pGuide-IT-ZsGreen1

保存条件 ：-20℃

保质期 ：6个月

英文名 ：pGuide-IT-ZsGreen1

库存 ：大量

供应商 ：钰博生物

规格 ：1ug/5ug

pGuide-IT-ZsGreen1

载体基本信息

出品公司:	Ybscience
载体名称:	pGuide-it-ZsGreen1
质粒类型:	慢病毒载体；CRISPR/Cas9载体；荧光报告载体
高拷贝/低拷贝:	高拷贝
克隆方法:	限制性内切酶，多克隆位点
启动子:	Minimal CMV
载体大小:	8303 bp
5' 测序引物及序列:	--
3' 测序引物及序列:	--
载体标签:	--
载体抗性:	氨苄青霉素
筛选标记:	绿色荧光蛋白ZsGreen1
克隆菌株:	Stbl3、 Stellar
宿主细胞（系）:	常规细胞系（293、CV-1、CHO等）
备注:	慢病毒载体pGuide-it-ZsGreen1是CRISPR/Cas9载体；Clontech提供的是线性化的质粒，Biofeng提供的是环状质粒。
稳定性:	稳表达
组成型/诱导型:	组成型
病毒/非病毒:	慢病毒

 

载体质粒图谱和多克隆位点信息

载体简介

 

CRISPR/Cas9 技术The Guide-it CRISPR/Cas9 System (Green, Cat. No. 632601; Red, Cat. No. 632602) is a complete system for the cloning and expression of a target single guide RNA (sgRNA) for mammalian gene modification studies using CRISPR/Cas9 technology. The included vector is used to simultaneously express Cas9, a target-specific sgRNA, and an exceptionally bright fluorescent protein (either ZsGreen1 or tdTomato) for monitoring transfection efficiency and/or for further enriching/isolating transfected cells by flow cytometry. The vector is pre-linearized for simple insertion of a target sequence for sgRNA expression from the human U6 promoter; Cas9 nuclease and either ZsGreen1 or tdTomato are co-expressed from a bidirectional CMV promoter. Sufficient reagents are provided in this kit for construction of 10 different target (sgRNA) expression plasmids. To construct the vector, a pair of user-provided oligos corresponding to the target genomic sequence of interest are annealed to form a duplex. Then, the duplexed DNA is cloned into the pre-linearized vector using the included high-efficiency ligation mix. This kit also contains necessary controls and Stellar? Competent Cells.Cloning and Expression System for sgRNAsThe Guide-it CRISPR/Cas9 Systems are kits for the cloning and expression of target single guide RNAs (sgRNAs) for mammalian genome editing using CRISPR/Cas9 technology. The vector in this system simultaneously expresses Cas9 nuclease, a target-specific sgRNA, and an exceptionally bright fluorescent protein for monitoring transfection efficiency and/or for further enriching/isolating transfected cells by flow cytometry (ZsGreen1 and tdTomato versions are available). Generating a plasmid that expresses a sequence-specific sgRNA with this system is simple; a pair of user-provided oligos corresponding to the target genomic sequence of interest are annealed to form a duplex, and the duplexed oligos are inserted into the pre-linearized vector using the included high-efficiency ligation mix. The kit also includes Stellar competent cells to ensure high efficiency transformation.FeaturesA single vector expresses your target-specific sgRNA from the human U6 promoter, and Cas9 nuclease and a bright fluorescent marker (either ZsGreen1 or tdTomato) are expressed from a bidirectional CMV promoter tdTomato and ZsGreen1 are exceptionally bright fluorescent proteins, 6x and 2.5x brighter than EGFP, respectively; these markers can be used to monitor transfection  efficiency and/or to enrich/isolate transfected cells by flow cytometry Simple plasmid construction—design oligos specific to your target sequence, and clone into the pre-linearized plasmid using the included ligation reagents and high- efficiency competent cells Includes sufficient reagents for construction of 10 different target (sgRNA) expression plasmidsApplicationsCloning and expression of a target single guide RNA (sgRNA) for mammalian genome editing using CRISPR/Cas9 technology

