| 质粒名称: | pETconNK |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 81169 |
| 存储实验室: | Timothy Whitehead |
|
载体骨架基本信息 |
|
| 载体名称: | pETconNK |
| 载体抗性: | 卡那霉素 |
| 空载体大小: | 6137 bp |
| 载体修饰: | 从pETcon开始，将氨苄青霉素耐药基因与kanmycin进行交换，并将KanR基因的BbvCI切痕位点3′。缺口序列CCTCAGCAA位于编码链上。 |
| 载体类型: | 细菌表达，酵母表达 |
| 筛选标记: | TRP1 |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 启动子 | GAL1 |
| 标签/融合蛋白: | c-myc（主干上的c终端） |
| 克隆方法: | 限制性内切酶 |
| 5' 测序引物 | GGACAATAGCTCGACGATTGAAGGTAGATACCCATA |
| 3' 测序引物 | CAAGTCCTCTTCAGAAATAAGCTTTTGTTC |
|
文献引用方法 |
|
| 材料和方法部分: | pETconNK was a gift from Timothy Whitehead (Addgene plasmid # 81169 ; http://n2t.net/addgene:81169 ; RRID:Addgene_81169) |
| 参考文献部分: | Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning. Klesmith JR, Bacik JP, Wrenbeck EE, Michalczyk R, Whitehead TA. Proc Natl Acad Sci U S A. 2017 Feb 14. pii: 201614437. doi: 10.1073/pnas.1614437114. 10.1073/pnas.1614437114 PubMed 28196882 |
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 pETconNK 6137 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION pETconNK Empty Backbone.
ACCESSION .
VERSION .
KEYWORDS pETconNK
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6137)
AUTHORS Klesmith JR, Bacik JP, Wrenbeck EE, Michalczyk R, Whitehead TA
TITLE Trade-offs between enzyme fitness and solubility illuminated by deep
mutational scanning.
JOURNAL Proc Natl Acad Sci U S A. 2017 Feb 14. pii: 201614437. doi:
10.1073/pnas.1614437114.
PUBMED 28196882
REFERENCE 2 (bases 1 to 6137)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6137
/organism="synthetic DNA construct"
/mol_type="other DNA"
protein_bind 2..18
/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)."
primer_bind 7..29
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 26..42
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 26..42
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
promoter 63..81
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 315..756
/gene="S. cerevisiae GAL1"
/label=GAL1 promoter
/note="inducible promoter, regulated by Gal4"
protein_bind 315..432
/label=UAS
/bound_moiety="Gal4"
/note="upstream activating sequence mediating
Gal4-dependent induction"
primer_bind 727..750
/label=GAL1
/note="S. cerevisiae GAL1 promoter, forward primer"
CDS 1082..1093
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
CDS 1094..1120
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 1236..1265
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
primer_bind complement(1594..1613)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(1595..1613)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(1620..1637)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(1620..1636)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(1629..1651)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 1777..2232
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(1864..1883)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2074..2095
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(2335..3009)
/codon_start=1
/gene="S. cerevisiae TRP1"
/product="phosphoribosylanthranilate isomerase, required
for tryptophan biosynthesis"
/label=TRP1
/note="yeast auxotrophic marker"
/translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
KK"
promoter complement(3010..3290)
/gene="S. cerevisiae TRP1"
/label=TRP1 promoter
primer_bind complement(3311..3330)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 3430..3452
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(3490..3508)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
misc_feature 3549..4052
/label=CEN/ARS
/note="S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence"
promoter 4089..4193
/gene="bla"
/label=AmpR promoter
CDS 4194..5009
/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 complement(4267..4286)
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
rep_origin 5189..5777
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5678..5697
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 5931..5948
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 6065..6086
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 6101..6131
/label=lac promoter
/note="promoter for the E. coli lac operon"
ORIGIN
1 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc
61 gaaattaacc ctcactaaag ggaacaaaag ctggtaccaa ttccttgaat tttcaaaaat
121 tcttactttt tttttggatg gacgcaaaga agtttaataa tcatattaca tggcattacc
181 accatataca tatccatatc taatcttact tatatgttgt ggaaatgtaa agagccccat
241 tatcttagcc taaaaaaacc ttctctttgg aactttcagt aatacgctta actgctcatt
301 gctatattga agtacggatt agaagccgcc gagcgggtga cagccctccg aaggaagact
361 ctcctccgtg cgtcctcgtc ttcaccggtc gcgttcctga aacgcagatg tgcctcgcgc
421 cgcactgctc cgaacaataa agattctaca atactagctt ttatggttat gaagaggaaa
481 aattggcagt aacctggccc cacaaacctt caaatgaacg aatcaaatta acaaccatag
541 gatgataatg cgattagttt tttagcctta tttctggggt aattaatcag cgaagcgatg
601 atttttgatc tattaacaga tatataaatg caaaaactgc ataaccactt taactaatac
661 tttcaacatt ttcggtttgt attacttctt attcaaatgt aataaaagta tcaacaaaaa
721 attgttaata tacctctata ctttaacgtc aaggagaaaa aaccccggat cgaattccct
781 acttcataca ttttcaatta agatgcagtt acttcgctgt ttttcaatat tttctgttat
841 tgcttcagtt ttagcacagg aactgacaac tatatgcgag caaatcccct caccaacttt
901 agaatcgacg ccgtactctt tgtcaacgac tactattttg gccaacggga aggcaatgca
961 aggagttttt gaatattaca aatcagtaac gtttgtcagt aattgcggtt ctcacccctc
1021 aacaactagc aaaggcagcc ccataaacac acagtatgtt tttaaggaca atagctcgac
