagaagg accatatcca ccgagccctg gacaagatca cggacacttt gatccacctg
1921 atggccaagg caggcctgac cctgcagcag cagcaccagc ggctggccca gctcctcctc
1981 atcctctccc acatcaggca catgagtaac aaaggaatgg agcatctgta cagcatgaag
2041 tacaagaacg tggtgcccct ctatgacctg ctgctggaga tgctggacgc ccaccgccta
2101 catgcgggtg gttctggtgc tagccgcgtg caggcgttcg cggatgccct ggatgacaaa
2161 ttcctgcacg acatgctggc ggaaggactc cgctattccg tgatccgaga agtgctgcca
2221 acgcggcggg cacgaacgtt cgacctcgag gtcgaggaac tgcacaccct cgtcgccgaa
2281 ggggttgtcg tgcacaactg ccgacggcta gaaaacctga tcgcacaatt acccggagag
2341 aagaaaaatg ggttgttcgg taaccttata gcgctctcac taggcctgac accaaatttt
2401 aagtcgaact tcgacttagc tgaagatgcc aaattgcagc ttagtaagga cacgtacgat
2461 gacgatctcg acaatctact ggcacaaatt ggagatcagt atgcggactt atttttggct
2521 gccaaaaacc ttagcgatgc aatcctccta tctgacatac tgagagttaa tactgagatt
2581 accaaggcgc cgttatccgc ttcaatgatc aaaaggtacg atgaacatca ccaagacttg
2641 acacttctca aggccctagt ccgtcagcaa ctgcctgaga aatataagga aatattcttt
2701 gatcagtcga aaaacgggta cgcaggttat attgacggcg gagcgagtca agaggaattc
2761 tacaagttta tcaaacccat attagagaag atggatggga cggaagagtt gcttgtaaaa
2821 ctcaatcgcg aagatctact gcgaaagcag cggactttcg acaacggtag cattccacat
2881 caaatccact taggcgaatt gcatgctata cttagaaggc aggaggattt ttatccgttc
2941 ctcaaagaca atcgtgaaaa gattgagaaa atcctaacct ttcgcatacc ttactatgtg
3001 ggacccctgg cccgagggaa ctctcggttc gcatggatga caagaaagtc cgaagaaacg
3061 attactccat ggaattttga ggaagttgtc gataaaggtg cgtcagctca atcgttcatc
3121 gagaggatga ccaactttga caagaattta ccgaacgaaa aagtattgcc taagcacagt
3181 ttactttacg agtatttcac agtgtacaat gaactcacga aagttaagta tgtcactgag
3241 ggcatgcgta aacccgcctt tctaagcgga gaacagaaga aagcaatagt agatctgtta
3301 ttcaagacca accgcaaagt gacagttaag caattgaaag aggactactt taagaaaatt
3361 gaatgcttcg attctgtcga gatctccggg gtagaagatc gatttaatgc gtcacttggt
3421 acgtatcatg acctcctaaa gataattaaa gataaggact tcctggataa cgaagagaat
3481 gaagatatct tagaagatat agtgttgact cttaccctct ttgaagatcg ggaaatgatt
3541 gaggaaagac taaaaacata cgctcacctg ttcgacgata aggttatgaa acagttaaag
3601 aggcgtcgct atacgggctg gggacgattg tcgcggaaac ttatcaacgg gataagagac
3661 aagcaaagtg gtaaaactat tctcgatttt ctaaagagcg acggcttcgc caataggaac
3721 tttatgcagc tgatccatga tgactcttta accttcaaag aggatataca aaaggcacag
3781 gtttccggac aaggggactc attgcacgaa catattgcga atcttgctgg ttcgccagcc
3841 atcaaaaagg gcatactcca gacagtcaaa gtagtggatg agctagttaa ggtcatggga
3901 cgtcacaaac cggaaaacat tgtaatcgag atggcacgcg aaaatcaaac gactcagaag
3961 gggcaaaaaa acagtcgaga gcggatgaag agaatagaag agggtattaa agaactgggc
4021 agccagatct taaaggagca tcctgtggaa aatacccaat tgcagaacga gaaactttac
4081 ctctattacc tacaaaatgg aagggacatg tatgttgatc aggaactgga cataaaccgt
4141 ttatctgatt acgacgtcga tcacattgta ccccaatcct ttttgaagga cgattcaatc
4201 gacaataaag tgcttacacg ctcggataag aaccgaggga aaagtgacaa tgttccaagc
4261 gaggaagtcg taaagaaaat gaagaactat tggcggcagc tcctaaatgc gaaactgata
4321 acgcaaagaa agttcgataa cttaactaaa gctgagaggg gtggcttgtc tgaacttgac
4381 aaggccggat ttattaaacg tcagctcgtg gaaacccgcc aaatcacaaa gcatgttgca
4441 cagatactag attcccgaat gaatacgaaa tacgacgaga acgataagct gattcgggaa
4501 gtcaaagtaa tcactttaaa gtcaaaattg gtgtcggact tcagaaagga ttttcaattc
4561 tataaagtta gggagataaa taactaccac catgcgcacg acgcttatct taatgccgtc
4621 gtagggaccg cactcattaa gaaatacccg aagctagaaa gtgagtttgt gtatggtgat
4681 tacaaagttt atgacgtccg taagatgatc gcgaaaagcg aacaggagat aggcaaggct
4741 acagccaaat acttctttta ttctaacatt atgaatttct ttaagacgga aatcactctg
4801 gcaaacggag agatacgcaa acgaccttta attgaaacca atggggagac aggtgaaatc
4861 gtatgggata agggccggga cttcgcgacg gtgagaaaag ttttgtccat gccccaagtc
4921 aacatagtaa agaaaactga ggtgcagacc ggagggtttt caaaggaatc gattcttcca
4981 aaaaggaata gtgataagct catcgctcgt aaaaaggact gggacccgaa aaagtacggt
5041 ggcttcgata gccctacagt tgcctattct gtcctagtag tggcaaaagt tgagaaggga
5101 aaatccaaga aactgaagtc agtcaaagaa ttattgggga taacgattat ggagcgctcg