 

载体序列

LOCUS       pGuide-it-ZsGreen1 Vector (Linear)           8307 bp ds-DNA    linear   SYN 05-DEC-2014COMMENT     Created by Clontech Laboratories Inc. http://www.clontech.comCOMMENT     Cat No. 632601, 632603SOURCE      synthetic DNA constructFEATURES             Location/Qualifiers     misc_feature    1..93                     /label="sgRNA"                     /note="single-guide RNA scaffold segment; user clones guide sequence"     misc_feature    complement(147..746)                     /label="pUC origin"                     /note="ori;high-copy-number ColE1/pMB1/pBR322/pUC origin of                     replication"     CDS             complement(908..1768)                     /note="AmpR"                     /note="beta-lactamase, confers resistance to ampicillin, carbenicillin, and                     related antibiotics"     polyA_signal    complement(1904..2091)                     /label="SV40 polyA signal"     CDS             complement(2129..2821)                     /label="ZsGreen1"     promoter        complement(2839..2907)                     /label="Minimal CMV Promoter 2"     misc_feature    2971..3380                     /label="Enhancer"     promoter        3381..3506                     /label="Minimal CMV Promoter 1"     CDS             3551..7654                     /label="Cas9"     CDS             7670..7690                     /label="NLS"                     /note="nuclear localization signal of SV40 large T antigen"     CDS             7691..7714                     /label="DYKDDDDK tag"     misc_feature    7753..7940                     /label="SV40 polyA signal"     promoter        8057..8297                     /label="U6 Promoter"                     /note="U6 promoter;RNA polymerase III promoter for human U6                     snRNA"ORIGIN        1 gtttaagagc tatgctggaa acagcatagc aagtttaaat aaggctagtc cgttatcaac       61 ttgaaaaagt ggcaccgagt cggtgctttt tttgagcaaa aggccagcaa aaggccagga      121 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc      181 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg      241 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat      301 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt      361 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc      421 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg      481 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg      541 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg      601 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg      661 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca      721 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga      781 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga      841 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt      901 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt      961 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat     1021 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag     1081 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct     1141 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt     1201 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg     1261 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca     1321 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt     1381 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat     1441 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac     1501 cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa     1561 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt     1621 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt     1681 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa     1741 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt     1801 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa     1861 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcggcagt gaaaaaaatg     