| 质粒名称: | MSP1673 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 65769 |
| 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 Streptococcus thermophilus1 Cas9-NLS, and St1Cas9 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（插入件上的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: | MSP1673 was a gift from Keith Joung (Addgene plasmid # 65769 ; http://n2t.net/addgene:65769 ; RRID:Addgene_65769) |
| 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 MSP1673 7309 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Bacterial expression plasmid for S. thermophilus1 Cas9.
ACCESSION .
VERSION .
KEYWORDS MSP1673
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7309)
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 7309)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7309
/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..3436
/codon_start=1
/product="Cas9 endonuclease from the Streptococcus
thermophilus Type II CRISPR/Cas system (Esvelt et al.,
2013)"
/label=St1Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="MGSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNL
VRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFTKISINLNPYQLRVKGLTDELSNE
ELFIALKNMVKHRGISYLDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQTY
GQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQQEFNPQITDEFINRYLEILTG
KRKYYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNL
LNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVADIKGYRI
DKSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFAD
GSFSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELYETSEEQMTILTRLGKQK
TTSSSNKTKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNE
DDEKKAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGER
CLYTGKTISIHDLINNSNQFEVDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQAL
DSMDDAWSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFIERNLVDTRYASRV
VLNALQEHFRAHKIDTKVSVVRGQFTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQLN
LWKKQKNTLVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFS
YQVDSKFNRKISDATIYATRQAKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDKS
KFLMYRHDPQTFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYIRKYSKKGNG
PEIKSLKYYDSKLGNHIDITPKDSNNKVVLQSVSPWRADVYFNKTTGKYEILGLKYADL
QFEKGTGTYKISQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQQLFRFLSR
TMPKQKHYVELKPYDKQKFEGGEALIKVLGNVANSGQCKKGLGKSNISIYKVRTDVLGN
QHIIKNEGDKPKLDF"
CDS 3449..3469
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
promoter 3538..3556
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
misc_RNA 3618..3742
/label=St1 gRNA scaffold
/note="guide RNA scaffold for the Streptococcus
thermophilus CRISPR/Cas9 system (Esvelt et al., 2013)"
primer_bind complement(3747..3765)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 3761..3809
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(4030..4689)
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
primer_bind 4604..4623
/label=CAT-R
/note="Chloramphenicol resistance gene, reverse primer"
promoter complement(4690..4792)
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
primer_bind complement(5184..5201)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5318..5862)
/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(5398..5417)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6087..7169)
/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 7131..7150
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(7170..7247)
/gene="lacI"
/label=lacI promoter
primer_bind 7293..3
/label=T7
/note="T7 promoter, forward primer"
promoter 7293..2
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
61 gagatatacc atgggcagcg acctggtgct gggcctggac atcggcatcg gcagcgtggg
121 cgtgggcatc ctgaacaagg tgaccggcga gatcatccac aagaacagtc gcatcttccc
181 tgctgctcag gctgagaaca acctggtgcg ccgcaccaac cgccagggtc gccggcttgc
241 tcgccgcaag aagcaccggc gcgtgcgcct gaaccgcctg ttcgaggaga gcggcctgat
301 caccgacttc accaagatca gcatcaacct gaacccctac cagctgcgcg tgaagggcct
