| 质粒名称: | pT7-SpCas9_sgRNA-site2 (RTW448) |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 160137 |
| 存储实验室: | Keith Joung, Benjamin Kleinstiver |
|
载体骨架基本信息 |
|
| 载体名称: | DR274 (Addgene plasmid #42250) |
| 载体类型: | T7启动子sgRNA的体外转录 |
| 载体抗性: | 卡那霉素50 μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | SpCas9 sgRNA with spacer #2 (spacer=GTCGCCCTCGAACTTCACCT),RTW448 |
| 物种来源: | Synthetic |
| 启动子: | T7 |
| 克隆方法: | 限制性内切酶 |
| 5’测序引物 | M13F - TGTAAAACGACGGCCAGT |
|
文献引用方法 |
|
| 材料和方法部分: | pT7-SpCas9_sgRNA-site2 (RTW448) was a gift from Keith Joung & Benjamin Kleinstiver (Addgene plasmid # 160137 ; http://n2t.net/addgene:160137 ; RRID:Addgene_160137) |
| 参考文献部分: | Scalable characterization of the PAM requirements of CRISPR-Cas enzymes using HT-PAMDA. Walton RT, Hsu JY, Joung JK, Kleinstiver BP. Nat Protoc. 2021 Feb 5. pii: 10.1038/s41596-020-00465-2. doi: 10.1038/s41596-020-00465-2. 10.1038/s41596-020-00465-2 PubMed 33547443 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
质粒图谱.png)
载体简介
载体序列
LOCUS pT7-SpCas9_sgRNA-site2 (RTW448) 2144 bp ds-DNA circular SYN 25-MAY-2021
DEFINITION T7 promoter expression plasmid for in vitro transcription of SpCas9
sgRNA with spacer #2.
ACCESSION .
VERSION .
KEYWORDS pT7-SpCas9_sgRNA-site2 (RTW448)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 2144)
AUTHORS Walton RT, Hsu JY, Joung JK, Kleinstiver BP
TITLE Scalable characterization of the PAM requirements of CRISPR-Cas
enzymes using HT-PAMDA.
JOURNAL Nat Protoc. 2021 Feb 5. pii: 10.1038/s41596-020-00465-2. doi:
10.1038/s41596-020-00465-2.
PUBMED 33547443
REFERENCE 2 (bases 1 to 2144)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 25, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..2144
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 20..36
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 20..36
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
promoter 165..182
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
misc_RNA 202..277
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
primer_bind complement(459..476)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(459..475)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 516..1103
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 1004..1023
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
terminator 1115..1142
/label=T7Te terminator
/note="phage T7 early transcription terminator"
CDS complement(1166..1981)
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCLKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
primer_bind 1889..1908
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
promoter complement(1982..2084)
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
terminator 2085..2116
/label=tonB terminator
/note="bidirectional E. coli tonB-P14 transcription
terminator"
ORIGIN
1 aatggtcagt attgagcctc aggaaacagc tatgacatca agctgactag ataatctagc
61 tgatcgtgga ccgatcatac gtataatgcc gtaagatcac gggtcgcagc acagctcgcg
121 gtccagtagt gatcgacact gctcgatccg ctcgcaccgc tagctaatac gactcactat
181 agtcgccctc gaacttcacc tgttttagag ctagaaatag caagttaaaa taaggctagt
241 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt taaaaagctt ggatcgacga
301 gagcagcgcg actggatctg tcgcccgtct caaacgcaac cctccggcgg tcgcatatca
361 ttcaggacga gcctcagact ccagcgtaac tggactgcaa tcaactcact ggctcacctt
421 ccggtccacg atcagctaga atcaagctga ctagataaac tggccgtcgt tttacacggg
481 tgggcctttc ttcggtagaa aatcaaagga tcttcttgag atcctttttt tctgcgcgta
541 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
601 gagctaccaa ctctttttcc gaggtaactg gcttcagcag agcgcagata ccaaatactg
661 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
721 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
781 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
841 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
901 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
961 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
1021 tttatagtcc tgtcgggttt cgccacctct gacttgagca tcgatttttg tgatgctcgt
1081 caggggggcg gagcctatgg aaaaacgcca gcaacgcaga aaggcccacc cgaaggtgag
1141 ccaggtgatt acatttaggt cctcattaga aaaactcatc gagcatcaag tgaaactgca
1201 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
1261 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
1321 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
1381 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaagagt ttatgcattt
1441 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcaccaa
1501 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgccgttaa
1561 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
1621 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttccctggga
1681 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
1741 gaggcataaa ttccgtcagc cagtttagcc tgaccatctc atctgtaaca tcattggcaa
1801 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
1861 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
1921 catccatgtt ggaatttaat cgcggcttca agcaagacgt ttcccgttga atatggctca
1981 ttttagcttc cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc
2041 ttatttcatt atggtgaaag ttggaacctc ttacgtgccg atcaagtcaa aagcctccgg
2101 tcggaggctt ttgactttct gctatggagg tcaggtatga ttta
//
