pGuide-IT-tdTomato

pGuide-IT-tdTomato

载体基本信息

出品公司:	Ybscience
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。
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 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

 

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