1921 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa     1981 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga     2041 ggttttttaa agcaagtaaa acctctacaa atgtggtatg gctgattatg atcctctaga     2101 ctgcagcctc aggagatctg ggccctcagg gcaaggcgga gccggaggcg atggcgtgct     2161 cggtcaggtg ccacttctgg ttcttggcgt cgctgcggtc ctcgcgggtc agcttgtgct     2221 ggatgaagtg ccagtcgggc atcttgcggg gcacggactt ggccttgtac acggtgtcga     2281 actggcagcg caagcggcca ccgtccttca gcagcaggta catgctcacg tcgcccttca     2341 agatgccctg cttgggcacg gggatgatct tctcgcagga gggctcccag ttgtcggtca     2401 tcttcttcat cacggggccg tcggcgggga agttcacgcc gtagaacttg gactcgtggt     2461 acatgcagtt ctcctccacg ctcacggtga tgtcggcgtt gcagatgcac acggcgccgt     2521 cctcgaacag gaaggagcgg tcccaggtgt agccggcggg gcaggagttc ttgaagtagt     2581 cgacgatgtc ctgggggtac tcggtgaaca cgcggttgcc gtacatgaag gcggcggaca     2641 agatgtcctc ggcgaagggc aaggggccgc cctccaccac gcacaggttg atggcctgct     2701 tgcccttgaa ggggtagccg atgccctcgc cggtgatcac gaacttgtgg ccgtccacgc     2761 agccctccat gcggtacttc atggtcatct ccttggtcag gccgtgcttg gactgggcca     2821 tggtggcgaa ttctccaggc gatctgacgg ttcactaaac gagctctgct tatataggcc     2881 tcccaccgta cacgccacct cgacatactc gagtagttat taatagtaat caattacggg     2941 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc     3001 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat     3061 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc     3121 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga     3181 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg     3241 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat     3301 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt     3361 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc     3421 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc     3481 tggtttagtg aaccgtcaga tccgctaggg atcctctagt cagctgacgc gtgctagcgc     3541 ggccgccacc atggacaaga agtacagcat cggcctggac atcggtacca acagcgtggg     3601 ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aagttcaagg tgctgggcaa     3661 caccgaccgc cacagcatca agaagaacct gatcggcgcc ctgctgttcg acagcggcga     3721 gaccgccgag gccacccgcc tgaagcgcac cgcccgccgc cgctacaccc gccgcaagaa     3781 ccgcatctgc tacctgcagg agatcttcag caacgagatg gccaaggtgg acgacagctt     3841 cttccaccgc ctggaggaga gcttcctggt ggaggaggac aagaagcacg agcgccaccc     3901 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca     3961 cctgcgcaag aagctggtgg acagcaccga caaggccgac ctgcgcctga tctacctggc     4021 cctggcccac atgatcaagt tccgcggcca cttcctgatc gagggcgacc tgaaccccga     4081 caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga     4141 ggagaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgagcg cccgcctgag     4201 caagagccgc cgcctggaga acctgatcgc ccagctgccc ggcgagaaga agaacggcct     4261 gttcggcaac ctgatcgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga     4321 cctggccgag gacgccaagc tgcagctgag caaggacacc tacgacgacg acctggacaa     4381 cctgctggcc cagatcggcg accagtacgc cgacctgttc ctggccgcca agaacctgag     4441 cgacgccatc ctgctgagcg acatcctgcg cgtgaacacc gagatcacca aggcccccct     4501 gagcgccagc atgatcaagc gctacgacga gcaccaccag gacctgaccc tgctgaaggc     4561 cctggtgcgc cagcagctgc ccgagaagta caaggagatc ttcttcgacc agagcaagaa     4621 cggctacgcc ggctacatcg acggcggcgc cagccaggag gagttctaca agttcatcaa     4681 gcccatcctg gagaagatgg acggcaccga ggagctgctg gtgaagctga accgcgagga     4741 cctgctgcgc aagcagcgca ccttcgacaa cggcagcatc ccccaccaga tccacctggg     4801 cgagctgcac gccatcctgc gccgccagga ggacttctac cccttcctga aggacaaccg     4861 cgagaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc ccctggcccg     4921 cggcaacagc cgcttcgcct ggatgacccg caagagcgag gagaccatca ccccctggaa     4981 cttcgaggag gtggtggaca agggcgccag cgcccagagc ttcatcgagc gcatgaccaa     5041 cttcgacaag aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta     5101 cttcaccgtg tacaacgagc tgaccaaggt