| 出品公司: | Clontech |
|---|---|
| 载体名称: | pGuide-it-tdTomato |
| 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: | 9038 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: | tdTomato红色荧光蛋白 |
| 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-tdTomato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: | 632604 |
| 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-tdTomato载体质粒基本信息
pGuide-IT-tdTomato质粒图谱载体图谱和pGuide-IT-tdTomato载体序列质粒序列多克隆位点信息
pGuide-IT-tdTomato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-tdTomato质粒序列载体序列
LOCUS pGuide-it-tdTomato Vector (Linear) 9042 bp ds-DNA linear SYN 05-DEC-2014
COMMENT Created by Clontech Laboratories Inc. http://www.clontech.com
COMMENT Cat No. 632602, 632604
SOURCE synthetic DNA construct
FEATURES Location/Qualifiers
misc_feature 1..93
/label="sgRNA"
/note="single-guide RNA scaffold segment; user clones guide sequence"
rep_origin complement(147..746)
/label="pUC origin"
/note="ori;high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(908..1768)
/label="AmpR"
/note="beta-lactamase, confers resistance to ampicillin, carbenicillin, and
related antibiotics"
polyA_signal complement(1904..2091)
/label="SV40 polyA signal"
CDS complement(2129..3556)
/label="tdTomato"
promoter complement(3574..3642)
/label="Minimal CMV Promoter 2"
misc_feature 3706..4115
/label="Enhancer"
promoter 4116..4241
/label="Minimal CMV Promoter 1"
CDS 4286..8389
/label="Cas9"
CDS 8405..8425
/label="NLS"
/note="nuclear localization signal of SV40 large T antigen"
CDS 8426..8449
/label="DYKDDDDK tag"
misc_feature 8488..8675
/label="SV40 polyA signal"
promoter 8792..9032
/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 ggcccctact tgtacagctc gtccatgccg tacaggaaca
2161 ggtggtggcg gccctcggag cgctcgtact gttccacgat ggtgtagtcc tcgttgtggg
2221 aggtgatgtc cagcttggtg tccacgtagt agtagccggg cagttgcacg ggcttcttgg
2281 ccatgtagat ggtcttgaac tccaccaggt agtggccgcc gtccttcagc ttcagggcct
2341 ggtggatctc gcccttcagc acgccgtcgc gggggtacag gcgctcggtg gaggcctccc
2401 agcccatggt cttcttctgc attacggggc cgtcgggggg gaagttggtg ccgcgcatct
2461 tcaccttgta gatcagcgtg ccgtcctgca gggaggagtc ctgggtcacg gtcaccagac
2521 cgccgtcctc gaagttcatc acgcgctccc acttgaagcc ctcggggaag gacagcttct
2581 tgtaatcggg gatgtcggcg gggtgcttca cgtacgcctt ggagccgtac atgaactggg
2641 gggacaggat gtcccaggcg aagggcaggg ggccgccctt ggtcaccttc agcttggcgg
2701 tctgggtgcc ctcgtagggg cggccctcgc cctcgccctc gatctcgaac tcgtggccgt
2761 tcatggagcc ctccatgcgc accttgaagc gcatgaactc tttgatgacg gccatgttgt
2821 tgtcctcgga ggaggcggtg ccggagctgc cgctgccggt gctgccggtg ccatgcccca
2881 ggaacaggtg gtggcggccc tcggagcgct cgtactgttc cacgatggtg tagtcctcgt
2941 tgtgggaggt gatgtccagc ttggtgtcca cgtagtagta gccgggcagt tgcacgggct
3001 tcttggccat gtagatggtc ttgaactcca ccaggtagtg gccgccgtcc ttcagcttca
3061 gggcctggtg gatctcgccc ttcagcacgc cgtcgcgggg gtacaggcgc tcggtggagg
3121 cctcccagcc catggtcttc ttctgcatta cggggccgtc gggggggaag ttggtgccgc
3181 gcatcttcac cttgtagatc agcgtgccgt cctgcaggga ggagtcctgg gtcacggtca
3241 ccagaccgcc gtcctcgaag ttcatcacgc gctcccactt gaagccctcg gggaaggaca
3301 gcttcttgta atcggggatg tcggcggggt gcttcacgta cgccttggag ccgtacatga
3361 actgggggga caggatgtcc caggcgaagg gcagggggcc gcccttggtc accttcagct
