| 质粒名称: | pKH3 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 12555 |
| 存储实验室: | Ian Macara |
|
载体骨架基本信息 |
|
| 载体名称: | pBR322 |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 4750 bp |
| 载体类型: | 哺乳细胞表达 |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 标签/融合蛋白: | 3xHA（主干网上的N个终端） |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | Xba1, BamH1(未破坏) |
| 3' 克隆位点: | EcoR1(未破坏) |
| 5' 测序引物 | CAGGTCCAACTGCACCTCGGTTC |
|
文献引用方法 |
|
| 材料和方法部分: | pKH3 was a gift from Ian Macara (Addgene plasmid # 12555 ; http://n2t.net/addgene:12555 ; RRID:Addgene_12555) |
| 参考文献部分: | Phosphorylation-dependent activation of the Ras-GRF/CDC25Mm exchange factor by muscarinic receptors and G-protein beta gamma subunits. Mattingly RR, Macara IG. Nature. 1996 Jul 18. 382(6588):268-72. 10.1038/382268a0 PubMed 8717044 |
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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 pKH3 4768 bp ds-DNA circular SYN 22-AUG-2018
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pKH3
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4768)
AUTHORS Mattingly RR, Macara IG
TITLE Phosphorylation-dependent activation of the Ras-GRF/CDC25Mm exchange
factor by muscarinic receptors and G-protein beta gamma subunits.
JOURNAL Nature. 1996 Jul 18. 382(6588):268-72.
PUBMED 8717044
REFERENCE 2 (bases 1 to 4768)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 22, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..4768
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 94..111
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 228..249
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 264..294
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 302..318
/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 307..329
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 326..342
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 326..342
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
enhancer 371..750
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 751..954
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 905..925
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 951..975
/label=LNCX
/note="Human CMV promoter, forward primer"
promoter 1185..1203
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind 1185..1202
/label=SP6
/note="SP6 promoter, forward primer"
primer_bind 1216..1235
/label=pMT2-F
/note="Synthetic intron, forward primer"
CDS 1296..1322
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 1329..1355
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 1362..1388
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
regulatory 1425..1434
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
polyA_signal 1438..1572
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(1475..1494)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 1529..1548
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
promoter 1641..1998
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
primer_bind complement(1664..1684)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 1849..1984
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 1911..1930
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
primer_bind complement(2018..2035)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(2018..2034)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2027..2049)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 2247..2702
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2334..2353)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2544..2565
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(2719..2738)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 2838..2860
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(2898..2916)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 2984..3088
/gene="bla"
/label=AmpR promoter
CDS 3089..3949
/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 complement(3307..3326)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 4120..4708
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4609..4628
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
61 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
121 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
181 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
241 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
301 attgtgagcg gataacaatt tcacacagga aacagctatg acatgattac gaattaattc
361 gagctcgccc gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
421 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
481 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
541 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
601 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
661 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
721 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
781 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
841 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
901 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta
961 gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac
1021 cgggaccgat ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc
1081 aagagtgacg taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa
1141 cgcggctaca attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat
1201 agaataacat ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc
1261 tcggttctaa gcttctgcag gtcgactcta gaatgtaccc atacgatgtt ccagattacg
1321 ctggatctta cccatacgat gttccagatt acgctggatc ttacccatac gatgttccag
1381 attacgctgg atccgaattc aatcgataga tagctgagtc gatggccgcc atggcccaac
1441 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
1501 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
1561 catgtctgga tcgggaatta attcggcgca gcaccatggc ctgaaataac ctctgaaaga
1621 ggaacttggt taggtacctt ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta
1681 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
1741 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
1801 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
1861 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
1921 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
1981 ggcctaggct tttgcaaaaa gctgttaaca gcttggcact ggccgtcgtt ttacaacgtc
2041 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
2101 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
2161 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
2221 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
2281 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
2341 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
2401 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
2461 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
2521 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
2581 tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
2641 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
2701 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
2761 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
2821 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
2881 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
2941 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
3001 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
3061 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
3121 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
3181 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
3241 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
3301 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
3361 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
3421 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
3481 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
3541 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
3601 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
3661 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
3721 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
3781 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
3841 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
3901 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
3961 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
4021 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
4081 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
4141 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
4201 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
4261 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
4321 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
4381 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
4441 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
4501 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
4561 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
4621 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
4681 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
4741 ggccttttgc tggccttttg ctcacatg
//
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