gaagtacgtg accgagggca tgcgcaagcc     5161 cgccttcctg agcggcgagc agaagaaggc catcgtggac ctgctgttca agaccaaccg     5221 caaggtgacc gtgaagcagc tgaaggagga ctacttcaag aagatcgagt gcttcgacag     5281 cgtggagatc agcggcgtgg aggaccgctt caacgccagc ctgggcacct accacgacct     5341 gctgaagatc atcaaggaca aggacttcct ggacaacgag gagaacgagg acatcctgga     5401 ggacatcgtg ctgaccctga ccctgttcga ggaccgcgag atgatcgagg agcgcctgaa     5461 gacctacgcc cacctgttcg acgacaaggt gatgaagcag ctgaagcgcc gccgctacac     5521 cggctggggc cgcctgagcc gcaagcttat caacggcatc cgcgacaagc agagcggcaa     5581 gaccatcctg gacttcctga agagcgacgg cttcgccaac cgcaacttca tgcagctgat     5641 ccacgacgac agcctgacct tcaaggagga catccagaag gcccaggtga gcggccaggg     5701 cgacagcctg cacgagcaca tcgccaacct ggccggcagc cccgccatca agaagggcat     5761 cctgcagacc gtgaaggtgg tggacgagct ggtgaaggtg atgggccgcc acaagcccga     5821 gaacatcgtg atcgagatgg cccgcgagaa ccagaccacc cagaagggcc agaagaacag     5881 ccgcgagcgc atgaagcgca tcgaggaggg catcaaggag ctgggcagcc agatcctgaa     5941 ggagcacccc gtggagaaca cccagctgca gaacgagaag ctgtacctgt actacctgca     6001 gaacggccgc gacatgtacg tggaccagga gctggacatc aaccgcctga gcgactacga     6061 cgtggaccac atcgtgcccc agagcttcct gaaggacgac agcatcgaca acaaggtgct     6121 gacccgcagc gacaagaacc gcggcaagag cgacaacgtg cccagcgagg aggtggtgaa     6181 gaagatgaag aactactggc gccagctgct gaacgccaag ctgatcaccc agcgcaagtt     6241 cgacaacctg accaaggccg agcgcggcgg cctgagcgag ctggacaagg ccggcttcat     6301 caagcgccag ctggtggaga cccgccagat caccaagcac gtggcccaga tcctggacag     6361 ccgcatgaac accaagtacg acgagaacga caagctgatc cgcgaggtga aggtgatcac     6421 cctgaagagc aagctggtga gcgacttccg caaggacttc cagttctaca aggtgcgcga     6481 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtggtgg gcaccgccct     6541 gatcaagaag taccccaagc tggagagcga gttcgtgtac ggcgactaca aggtgtacga     6601 cgtgcgcaag atgatcgcca agagcgagca ggagatcggc aaggccaccg ccaagtactt     6661 cttctacagc aacatcatga acttcttcaa gaccgagatc accctggcca acggcgagat     6721 ccgcaagcgc cccctgatcg agaccaacgg cgagaccggc gagatcgtgt gggacaaggg     6781 ccgcgacttc gccaccgtgc gcaaggtgct gagcatgccc caggtgaaca tcgtgaagaa     6841 gaccgaggtg cagaccggcg gcttcagcaa ggagagcatc ctgcccaagc gcaacagcga     6901 caagctgatc gcccgcaaga aggactggga ccccaagaag tacggcggct tcgacagccc     6961 caccgtggcc tacagcgtgc tggtggtggc caaggtggag aagggcaaga gcaagaagct     7021 gaagagcgtg aaggagctgc tgggcatcac catcatggag cgcagcagct tcgagaagaa     7081 ccccatcgac ttcctggagg ccaagggcta caaggaggtg aagaaggacc tgatcatcaa     7141 gctgcccaag tacagcctgt tcgagctgga gaacggccgc aagcgcatgc tggccagcgc     7201 cggcgagctg cagaagggca acgagctggc cctgcccagc aagtacgtga acttcctgta     7261 cctggccagc cactacgaga agctgaaggg cagccccgag gacaacgagc agaagcagct     7321 gttcgtggag cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttcag     7381 caagcgcgtg atcctggccg acgccaacct ggacaaggtg ctgagcgcct acaacaagca     7441 ccgcgacaag cccatccgcg agcaggccga gaacatcatc cacctgttca ccctgaccaa     7501 cctgggcgcc cccgccgcct tcaagtactt cgacaccacc atcgaccgca agcgctacac     7561 cagcaccaag gaggtgctgg acgccaccct gatccaccag agcatcaccg gtctgtacga     7621 gacccgcatc gacctgagcc agctgggcgg cgacggcggc tccggacctc caaagaaaaa     7681 gagaaaagta gactacaagg acgacgatga caagtaaatc gataagcttg tcgacgatat     7741 ctccagagga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac     7801 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg     7861 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa     7921 gcattttttt cactgccccg agcttcctcg ctcactgact cgctgcgctc ggtcgttcgg     7981 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg     8041 gataacgcag gaaagagagg gcctatttcc catgattcct tcatatttgc atatacgata     8101 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca     8161 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt     8221 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata     8281 tatcttgtgg aaaggacgaa acaccgg