| 质粒名称: | pKAR2-Br512_Mut235 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 179278 |
| 存储实验室: | Andrew Ellington |
|
载体骨架基本信息 |
|
| 载体名称: | pKAR2 |
| 载体出品公司: | In-house made |
| 空载体大小: | 5187 bp |
| 完整质粒大小: | 6218 bp |
| 载体类型: | 细菌表达 |
| 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: | Engineered Bst DNA Polymerase I Large Fragment,Br512g2.2, Br512_Mut235, Mut235 |
| 物种来源: | Geobacillus stearothermophilus |
| 插入大小: | 1932 bp |
| GenBank编号 | 4O0I_A |
| 启动子: | T7 |
| 标签/融合蛋白: | 8x His tag; Villin Headpiece 47 amino acid (HP47) ; Linker（插接件上的N端子） |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | BsaI（如果已销毁，则未知） |
| 3’克隆位点: | BsaI（如果已销毁，则未知） |
| 5’测序引物 | CCTATAAAAATAGGCGTATCACGAGGC |
| 3’测序引物 | ACCCGTTTAGAGGCCCCAAGG |
|
文献引用方法 |
|
| 材料和方法部分: | pKAR2-Br512_Mut235 was a gift from Andrew Ellington (Addgene plasmid # 179278 ; http://n2t.net/addgene:179278 ; RRID:Addgene_179278) |
| 参考文献部分: | Improved Bst DNA Polymerase Variants Derived via a Machine Learning Approach. Paik I, Ngo PHT, Shroff R, Diaz DJ, Maranhao AC, Walker DJF, Bhadra S, Ellington AD. Biochemistry. 2021 Nov 11. doi: 10.1021/acs.biochem.1c00451. 10.1021/acs.biochem.1c00451 PubMed 34762799 |
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 pKAR2-Br512_Mut235 6218 bp ds-DNA circular SYN 16-MAY-2022
DEFINITION Expresses Br512_Mut235 polymerase in E.coli.
ACCESSION .
VERSION .
KEYWORDS pKAR2-Br512_Mut235
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6218)
AUTHORS Paik I, Ngo PHT, Shroff R, Diaz DJ, Maranhao AC, Walker DJF, Bhadra
S, Ellington AD
TITLE Improved Bst DNA Polymerase Variants Derived via a Machine Learning
Approach.
JOURNAL Biochemistry. 2021 Nov 11. doi: 10.1021/acs.biochem.1c00451.
PUBMED 34762799
REFERENCE 2 (bases 1 to 6218)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 16, 2022 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6218
/organism="synthetic DNA construct"
/mol_type="other DNA"
terminator 16..62
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(359..985)
/codon_start=1
/gene="tetR"
/product="tetracycline repressor TetR"
/label=TetR
/note="TetR binds to the tetracycline operator tetO to
inhibit transcription. This inhibition can be relieved by
adding tetracycline or doxycycline."
/translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
primer_bind 890..909
/label=Tet-R
/note="Tetracycline resistance gene, reverse primer"
CDS complement(1014..1874)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind 1637..1656
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(1875..1946)
/gene="bla"
/label=AmpR promoter
terminator 2023..2109
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
primer_bind complement(2253..2270)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(2424..3012)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(2504..2523)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 3822..3841
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 3906..3925
/label=pENTR-R
/note="pENTR vectors, reverse primer"
primer_bind 4069..4087
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
protein_bind 4108..4126
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O1 for the tetR and tetA genes"
primer_bind 4127..4146
/label=T7
/note="T7 promoter, forward primer"
promoter 4127..4145
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 4147..4165
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
misc_RNA 4185..4234
/label=sTRSV HHRz
/note="hammerhead ribozyme from the tobacco ringspot virus
satellite RNA (Khvorova et al., 2003)"
RBS 4268..4276
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
ORIGIN
1 atcctaagct taatttagca taaccccttg gggcctctaa acgggtcttg aggggttttt
61 tgagctgagc ttggactcct gttgatagat ccggccggta atgacctcag aactccatct
121 gcctaatgga gtgattctag ccggtcgtta cacgtggaac gggaactgcc agacatcaaa
181 taaaacaaaa ggctcagtcg gaagactggg ccttttgttt tatctgttgt ttgtcggtga
241 acactctccc ggaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
301 tcacctagat ccttttaaac tagtgaagtt accatcacgg aaaaaggtta tgctgctttt
361 aagacccact ttcacattta agttgttttt ctaatccgca aatgatcaat tcaaggccga
421 ataagaaggc tggctctgca ccttggtgat caaataattc gatagcttgt cgtaataatg
481 gcggcatact atcagtagta ggtgtttccc tttcttcttt agcgacttga tgctcttgat
541 cttccaatac gcaacctaaa gtaaaatgcc ccactgcact gagtgcatat aatgcattct
601 ctagtgaaaa accttgttgg cataaaaagg ctaattgatt ttcgagagtt tcatactgtt
661 tttctgtagg ccgtgtacct aaatgtactt ttgctccatc gcgatgactt agtaaagcac
721 atctaaaact tttagcgtta ttacgtaaaa aatcttgcca gctttcccct tctaaagggc
781 aaaagtgagt atggtgccta tctaacatct caatggctaa ggcgtcgagc aaagcccgct
841 tattttttac atgccaatac aatgtaggct gctctacacc tagcttctgg gcgagtttac
901 gggttgttaa accttcgatt ccgacctcat taagcagctc taatgcgctg ttaatcactt
961 tacttttatc taaacgagac atcattaacc tcctcaagag gatcgaatag ttattaccaa
1021 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
1081 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
1141 gcaatgatac cgcgagagcc acgctcaccg gctccagatt tatcagcaat aaaccagcca
1201 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatc
1261 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
1321 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
1381 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
