| 质粒名称: | pET_His6-SUMO-TEV-TtCsm6 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 172486 |
| 存储实验室: | Jennifer Doudna |
|
载体骨架基本信息 |
|
| 载体名称: | pET |
| 完整质粒大小: | 7060 bp |
| 载体类型: | 细菌表达 |
| 载体抗性: | 卡那霉素50 μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
|
插入基因信息 |
|
| 插入基因名称: | T. thermophilus Csm6 (codon optimized),TTHB152 |
| 物种来源: | Synthetic |
| 启动子: | T7 promoter and lac operator |
| 标签/融合蛋白: | His-SUMO-TEV（主干线上的N终端） |
| 克隆方法: | Ligation Independent克隆 |
| 5’测序引物 | SUMO forward (5'-ttattgaggctcacagagaac-3') |
| 3’测序引物 | T7 reverse (5'-GCTAGTTATTGCTCAGCGG-3') |
|
文献引用方法 |
|
| 材料和方法部分: | pET_His6-SUMO-TEV-TtCsm6 was a gift from Jennifer Doudna (Addgene plasmid # 172486 ; http://n2t.net/addgene:172486 ; RRID:Addgene_172486) |
| 参考文献部分: | Accelerated RNA detection using tandem CRISPR nucleases. Liu TY, Knott GJ, Smock DCJ, Desmarais JJ, Son S, Bhuiya A, Jakhanwal S, Prywes N, Agrawal S, Diaz de Leon Derby M, Switz NA, Armstrong M, Harris AR, Charles EJ, Thornton BW, Fozouni P, Shu J, Stephens SI, Kumar GR, Zhao C, Mok A, Iavarone AT, Escajeda AM, McIntosh R, Kim S, Dugan EJ, Pollard KS, Tan MX, Ott M, Fletcher DA, Lareau LF, Hsu PD, Savage DF, Doudna JA. Nat Chem Biol. 2021 Sep;17(9):982-988. doi: 10.1038/s41589-021-00842-2. Epub 2021 Aug 5. 10.1038/s41589-021-00842-2 PubMed 34354262 |
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
载体简介
载体序列
LOCUS pET_His6-SUMO-TEV-TtCsm6 7057 bp ds-DNA circular SYN 17-AUG-2021
DEFINITION Bacterial expression of codon-optimized His-SUMO-TtCsm6 with a TEV
protease cleavage site..
ACCESSION .
VERSION .
KEYWORDS pET_His6-SUMO-TEV-TtCsm6
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7057)
AUTHORS Liu TY, Knott GJ, Smock DCJ, Desmarais JJ, Son S, Bhuiya A,
Jakhanwal S, Prywes N, Agrawal S, Diaz de Leon Derby M, Switz NA,
Armstrong M, Harris AR, Charles EJ, Thornton BW, Fozouni P, Shu J,
Stephens SI, Kumar GR, Zhao C, Mok A, Iavarone AT, Escajeda AM,
McIntosh R, Kim S, Dugan EJ, Hamilton JR, Lin-Shiao E, Stahl EC,
Tsuchida CA, Moehle EA, Giannikopoulos P, McElroy M, McDevitt S, Zur
A, Sylvain I, Ciling A, Zhu M, Williams C, Baldwin A, Pollard KS,
Tan MX, Ott M, Fletcher DA, Lareau LF, Hsu PD, Savage DF, Doudna JA
TITLE Accelerated RNA detection using tandem CRISPR nucleases.