3421 tggcggtctg ggtgccctcg taggggcggc cctcgccctc gccctcgatc tcgaactcgt
3481 ggccgttcat ggagccctcc atgcgcacct tgaagcgcat gaactctttg atgacctcct
3541 cgcccttgct caccatggtg gcgaattctc caggcgatct gacggttcac taaacgagct
3601 ctgcttatat aggcctccca ccgtacacgc cacctcgaca tactcgagta gttattaata
3661 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
3721 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
3781 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
3841 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
3901 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
3961 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
4021 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
4081 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
4141 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
4201 ctatataagc agagctggtt tagtgaaccg tcagatccgc tagggatcct ctagtcagct
4261 gacgcgtgct agcgcggccg ccaccatgga caagaagtac agcatcggcc tggacatcgg
4321 taccaacagc gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaagtt
4381 caaggtgctg ggcaacaccg accgccacag catcaagaag aacctgatcg gcgccctgct
4441 gttcgacagc ggcgagaccg ccgaggccac ccgcctgaag cgcaccgccc gccgccgcta
4501 cacccgccgc aagaaccgca tctgctacct gcaggagatc ttcagcaacg agatggccaa
4561 ggtggacgac agcttcttcc accgcctgga ggagagcttc ctggtggagg aggacaagaa
4621 gcacgagcgc caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
4681 ccccaccatc taccacctgc gcaagaagct ggtggacagc accgacaagg ccgacctgcg
4741 cctgatctac ctggccctgg cccacatgat caagttccgc ggccacttcc tgatcgaggg
4801 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
4861 caaccagctg ttcgaggaga accccatcaa cgccagcggc gtggacgcca aggccatcct
4921 gagcgcccgc ctgagcaaga gccgccgcct ggagaacctg atcgcccagc tgcccggcga
4981 gaagaagaac ggcctgttcg gcaacctgat cgccctgagc ctgggcctga cccccaactt
5041 caagagcaac ttcgacctgg ccgaggacgc caagctgcag ctgagcaagg acacctacga
5101 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgttcctggc
5161 cgccaagaac ctgagcgacg ccatcctgct gagcgacatc ctgcgcgtga acaccgagat
5221 caccaaggcc cccctgagcg ccagcatgat caagcgctac gacgagcacc accaggacct
5281 gaccctgctg aaggccctgg tgcgccagca gctgcccgag aagtacaagg agatcttctt
5341 cgaccagagc aagaacggct acgccggcta catcgacggc ggcgccagcc aggaggagtt
5401 ctacaagttc atcaagccca tcctggagaa gatggacggc accgaggagc tgctggtgaa
5461 gctgaaccgc gaggacctgc tgcgcaagca gcgcaccttc gacaacggca gcatccccca
5521 ccagatccac ctgggcgagc tgcacgccat cctgcgccgc caggaggact tctacccctt
5581 cctgaaggac aaccgcgaga agatcgagaa gatcctgacc ttccgcatcc cctactacgt
5641 gggccccctg gcccgcggca acagccgctt cgcctggatg acccgcaaga gcgaggagac
5701 catcaccccc tggaacttcg aggaggtggt ggacaagggc gccagcgccc agagcttcat
5761 cgagcgcatg accaacttcg acaagaacct gcccaacgag aaggtgctgc ccaagcacag
5821 cctgctgtac gagtacttca ccgtgtacaa cgagctgacc aaggtgaagt acgtgaccga
5881 gggcatgcgc aagcccgcct tcctgagcgg cgagcagaag aaggccatcg tggacctgct
5941 gttcaagacc aaccgcaagg tgaccgtgaa gcagctgaag gaggactact tcaagaagat
6001 cgagtgcttc gacagcgtgg agatcagcgg cgtggaggac cgcttcaacg ccagcctggg
6061 cacctaccac gacctgctga agatcatcaa ggacaaggac ttcctggaca acgaggagaa
6121 cgaggacatc ctggaggaca tcgtgctgac cctgaccctg ttcgaggacc gcgagatgat
6181 cgaggagcgc ctgaagacct acgcccacct gttcgacgac aaggtgatga agcagctgaa
6241 gcgccgccgc tacaccggct ggggccgcct gagccgcaag cttatcaacg gcatccgcga