1081 gattgaaggt agatacccat acgacgttcc agactacgct ctgcaggcta gtggtggagg
1141 aggctctggt ggaggcggta gcggaggcgg agggtcggct agccatatgt gaagcatgct
1201 taattaaggc gccttatctc gaggggggcg gatccgaaca aaagcttatt tctgaagagg
1261 acttgtaata gagatctgat aacaacagtg tagatgtaac aaaatcgact ttgttcccac
1321 tgtactttta gctcgtacaa aatacaatat acttttcatt tctccgtaaa caacatgttt
1381 tcccatgtaa tatccttttc tatttttcgt tccgttacca actttacaca tactttatat
1441 agctattcac ttctatacac taaaaaacta agacaatttt aattttgctg cctgccatat
1501 ttcaatttgt tataaattcc tataatttat cctattagta gctaaaaaaa gatgaatgtg
1561 aatcgaatcc taagagaatt gagctccaat tcgccctata gtgagtcgta ttacaattca
1621 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
1681 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
1741 ctttcccaac agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa
1801 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
1861 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
1921 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
1981 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
2041 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
2101 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
2161 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
2221 cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
2281 catagatcgg caagtgcaca aacaatactt aaataaatac tactcagtaa taacctattt
2341 cttagcattt ttgacgaaat ttgctatttt gttagagtct tttacaccat ttgtctccac
2401 acctccgctt acatcaacac caataacgcc atttaatcta agcgcatcac caacattttc
2461 tggcgtcagt ccaccagcta acataaaatg taagctttcg gggctctctt gccttccaac
2521 ccagtcagaa atcgagttcc aatccaaaag ttcacctgtc ccacctgctt ctgaatcaaa
2581 caagggaata aacgaatgag gtttctgtga agctgcactg agtagtatgt tgcagtcttt
2641 tggaaatacg agtcttttaa taactggcaa accgaggaac tcttggtatt cttgccacga
2701 ctcatctcca tgcagttgga cgatatcaat gccgtaatca ttgaccagag ccaaaacatc
2761 ctccttaggt tgattacgaa acacgccaac caagtatttc ggagtgcctg aactattttt
2821 atatgctttt acaagacttg aaattttcct tgcaataacc gggtcaattg ttctctttct
2881 attgggcaca catataatac ccagcaagtc agcatcggaa tctagagcac attctgcggc
2941 ctctgtgctc tgcaagccgc aaactttcac caatggacca gaactacctg tgaaattaat
3001 aacagacata ctccaagctg cctttgtgtg cttaatcacg tatactcacg tgctcaatag
3061 tcaccaatgc cctccctctt ggccctctcc ttttcttttt tcgaccgaat taattcttaa
3121 tcggcaaaaa aagaaaagct ccggatcaag attgtacgta aggtgacaag ctatttttca
3181 ataaagaata tcttccacta ctgccatctg gcgtcataac tgcaaagtac acatatatta
3241 cgatgctgtt ctattaaatg cttcctatat tatatatata gtaatgtcgt gatctatggt
3301 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
3361 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
3421 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
3481 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
3541 cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa
3601 tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta
3661 aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa
3721 tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag
3781 atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt
3841 ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa
3901 aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta
3961 ttttttcttt aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa
4021 tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg
4081 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
4141 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagcc
4201 atattcaacg ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg gatgctgatt
4261 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat
4321 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
4381 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
4441 ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg
4501 ggaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
4561 cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
4621 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
4681 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
4741 ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
4801 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
4861 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
4921 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
4981 ttcatttgat gctcgatgag tttttctaac ctcagcaact gtcagaccaa gtttactcat
5041 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
5101 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
5161 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
5221 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
5281 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
5341 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
5401 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
5461 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
5521 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
5581 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
5641 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
5701 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
5761 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
5821 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
5881 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
5941 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
6001 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
6061 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
6121 ctcgtatgtt gtgtgga
//
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