| 出品公司: | Clontech |
|---|---|
| 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。 |
| 产品目录号: | 632603 |
| 稳定性: | 稳表达 |
| 组成型/诱导型: | 组成型 |
| 病毒/非病毒: | 慢病毒 |
买家导航
pGuide-IT-ZsGreen1载体质粒基本信息
pGuide-IT-ZsGreen1质粒图谱载体图谱和pGuide-IT-ZsGreen1载体序列质粒序列多克隆位点信息
pGuide-IT-ZsGreen1质粒载体简介
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 sgRNAs
The 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.
Features
A 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 plasmids
Applications
Cloning and expression of a target single guide RNA (sgRNA) for mammalian genome editing using CRISPR/Cas9 technology
pGuide-IT-ZsGreen1质粒序列载体序列
LOCUS pGuide-it-ZsGreen1 Vector (Linear) 8307 bp ds-DNA linear SYN 05-DEC-2014
COMMENT Created by Clontech Laboratories Inc. http://www.clontech.com
COMMENT Cat No. 632601, 632603
SOURCE synthetic DNA construct
FEATURES 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
//
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