6301 caagcagagc ggcaagacca tcctggactt cctgaagagc gacggcttcg ccaaccgcaa
6361 cttcatgcag ctgatccacg acgacagcct gaccttcaag gaggacatcc agaaggccca
6421 ggtgagcggc cagggcgaca gcctgcacga gcacatcgcc aacctggccg gcagccccgc
6481 catcaagaag ggcatcctgc agaccgtgaa ggtggtggac gagctggtga aggtgatggg
6541 ccgccacaag cccgagaaca tcgtgatcga gatggcccgc gagaaccaga ccacccagaa
6601 gggccagaag aacagccgcg agcgcatgaa gcgcatcgag gagggcatca aggagctggg
6661 cagccagatc ctgaaggagc accccgtgga gaacacccag ctgcagaacg agaagctgta
6721 cctgtactac ctgcagaacg gccgcgacat gtacgtggac caggagctgg acatcaaccg
6781 cctgagcgac tacgacgtgg accacatcgt gccccagagc ttcctgaagg acgacagcat
6841 cgacaacaag gtgctgaccc gcagcgacaa gaaccgcggc aagagcgaca acgtgcccag
6901 cgaggaggtg gtgaagaaga tgaagaacta ctggcgccag ctgctgaacg ccaagctgat
6961 cacccagcgc aagttcgaca acctgaccaa ggccgagcgc ggcggcctga gcgagctgga
7021 caaggccggc ttcatcaagc gccagctggt ggagacccgc cagatcacca agcacgtggc
7081 ccagatcctg gacagccgca tgaacaccaa gtacgacgag aacgacaagc tgatccgcga
7141 ggtgaaggtg atcaccctga agagcaagct ggtgagcgac ttccgcaagg acttccagtt
7201 ctacaaggtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
7261 ggtgggcacc gccctgatca agaagtaccc caagctggag agcgagttcg tgtacggcga
7321 ctacaaggtg tacgacgtgc gcaagatgat cgccaagagc gagcaggaga tcggcaaggc
7381 caccgccaag tacttcttct acagcaacat catgaacttc ttcaagaccg agatcaccct
7441 ggccaacggc gagatccgca agcgccccct gatcgagacc aacggcgaga ccggcgagat
7501 cgtgtgggac aagggccgcg acttcgccac cgtgcgcaag gtgctgagca tgccccaggt
7561 gaacatcgtg aagaagaccg aggtgcagac cggcggcttc agcaaggaga gcatcctgcc
7621 caagcgcaac agcgacaagc tgatcgcccg caagaaggac tgggacccca agaagtacgg
7681 cggcttcgac agccccaccg tggcctacag cgtgctggtg gtggccaagg tggagaaggg
7741 caagagcaag aagctgaaga gcgtgaagga gctgctgggc atcaccatca tggagcgcag
7801 cagcttcgag aagaacccca tcgacttcct ggaggccaag ggctacaagg aggtgaagaa
7861 ggacctgatc atcaagctgc ccaagtacag cctgttcgag ctggagaacg gccgcaagcg
7921 catgctggcc agcgccggcg agctgcagaa gggcaacgag ctggccctgc ccagcaagta
7981 cgtgaacttc ctgtacctgg ccagccacta cgagaagctg aagggcagcc ccgaggacaa
8041 cgagcagaag cagctgttcg tggagcagca caagcactac ctggacgaga tcatcgagca
8101 gatcagcgag ttcagcaagc gcgtgatcct ggccgacgcc aacctggaca aggtgctgag
8161 cgcctacaac aagcaccgcg acaagcccat ccgcgagcag gccgagaaca tcatccacct
8221 gttcaccctg accaacctgg gcgcccccgc cgccttcaag tacttcgaca ccaccatcga
8281 ccgcaagcgc tacaccagca ccaaggaggt gctggacgcc accctgatcc accagagcat
8341 caccggtctg tacgagaccc gcatcgacct gagccagctg ggcggcgacg gcggctccgg
8401 acctccaaag aaaaagagaa aagtagacta caaggacgac gatgacaagt aaatcgataa
8461 gcttgtcgac gatatctcca gaggatcata atcagccata ccacatttgt agaggtttta
8521 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
8581 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
8641 aatttcacaa ataaagcatt tttttcactg ccccgagctt cctcgctcac tgactcgctg
8701 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
8761 tccacagaat caggggataa cgcaggaaag agagggccta tttcccatga ttccttcata
8821 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
8881 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
8941 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
9001 tttcttggct ttatatatct tgtggaaagg acgaaacacc gg
//
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