361 gaccgacgag ctgagcaacg aggagctgtt catcgccctg aagaacatgg tgaagcaccg
421 cggcatcagc tacctggacg acgccagcga cgacggcaac agcagcgtgg gcgactacgc
481 ccagatcgtg aaggagaaca gcaagcagct ggagaccaag acccccggcc agatccagct
541 ggagcgctac cagacctacg gccagctgcg cggcgacttc accgtggaga aggacggcaa
601 gaagcaccgc ctgatcaacg tgttccccac cagcgcctac cgcagcgagg ccctgcgcat
661 cctgcagacc cagcaggagt tcaaccccca gatcaccgac gagttcatca accgctacct
721 ggagatcctg accggcaagc gcaagtacta ccacggcccc ggcaacgaga agagccgcac
781 cgactacggc cgctaccgca ccagcggcga gaccctggac aacatcttcg gcatcctgat
841 cggcaagtgc accttctacc ccgacgagtt ccgcgccgcc aaggccagct acaccgccca
901 ggagttcaat ctgctgaacg acctgaacaa cctgaccgtg cccaccgaga ccaagaagct
961 gagcaaggag cagaagaacc agatcatcaa ctacgtgaag aacgagaagg ctatgggccc
1021 cgccaagctg ttcaagtaca tcgccaagct gctgagctgc gacgtggccg acatcaaggg
1081 ctaccgcatc gacaagagcg gcaaggccga gatccacacc ttcgaggcct accgcaagat
1141 gaagaccctg gagaccctgg acatcgagca gatggaccga gagaccctgg acaagctggc
1201 ctacgtgctg accctgaaca ccgagcgcga gggcatccag gaggccctgg agcacgagtt
1261 cgccgacggc agcttcagcc agaaacaggt ggacgagctg gtgcagttcc gcaaggccaa
1321 cagcagcatc ttcggcaagg gctggcacaa cttcagcgtg aagctgatga tggagctgat
1381 ccccgagctg tacgagacca gcgaggagca gatgaccatc ctgacccgcc tgggcaagca
1441 gaagaccacc agcagcagca acaagaccaa gtacatcgac gagaagctgc tgaccgagga
1501 gatctacaac cccgtggtgg ccaagagcgt gcgccaggcc atcaagatcg tgaacgccgc
1561 catcaaggag tacggcgact tcgacaacat cgtgatcgag atggcccgcg agaccaacga
1621 ggacgacgag aagaaggcca tccagaagat ccagaaggcc aacaaggacg agaaggacgc
1681 cgccatgctg aaggccgcca accagtacaa cggcaaggcc gagctgcccc acagcgtgtt
1741 ccacggccac aagcagctgg ccaccaagat ccgcctgtgg caccagcagg gcgagcgctg
1801 cctgtacacc ggcaagacca tcagcatcca cgacctgatc aacaacagca accagttcga
1861 ggtggaccac atcctgcccc tgagcatcac cttcgacgac agcctggcca acaaggtgct
1921 ggtgtacgcc accgccaacc aggagaaggg ccagcgcacc ccctaccagg ccctggacag
1981 catggacgac gcctggagct tccgcgagct gaaggccttc gtgcgcgaga gcaagaccct
2041 gagcaacaag aagaaggagt atctgctgac cgaggaggac atcagcaagt tcgacgtgcg
2101 caagaagttc atcgagcgca acctggtgga cacccgctac gccagccgcg tggtgctgaa
2161 cgccctgcag gagcacttcc gcgcccacaa gatcgacacc aaggtgagcg tggtgcgcgg
2221 ccagttcacc agccagctgc gccgccactg gggcatcgag aagacccgcg acacctacca
2281 ccaccacgcc gtggacgccc tgatcattgc ggcttctagc cagctgaacc tgtggaagaa
2341 gcagaagaac accctggtga gctacagcga ggaccagctg ctggacatcg agaccggcga
2401 gctgatcagc gacgacgagt acaaggagag cgtgttcaag gccccctacc agcacttcgt
2461 ggacaccctg aagagcaagg agttcgagga cagcatcctg ttcagctacc aggtggacag
2521 caagttcaac cgcaagatca gcgacgccac catctacgcc acccgccagg ccaaggtggg
2581 caaggacaag gccgacgaga cctacgtgct gggcaagatc aaggacatct acacccagga
2641 cggctacgac gccttcatga agatctacaa gaaggacaag agcaagttcc tgatgtaccg
2701 ccacgacccc cagaccttcg agaaggtgat cgagcccatc ctggagaact accccaacaa
2761 gcagatcaac gagaaaggca aggaggtgcc ctgcaacccc ttcctgaagt acaaggagga
2821 gcacggctac atccgcaagt acagcaagaa gggcaacggc cccgagatca agagcctgaa
2881 gtactacgac agcaagctgg gcaaccacat cgacatcacc cccaaggaca gcaacaacaa
2941 ggtggtgctg cagagcgtga gcccctggcg cgccgacgtg tacttcaaca agaccaccgg
3001 caagtacgag atcctggggc tgaagtacgc cgatctgcag tttgagaaag gcacaggcac
3061 ctacaagatc agccaggaga agtacaacga catcaagaag aaggagggcg tggacagcga
3121 cagcgagttc aagttcaccc tgtacaagaa cgaccttctg ctggtgaagg acaccgagac
3181 caaggagcaa cagctgttcc gcttcctgag ccgcaccatg cccaagcaga agcactacgt
3241 ggagctgaag ccctacgaca agcagaagtt cgagggcggc gaggccctga tcaaggtgct
3301 gggcaacgtg gccaacagcg gccagtgcaa gaagggcctg ggcaagagca acatcagcat
3361 ctacaaggtg cgcaccgacg tgctgggcaa ccagcacatc atcaagaacg agggcgacaa
3421 gcccaagttg gacttcagca gggctgaccc caagaagaag aggaaggtgt gagcggccgc
3481 ataatgctta agtcgaacag aaagtaatcg tattgtacac ggccgcataa tcgaaattaa
3541 tacgactcac tataggactg caggtcatgc catagcgttg ttcggaacag attcaccaac
3601 acctagtacc tgcactcgtt tttgtactct caagatttaa gtaactgtac aacgaaactt
3661 acacagttac ttaaatcttg cagaagctac aaagataagg cttcatgccg aaatcaacac
3721 cctgtcattt tatggcaggg tgttttccgc tgagcaataa ctagcataac ccccttgggg
