pGuide-IT-ZsGreen1

载体基本信息

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