1441 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
1501 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
1561 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
1621 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
1681 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
1741 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
1801 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
1861 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
1921 ctcatgagcg gatacatatt tgaatgtatt tagaaaaatt ttttaaggca gttattggtg
1981 ccgcttaaac gcctggggta atgactctct agcttgaggc atcaaataaa acgaaaggct
2041 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
2101 aggacaaatc cgccctctag attacgtgca gtcgatgata agctgtcaaa cggaatttcg
2161 ggcagcgttg ggtcctggcc acgggtgcgc cggtgtgaaa taccgcacag atgcgtaagg
2221 agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
2281 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
2341 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
2401 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
2461 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
2521 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
2581 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
2641 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
2701 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
2761 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
2821 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
2881 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
2941 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
3001 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
3061 aactcacgtt aagggatttt ggtcatggaa ttaattctta gaaaaactca tcgagcatca
3121 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
3181 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
3241 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
3301 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
3361 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
3421 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaag acgaaatacg
3481 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
3541 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
3601 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
3661 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
3721 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
3781 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
3841 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
3901 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtg
3961 taatcgttaa tccgcaaata acgtaaaaac ccgcttcggc gggttttttt atggggggag
4021 tttagggaaa gagcatttgt catcatgacc atgacattaa cctataaaaa taggcgtatc
4081 acgaggccct ttccctaggg tcttcacact ctatcattga tagagttaat acgactcact
4141 atagggtccc tatcagtgat agagagaatt cgtactgagc acagctgtca ccggatgtgc
4201 tttccggtct gatgagtccg tgaggacgaa acagcctcta caaataattt tgtttaaact
4261 agttagataa ggaggttaca tatgcaccat catcacggtc atcaccacca tccgcgtggt
4321 gttgacccga gccgtaagga gaaccacctg tctgacgaag acttcaaggc ggtgttcggt
4381 atgacccgtt ctgcgttcgc gaacctgccg ctgtggaaac aacagaacct gaagaaggag
4441 aaaggtctgt tcggttctgg aagcgcagca gcacctaaga tggcattcac attggccgat
4501 cgtgtcaccg aagagatgct ggcagacaag gcagccttgg tcgtggaggt agttgaggag
4561 aactatcacg acgcaccgat tgttggaatc gccgtggtca atgaacatgg tcgcttcttc
4621 ttgcgccctg agactgcgtt ggccgaccca caattcgtgg cctggttagg agatgaaacg
4681 aagaagaagt caatgttcga cagcaaacgc gcagccgtag ctctgaagtg gaaaggaatt
4741 gagctgtgtg gtgtgagttt cgaccttctc ttagcagcgt acttgcttga tcccgctcaa
4801 ggcgtcgacg acgtggcagc cgctgccaag atgaagcaat atgaagcggt gcgtccggat
4861 gaggctgtgt acgggaaggg agctaaacgc gcggtgcctg atgaacccgt gcttgctgag
4921 cacttggtac gcaaggctgc ggctatctgg gagctggagc gtcccttcct ggatgagttg
4981 cgtcgcaacg agcaggaccg cctgcttgta gagttagaac agcctcttag ctctattctt
5041 gccgagatgg agttcgctgg tgtcaaagta gataccaagc gccttgagca aatgggtaag
5101 gagttggctg aacaactggg cacagtggaa cagcgtatct acgaactggc cggtcaggag
5161 ttcaacatca acagccccaa gcagctggga gtgatcctgt tcgagaagtt gcagctgcca
5221 gtattgaaga agactaagac tggctacagt acctcggctg acgtactgga gaagctggct
5281 ccttaccatg agatcgtgga gaacatcttg cactaccgcc agctgggcaa gctgcagtct
5341 acctacattg agggtctgtt aaaggtcgtg cgtccagaca cgaagaaggt gcatacgatc
5401 ttcaatcagg cgctgaccca aactggtcgt ttgtcgtcca cagagcccaa tcttcagaat
5461 atccctattc gtcttgagga aggccgcaag attcgccagg ccttcgttcc ttcggaatcg
5521 gactggctga tcttcgcagc agattacgat cagatcgagc ttcgcgtgtt ggcacatatc
5581 gcggaggatg acaacttaat ggaggcgttc cgccgcgatc tggatatcca tactaagacc
5641 gcgatggata tcttccaagt gtcagaagac gaggtaacac cgaacatgcg acgccaggcg
5701 aaagcggtta acttcggcat cgtctacggc atcagcgact atggcctggc ccagaacttg
5761 aacatcagcc gcaaggaggc agccgagttc atcgagcgct acttcgagag tttcccaggt
5821 gtgaagcgtt atatggagaa tatcgtacaa gaggcgaagc agaaaggcta cgtgaccacg
5881 ctgttacacc gtcgtcgcta ccttcctgat atcaacagcc gtaacttcaa tgtacgttcc
5941 ttcgccgaac gcatggcgat gaataccccc atccaggggt cagctgcaga tatcatcaag
6001 aaagctatga tcgacttaaa cgctcgtctg aaggaagaac gcttacaggc gcacctctta
6061 ctgcaagtcc atgacgaatt gatccttgag ggccccaagg aagagatgga gcgtctttgc
6121 cgtctggtgc cggaagtaat ggaacaggcc gtcacgctgc gcgtacctct gaaagtcgat
6181 taccactacg gctccacctg gtatgacgcc aagtaagg
//