5161 tcttttgaaa agaaccccat cgacttcctt gaggcgaaag gttacaagga agtaaaaaag
5221 gatctcataa ttaaactacc aaagtatagt ctgtttgagt tagaaaatgg ccgaaaacgg
5281 atgttggcta gcgccggaga gcttcaaaag gggaacgaac tcgcactacc gtctaaatac
5341 gtgaatttcc tgtatttagc gtcccattac gagaagttga aaggttcacc tgaagataac
5401 gaacagaagc aactttttgt tgagcagcac aaacattatc tcgacgaaat catagagcaa
5461 atttcggaat tcagtaagag agtcatccta gctgatgcca atctggacaa agtattaagc
5521 gcatacaaca agcacaggga taaacccata cgtgagcagg cggaaaatat tatccatttg
5581 tttactctta ccaacctcgg cgctccagcc gcattcaagt attttgacac aacgatagat
5641 cgcaaacgat acacttctac caaggaggtg ctagacgcga cactgattca ccaatccatc
5701 acgggattat atgaaactcg gatagatttg tcacagcttg ggggtgacgg atcccccaag
5761 aagaagagga aagtctcgag cgactacaaa gaccatgacg gtgattataa agatcatgac
5821 atcgattaca aggatgacga tgacaaggct gcaggatgac cggtcatcat caccatcacc
5881 attgagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
5941 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
6001 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
6061 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
6121 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctcgata ccgtcgacct
6181 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
6241 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctag ggtgcctaat
6301 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
6361 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
6421 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
6481 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
6541 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
6601 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
6661 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
6721 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
6781 cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg
6841 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
6901 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
6961 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
7021 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
7081 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
7141 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
7201 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
7261 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
7321 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
7381 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
7441 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
7501 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
7561 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
7621 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
7681 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
7741 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
7801 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
7861 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
7921 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
7981 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
8041 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
8101 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
8161 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
8221 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
8281 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
8341 cccgaaaagt gccacctgac gtcgacggat cgggagatcg atctcccgat cccctagggt
8401 cgactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
8461 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
8521 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
8581 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
8641 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
8701 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
8761 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
8821 ctgcccactt ggcagtacat caagtgtatc
//