JOURNAL Nat Chem Biol
REFERENCE 2 (bases 1 to 7057)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 17, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7057
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 973..992
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(1012..1089)
/gene="lacI"
/label=lacI promoter
primer_bind 1295..1314
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 1398..1417
/label=T7
/note="T7 promoter, forward primer"
promoter 1398..1416
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 1417..1441
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
RBS 1456..1478
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 1497..1514
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 1533..1823
/codon_start=1
/gene="S. cerevisiae SMT3 (truncated)"
/product="cleavable ubiquitin-like protein tag"
/label=SUMO
/translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG"
CDS 1833..1853
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQS"
CDS 3318..3335
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(3388..3406)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 3402..3449
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 3486..3941
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3573..3592)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 3783..3804
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(4034..4849)
/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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
primer_bind 4757..4776
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 4841..4860
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 4971..5559
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5460..5479
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 5713..5730
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 5745..5884
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(5810..5829)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5970..5992
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(5986..6177)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(6986..1011)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/label=lacI
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
ORIGIN
1 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
61 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
121 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
181 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
241 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
301 gataccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
361 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
421 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
481 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
541 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
601 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
661 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
721 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
781 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
841 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
901 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
961 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
1021 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
1081 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
1141 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
1201 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
1261 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
1321 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
1381 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
1441 ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggt tcttctcacc
1501 atcaccatca ccatggttct tctatggcta gcatgtcgga ctcagaagtc aatcaagaag
1561 ctaagccaga ggtcaagcca gaagtcaagc ctgagactca catcaattta aaggtgtccg
1621 atggatcttc agagatcttc ttcaagatca aaaagaccac tcctttaaga aggctgatgg
1681 aagcgttcgc taaaagacag ggtaaggaaa tggactcctt aagattcttg tacgacggta
1741 ttagaattca agctgatcag acccctgaag atttggacat ggaggataac gatattattg
1801 aggctcacag agaacagatt ggtgggatcg aggaaaacct gtacttccaa tccaatgcag
1861 aggacctgga tgcgctgtgg gaacgctatc gtgaggcggt tcgtgcgggt ggtaatccgc
1921 aggcgctgta tcaagagatg gtgtggccgg cgctgctggc gctgtggcgt gaaaagccgc
1981 gtgtttaccc gtttccgcag gcgttcgctg tgagcgttca caccctgggt accagcccgg
2041 aggcgaccgc gctggcgatc ctgggtgcgg gcgcggagcg tgtgtatgtt ctgcacaccc
2101 cggaaagcgc gcgttttctg ccgcgtctgc gtcaagacac cggcaaggat ctgtacccgg
2161 tggaaatcgg taaaagcgac gttgaggcga tttatcgtga agtgaagcgt ctgctggaga
2221 aacacccgga agtgccggtt gcgctggatc tgaccagcgg taccaaagcg atgagcgcgg
2281 gtctggcggc ggcgggtttc tttttccagc gtttttaccc gaaagtgcgt gtggtttacg
2341 ttgacaacga ggactatgat ccggaactgc gtcgtccgcg tgcgggtacc gagaaactgc
2401 gtattctgcc gaacccgcac gaggcgctgg cggaagttga tgcgctgttc gcgaaggagc
2461 tgtacggtaa aggcgaattt ggtcaggcgg cggcgtattt ccgtggtatg gtgggccgta
2521 ccggtaacca agcgtacgcg ctgtatgcgc tgctggcgga gatgtaccgt gcgtggcgtg
2581 cgctggactt cggtgaagcg ctgaaggcgg gccgtaaact gctgggtcag ctgagccaaa
2641 acgtttggct gaaccacccg ctgaacgcgc gtcgtgaggc gctggaagcg caagtggcgc
2701 tgctggaggc ggttgaccgt tttctgaagg cgcgtgattt cgcgctgaaa gaaggcgttt
2761 atggtctggc gcgtaccctg ctgcacctgg cgcaggaagc gaaggaagag gcggcggtgc
2821 tggcggcgct gtatgcgtat cgtgcgctgg agctgctgct gcaagaacgt ctggcgctgc
2881 tgggtcgtcg tgcggaagcg ccgggtctga gcccggagga agcggaggcg ctgcgtaaag
2941 cgctggcgga actgctgggc gtgctgccgg aggaagttcg tctgccggcg aagctgggtc
3001 tgctggacct gctggcgttt ctgcgtctga aaggcgatga ggcgctgggt cgtctgagcc
3061 tggcggaact gcgtggcctg gcgggtgcgc tgaaaggtcg taacagcgcg ctgctggtgc
3121 acggtttcga tgttccgagc ccgaaagcgg tggagggtat tgcgcgtctg gcgcagggtc
3181 tgctgcaaga tctggaggcg cgtaccgcgc tgggtccgct gagcccggag ccggtgccgc
3241 tgggttttta ataacattgg aagtggataa cggatccgaa ttcgagcgcc gtcgacaagc
3301 ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
3361 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
3421 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
3481 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
3541 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
3601 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
3661 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
3721 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
3781 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
3841 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
3901 atttaacgcg aattttaaca aactagtaac gtttacaatt tcaggtggca cttttcgggg
3961 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
4021 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
4081 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
4141 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
4201 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
4261 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt
4321 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
4381 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
4441 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atgttttcac
4501 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
4561 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
4621 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
4681 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
4741 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
4801 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg
4861 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag
4921 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
4981 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
5041 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
5101 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
5161 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
5221 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
5281 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
5341 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
5401 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
5461 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
5521 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
5581 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
5641 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
5701 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
5761 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
5821 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
5881 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
5941 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
6001 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
6061 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
6121 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
6181 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
6241 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
6301 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
6361 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
6421 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
6481 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
6541 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
6601 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
6661 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
6721 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
6781 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
6841 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
6901 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
6961 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
7021 ccagctgcat taatgaatcg gccaacgcgc ggggaga
//
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