3781 cctctaaacg ggtcttgagg ggttttttgc tgaaacctca ggcattttga gaagcacacg
3841 gtcacactgc ttccggtagt caataaaccg gtaaaccagc aatagacata agcggctatt
3901 taacgaccct gccctgaacc gacgaccggg tcgaatttgc tttcgaattt ctgccattca
3961 tccgcttatt atcacttatt caggcgtagc accaggcgtt taagggcacc aataactgcc
4021 ttaaaaaaat tacgccccgc cctgccactc atcgcagtac tgttgtaatt cattaagcat
4081 tctgccgaca tggaagccat cacagacggc atgatgaacc tgaatcgcca gcggcatcag
4141 caccttgtcg ccttgcgtat aatatttgcc catagtgaaa acgggggcga agaagttgtc
4201 catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg ctgagacgaa
4261 aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt aacacgccac
4321 atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac tccagagcga
4381 tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac tatcccatat
4441 caccagctca ccgtctttca ttgccatacg gaactccgga tgagcattca tcaggcgggc
4501 aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg tctttaaaaa
4561 ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg actgaaatgc
4621 ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc cagtgatttt
4681 tttctccatt ttagcttcct tagctcctga aaatctcgat aactcaaaaa atacgcccgg
4741 tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtgccgat caacgtctca
4801 ttttcgccaa aagttggccc agggcttccc ggtatcaaca gggacaccag gatttattta
4861 ttctgcgaag tgatcttccg tcacaggtat ttattcggcg caaagtgcgt cgggtgatgc
4921 tgccaactta ctgatttagt gtatgatggt gtttttgagg tgctccagtg gcttctgttt
4981 ctatcagctg tccctcctgt tcagctactg acggggtggt gcgtaacggc aaaagcaccg
5041 ccggacatca gcgctagcgg agtgtatact ggcttactat gttggcactg atgagggtgt
5101 cagtgaagtg cttcatgtgg caggagaaaa aaggctgcac cggtgcgtca gcagaatatg
5161 tgatacagga tatattccgc ttcctcgctc actgactcgc tacgctcggt cgttcgactg
5221 cggcgagcgg aaatggctta cgaacggggc ggagatttcc tggaagatgc caggaagata
5281 cttaacaggg aagtgagagg gccgcggcaa agccgttttt ccataggctc cgcccccctg
5341 acaagcatca cgaaatctga cgctcaaatc agtggtggcg aaacccgaca ggactataaa
5401 gataccaggc gtttcccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt
5461 taccggtgtc attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt
5521 ccgggtaggc agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc
5581 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac
5641 cactggcagc agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt
5701 taaggctaaa ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg
5761 ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg
5821 ttttcagagc aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaat
5881 cagataaaat atttctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
5941 agccccatac gatataagtt gtaattctca tgttagtcat gccccgcgcc caccggaagg
6001 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
6061 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
6121 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
6181 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
6241 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
6301 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
6361 accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
6421 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
6481 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
6541 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
6601 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
6661 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
6721 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
6781 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
6841 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
6901 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
6961 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
7021 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
7081 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
7141 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
7201 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
7261 ctctccctta tgcgactcct gcattaggaa attaatacga ctcactata
//
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