| 质粒名称: | pET28a-gp32-H6 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 163912 |
| 存储实验室: | Paul Freemont |
|
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: | 5369 bp |
| 完整质粒大小: | 6121 bp |
| 载体修饰: | 移除N端子His标签 |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 载体抗性: | 卡那霉素，50μg/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: | gp32 |
| 物种来源: | 肠道细菌噬菌体T4 |
| 插入大小: | 903 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子: | T7 promoter |
| 标签/融合蛋白: | His（插入C端子） |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | BsaI (破坏) |
| 3’克隆位点: | BsaI (破坏) |
| 5’测序引物 | TAATACGACTCACTATAGGG |
| 3’测序引物 | GCTAGTTATTGCTCAGCGG |
|
文献引用方法 |
|
| 材料和方法部分: | pET28a-gp32-H6 was a gift from Paul Freemont (Addgene plasmid # 163912 ; http://n2t.net/addgene:163912 ; RRID:Addgene_163912) |
| 参考文献部分: | A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for Simultaneous COVID-19 Diagnosis and Variant Surveillance. Patchsung M, Homchan A, Aphicho K, Suraritdechachai S, Wanitchanon T, Pattama A, Sappakhaw K, Meesawat P, Wongsatit T, Athipanyasilp A, Jantarug K, Athipanyasilp N, Buahom J, Visanpattanasin S, Niljianskul N, Chaiyen P, Tinikul R, Wichukchinda N, Mahasirimongkol S, Sirijatuphat R, Angkasekwinai N, Crone MA, Freemont PS, Joung J, Ladha A, Abudayyeh O, Gootenberg J, Zhang F, Chewapreecha C, Chanarat S, Horthongkham N, Pakotiprapha D, Uttamapinant C. CRISPR J. 2022 Nov 11. doi: 10.1089/crispr.2022.0048. 10.1089/crispr.2022.0048 PubMed 36367987 |
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-gp32-H6 6121 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Expresses T4 gp32 for bacterial expression and affinity
purification.
ACCESSION .
VERSION .
KEYWORDS pET28a-gp32-H6
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6121)
TITLE One Pot CRISPR RPA
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 6121)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..6121
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(646..1728)
/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 1690..1709
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(1729..1806)
/gene="lacI"
/label=lacI promoter
primer_bind 2012..2031
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 2115..2134
/label=T7
/note="T7 promoter, forward primer"
promoter 2115..2133
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 2134..2158
/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)."
promoter 2167..2196
/label=lac promoter
/note="promoter for the E. coli lac operon"
RBS 2216..2238
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 3149..3166
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(3170..3188)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 3184..3231
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 3268..3723
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3355..3374)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 3565..3586
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(3815..4630)
/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 4538..4557
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 4622..4641
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 4752..5340
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5241..5260
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 5494..5511
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 5526..5665
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(5591..5610)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5751..5773
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(5767..5958)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
ORIGIN
1 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
61 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
121 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
181 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
241 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
301 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
361 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
421 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
481 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
541 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
601 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
661 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
721 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
781 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
841 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
901 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
961 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
1021 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
1081 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
1141 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
1201 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
1261 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
1321 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
1381 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
1441 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
1501 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
1561 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
1621 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
1681 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
1741 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
1801 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
1861 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
1921 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
1981 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
2041 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
2101 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
2161 tctagattta cactttatgc ttccggctcg tatgtttcta gagaattcaa ataattttgt
2221 ttaactttaa gaaggagatt taaatatgtt caaacgtaaa agcaccgcag aactggcagc
2281 acagatggca aaactgaatg gtaataaagg ttttagcagc gaggataaag gtgagtggaa
2341 actgaaactg gataatgccg gtaatggtca ggcagttatt cgttttctgc cgagcaaaaa
2401 tgatgaacag gcaccgtttg caattctggt gaatcatggt ttcaaaaaga acggcaaatg
2461 gtacattgaa acctgtagca gcacccatgg tgattatgat agctgtccgg tttgtcagta
2521 tatcagcaaa aacgatctgt acaacaccga taacaaagaa tacagcctgg ttaaacgcaa
2581 aaccagctat tgggcaaata ttctggttgt taaagatccg gcagcaccgg aaaatgaagg
2641 taaagttttc aaatatcgct ttggcaagaa gatctgggat aaaatcaatg ccatgattgc
2701 cgttgatgtg gaaatgggtg aaacaccggt tgatgttacc tgtccgtggg aaggtgcaaa
2761 ttttgttctg aaagttaaac aggtgagcgg cttcagcaat tatgatgaaa gcaaatttct
2821 gaaccagagc gccattccga atatcgatga tgaatcattt cagaaagaac tgttcgagca
2881 gatggttgat ctgagcgaaa tgaccagcaa agacaaattc aaaagcttcg aagaactgaa
2941 cacgaaattt ggtcaggtta tgggcaccgc agttatgggt ggtgcagcag caaccgcagc
3001 aaaaaaagca gataaagttg cagatgatct ggatgccttt aacgtggatg attttaacac
3061 caaaaccgag gatgatttca tgagcagcag cagcggtagc agcagctcag cagatgatac
3121 cgatctggac gatctgctga atgatctgca ccatcaccat caccactaac cgctgagcaa
3181 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
3241 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
3301 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
3361 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
3421 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
3481 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
3541 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
3601 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
3661 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
3721 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
3781 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
3841 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
3901 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
3961 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
4021 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
4081 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
4141 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
4201 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
4261 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
4321 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
4381 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
4441 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
4501 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
4561 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
4621 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
4681 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
4741 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
4801 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
4861 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
4921 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
4981 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
5041 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
5101 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
5161 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
5221 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
5281 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
5341 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
5401 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
5461 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
5521 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
5581 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
5641 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
5701 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
5761 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
5821 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
5881 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
5941 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
6001 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
6061 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
6121 a
//
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