| 质粒名称: | pET28a-At-TRL |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 32242 |
| 存储实验室: | Stan Fields, Jay Hesselberth |
|
载体骨架基本信息 |
|
| 载体名称: | pET28a |
| 载体出品公司: | Novagen |
| 空载体大小: | 5255 bp |
| 完整质粒大小: | 8638bp |
| 载体类型: | 哺乳细胞表达 |
| 载体抗性: | Kanamycin |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 低拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | A. thaliana tRNA ligase |
| 物种来源: | A. thaliana (mustard weed) |
| 插入大小: | 3300 bp |
| 启动子: | T7 RNA Polmyerase |
| 标签/融合蛋白: | 6xHistidine (N terminal on backbone) |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | NheI (not destroyed) |
| 3’克隆位点: | SalI (not destroyed) |
| 5’测序引物 | T7 Promoter |
| 3’测序引物 | T7 Terminator |
|
文献引用方法 |
|
| 材料和方法部分: | pET28a-At-TRL was a gift from Stan Fields & Jay Hesselberth (Addgene plasmid # 32242 ; http://n2t.net/addgene:32242 ; RRID:Addgene_32242) |
| 参考文献部分: | Capture and sequence analysis of RNAs with terminal 2',3'-cyclic phosphates. Schutz K, Hesselberth JR, Fields S. RNA. 2010 Mar;16(3):621-31. Epub 2010 Jan 14. 10.1261/rna.1934910 PubMed 20075163 |
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 pET28a-At-TRL 8638 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pET28a-At-TRL
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8638)
AUTHORS Schutz K, Hesselberth JR, Fields S
TITLE Capture and sequence analysis of RNAs with terminal 2',3'-cyclic
phosphates.
JOURNAL RNA. 2010 Mar;16(3):621-31. Epub 2010 Jan 14.
PUBMED 20075163
REFERENCE 2 (bases 1 to 8638)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..8638
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 12..467
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(99..118)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 309..330
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(560..1375)
/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 1283..1302
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 1367..1386
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 1497..2085
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 1986..2005
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 2239..2256
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 2271..2413
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(2339..2358)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 2499..2521
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(2515..2706)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(3515..4597)
/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"
primer_bind 4559..4578
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(4598..4675)
/gene="lacI"
/label=lacI promoter
primer_bind 4881..4900
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 4984..5003
/label=T7
/note="T7 promoter, forward primer"
promoter 4984..5002
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 5003..5027
/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 5042..5064
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 5083..5100
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 5110..5127
/codon_start=1
/product="thrombin recognition and cleavage site"
/label=thrombin site
/translation="LVPRGS"
CDS 8482..8499
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(8552..8570)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 8566..8613
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
ORIGIN
1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt
481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
541 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
601 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
661 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
721 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
781 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
841 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
901 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
961 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
1021 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
1081 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
1141 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
1201 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
1261 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
1321 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
1381 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
1441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
1501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
1561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
1621 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
1681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
1741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
1801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
1861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
1921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
1981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
2041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
2101 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
2161 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
2221 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
2281 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta
2341 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
2401 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
2461 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
2521 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
2581 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
2641 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
2701 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
2761 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
2821 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
2881 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
2941 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
3001 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
3061 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
3121 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
3181 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
3241 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
3301 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
3361 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
3421 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
3481 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
3541 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
3601 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
3661 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
3721 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
3781 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
3841 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
3901 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
3961 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
4021 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
4081 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
4141 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
4201 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
4261 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
4321 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
4381 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
4441 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
4501 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
4561 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
4621 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
4681 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
4741 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
4801 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
4861 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
4921 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
4981 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
5041 ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac
5101 agcagcggcc tggtgccgcg cggcagccat atggctagca tggatgctcc atttgaatcc
5161 ggtgatagtt cggcaacagt tgttgcagaa gcagtaaaca accaatttgg cgggttaagt
5221 cttaaggaaa gcaacactaa tgcgccagtc ttaccgagtc aaactacaag taaccaccgt
5281 gttcaaaatc tagtatggaa acctaagtca tatggaacag ttagtggatc atcttcggca
5341 acagaagttg gtaaaacatc ggctgtctca cagataggtt cttctggaga tacaaaagtt
5401 ggtctcaatc tgagcaaaat tttcggaggt aatttgttgg aaaaattttc ggtggacaaa
5461 tcgacttatt gtcatgctca gatcagagcc actttctatc caaaatttga gaacgaaaag
5521 actgaccaag agataagaac aagaatgatt gagatggtat ctaaaggatt ggcaacactg
5581 gaggtatctc ttaagcattc aggctctctc tttatgtacg ccggtcacaa ggggggagca
5641 tatgcaaaga acagttttgg taatatttat actgcggttg gtgtttttgt tctttcacgg
5701 atgttcaggg aggcttgggg aactaaggct ccaaagaaag aagcagagtt caatgatttt
5761 cttgagaaaa atcgcatgtg catatctatg gaactggtaa ccgctattct tggagatcat
5821 ggtcaacgcc cactggatga ttatgtggta gtgactgctg ttaccgagtt aggtaatggg
5881 aagccccagt tctattcaac ttccgaaata atttcatttt gccggaaatg gcgtctaccc
5941 acaaatcatg tttggctgtt ttccacaagg aaatcagtga cctctttttt cgcggcgttt
6001 gatgcactgt gtgaagaagg gatagcaacc tcggtctgta gagctcttga tgaagtagct
6061 gatatatcag tcccagcctc aaaggaccat gttaaggtgc agggtgagat attagagggt
6121 cttgttgctc gtatcgtgag cagtcagagc tcaagagata tggaaaatgt cttgagagat
6181 catcctccac caccctgtga tggagctaat cttgatctgg gactcagtct aagagagata
6241 tgtgctgccc atagatctaa cgagaaacag caaatgagag cacttttaag gagtgttggg
6301 ccaagctttt gccccagtga cgtggaatgg ttcggagatg aatctcatcc gaaaagtgct
6361 gacaaatctg ttataacaaa attcctgcaa tctcagcctg cagattattc aactagtaaa
6421 ttacaggaaa tggtacgctt gatgaaggaa aaacgtcttc cagccgcatt caagtgttac
6481 cataattttc atagagctga ggacatatca cctgataacc ttttttataa attggttgtc
6541 catgtgcaca gcgattcagg atttaggcgt taccacaaag agatgaggca catgcctagt
6601 ttgtggcctt tatatcgagg tttctttgtt gatattaatt tgttcaagtc aaacaaaggg
6661 agggatctga tggctctgaa aagcattgat aatgccagtg aaaatgatgg tcgaggagaa
6721 aaggatggtt tggctgatga tgatgctaac ttaatgatta aaatgaagtt tctcacatac
6781 aagttgagaa cctttttgat ccgtaatggc ctatcaattc tatttaaaga cggggcagca
6841 gcttacaaaa cttactacct taggcaaatg aaaatctggg gtacgtcaga tggaaagcag
6901 aaagaacttt gcaagatgct tgatgaatgg gctgcttaca taagaagaaa gtgtgggaat
6961 gatcagctat cttcatcaac atacttaagt gaagctgagc cattcctgga gcagtacgcg
7021 aaacggagcc caaagaacca tattttgata gggtctgctg ggaacctagt tagaactgag
7081 gacttcttgg ccattgttga cggtgatcta gatgaagaag gcgatcttgt gaagaaacaa
7141 ggagtgacac cagctactcc tgagccagct gtgaaggaag ctgtccaaaa ggatgagggg
7201 ttaattgtat tctttccagg aattcctgga tctgctaaat ctgcactttg caaggagtta
7261 ttgaacgccc caggaggctt tggagatgat aggccagtgc atactctgat gggtgatctt
7321 gtcaaaggaa aatattggcc aaaggttgct gatgaacgtc gtaaaaaacc acaatcgatt
7381 atgttggctg acaaaaatgc cccaaatgaa gatgtctgga gacagattga agatatgtgt
7441 cggagaacta gggcttctgc agttccaatc gttgctgatt ctgaaggaac cgatacaaat
7501 ccatattcac ttgatgcctt ggctgtattc atgttccgtg tacttcaacg agttaaccat
7561 ccgggaaagc tcgacaagga atcttcaaat gcaggttatg tgctattgat gttttatcac
7621 ctttatgagg gaaagaaccg taatgaattt gagagtgaat tgattgagcg ctttggctcc
7681 ctcatcaaga tgccactatt gaaatcagac aggactcctt tacctgatcc tgttaaatca
7741 gttcttgagg agggcattga tttgttcaat ctccatagta gaagacatgg gagactcgag
7801 tcaacaaaag gaacatatgc agcggaatgg accaaatggg agaaacaact acgagatact
7861 ctagttgcaa attctgagta tctcagttct attcaggttc catttgagtc tatggttcat
7921 caagtgcgag aagagctaaa aacaatagcg aagggtgatt acaagccacc aagttcggag
7981 aaaagaaaac acgggtctat tgttttcgct gccatcaact tgcctgctac tcaagttcac
8041 agtcttcttg aaaagttggc tgcagcaaac ccaacaatga gatcttttct agagggaaag
8101 aaaaagagca tacaggaaaa acttgaacgg tctcacgtga cgctcgccca caagagaagc
8161 catggcgtag caactgtagc cagctatagt cagcacttga acagagaggt acccgtagag
8221 ctcaccgagc tcatctacaa cgacaagatg gctgctctaa cagcccatgt tggatctgtg
8281 gacggagaga ccgtagtctc caagaacgaa tggccacatg ttacattgtg gacagcggaa
8341 ggcgttactg cgaaagaggc caacacgtta cctcagcttt acttagaagg aaaggcgagc
8401 cgcttggtga tagatcctcc ggtgtcaatc tcaggtcctc tggagttttt ctaggtcgac
8461 aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
8521 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
8581 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggat
//
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