| 質粒名稱: | pCMV-T7-hAcrVA2-NLS(sv40) (AAS2283) |
|---|---|
| 出品公司: | addgene |
| 目錄編號: | 115138 |
| 存儲實驗室: | Joseph Bondy-Denomy, Keith Joung, Benjamin Kleinstiver |
|
載體骨架基本信息 |
|
| 載體名稱: | JDS246 (Plasmid #43861) |
| 載體出品公司: | Joung lab (Addgene Plasmid #43861) |
| 完整質粒大小: | 4416bp |
| 載體類型: | 哺乳細胞表達 |
| 載體抗性: | 氨芐霉素 |
| 生長溫度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷貝數: | 高拷貝 |
|
插入基因信息 |
|
| 插入基因名稱: | human codon optimized AcrVA2 anti-CRISPR protein / AAS2283 |
| 物種來源: | Synthetic |
| 標簽/融合蛋白: | NLS(SV40) (C terminal on insert) |
| 克隆方法: | Gibson Cloning |
| 5’測序引物 | CMV-F |
| 3’測序引物 | BGH-rev |
|
文獻引用方法 |
|
| 材料和方法部分: | pCMV-T7-hAcrVA2-NLS(sv40) (AAS2283) was a gift from Joseph Bondy-Denomy & Keith Joung & Benjamin Kleinstiver (Addgene plasmid # 115138 ; //n2t.net/addgene:115138 ; RRID:Addgene_115138) |
| 參考文獻部分: | Discovery of widespread Type I and Type V CRISPR-Cas inhibitors. Marino ND, Zhang JY, Borges AL, Sousa AA, Leon LM, Rauch BJ, Walton RT, Berry JD, Joung JK, Kleinstiver BP, Bondy-Denomy J. Science. 2018 Sep 6. pii: science.aau5174. doi: 10.1126/science.aau5174. 10.1126/science.aau5174 PubMed 30190308 |
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pCMV-T7-hAcrVA2-NLS(sv40) (AAS2283)載體質粒基本信息
pCMV-T7-hAcrVA2-NLS(sv40) (AAS2283)質粒圖譜載體圖譜和pCMV-T7-hAcrVA2-NLS(sv40) (AAS2283)載體序列質粒序列多克隆位點信息
 (AAS2283)質粒圖譜.png)
pCMV-T7-hAcrVA2-NLS(sv40) (AAS2283)質粒載體簡介
pCMV-T7-hAcrVA2-NLS(sv40) (AAS2283)質粒序列載體序列
LOCUS pCMV-T7-hAcrVA2-NLS(sv40) (AAS2283) 4416 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION CMV-T7 promoter expression plasmid for human codon optimized AcrVA2
anti-CRISPR protein with C-terminal NLS .
ACCESSION .
VERSION .
KEYWORDS pCMV-T7-hAcrVA2-NLS(sv40) (AAS2283)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4416)
AUTHORS Marino ND, Zhang JY, Borges AL, Sousa AA, Leon LM, Rauch BJ, Walton
RT, Berry JD, Joung JK, Kleinstiver BP, Bondy-Denomy J
TITLE Discovery of widespread Type I and Type V CRISPR-Cas inhibitors.
JOURNAL Science. 2018 Sep 6. pii: science.aau5174. doi:
10.1126/science.aau5174.
PUBMED 30190308
REFERENCE 2 (bases 1 to 4416)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
//gt12365.cn
FEATURES Location/Qualifiers
source 1..4416
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 994..1014
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1023..1040
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(1063..1080)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 1069..1293
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(1364..1380)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(1364..1380)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(1377..1399)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 1388..1404
/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 complement(1412..1442)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 1457..1478
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(1595..1612)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(1766..2354)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(1846..1865)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(2525..3385)
/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 3148..3167
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(3386..3490)
/gene="bla"
/label=AmpR promoter
primer_bind complement(3569..3588)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 3760..4139
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 4140..4343
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 4294..4314
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 4385..4404
/label=T7
/note="T7 promoter, forward primer"
promoter 4385..4403
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
regulatory 4411..4
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
ORIGIN
1 atgggccatc acaccatcgc ccgcatgaat gctttcaaca aggcttttgc aaacgctaaa
61 gactgctaca aaaaaatgca agcctggcac ctgttgaaca aaccgaaaca cgcattcttt
121 cctatgcaga acacgccagc cctggataac ggcctcgctg cgctttatga actccggggg
181 ggaaaggaag atgcacatat tttgtctatt ctcagcagat tgtacttgta cggagcgtgg
241 cggaatacat tgggcatata ccagttggac gaggagatca ttaaggactg caaggagctt
301 cccgacgata caccgacgag catattcctc aacttgcctg actggtgtgt atatgtagat
361 atatccagtg ctcagatcgc cactttcgac gacggcgtag cgaagcacat caaaggtttc
421 tgggccatat acgatattgt tgaaatgaat ggaatcaacc atgacgtatt ggacttcgtt
481 gtagatactg acaccgacga taatgtgtac gttccgcagc ctttcatttt gtcatctggt
541 caatccgtgg cagaagtgct tgactatgga gcaagtttgt tcgacgacga tactagcaat
601 acgcttatta agggcctcct gccgtacttg ctctggcttt gcgtagcaga acccgacatc
661 acttacaaag gtttgccagt ctcaagagag gaacttactc gaccgaaaca ttcaatcaat
721 aaaaaaacgg gcgcgtttgt gacacctagt gagccattca tctaccagat aggagaacgg
781 ctgggaagtg aggttaggag gtatcagagc attatagatg gggagcaaaa gcgcaatcga
841 ccacatacaa agcgccctca tattcgccgc ggtcactggc atggatattg gcaagggact
901 ggacaggcga aggagtttcg agttcgatgg caacccgctg tgttcgtgaa tagcggaaga
961 gtatcatccg gatccggcgg tggaggcagt gggcccaaga agaagaggaa agtctgaccg
1021 gtcatcatca ccatcaccat tgagtttaaa cccgctgatc agcctcgact gtgccttcta
1081 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
1141 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
1201 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
1261 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
1321 gctcgatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
1381 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
1441 aagcctaggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
1501 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
1561 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
1621 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
1681 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
1741 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
1801 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
1861 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
1921 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
1981 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
2041 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
2101 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
2161 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
2221 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
2281 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
2341 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
2401 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
2461 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
2521 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
2581 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
2641 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
2701 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
2761 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
2821 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
2881 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
2941 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
3001 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
3061 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
3121 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
3181 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
3241 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
3301 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
3361 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
3421 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
3481 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatcgat
3541 ctcccgatcc cctagggtcg actctcagta caatctgctc tgatgccgca tagttaagcc
3601 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
3661 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
3721 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt
3781 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
3841 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
3901 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
3961 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
4021 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
4081 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
4141 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
4201 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
4261 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
4321 gggaggtcta tataagcaga gctggtttag tgaaccgtca gatccgctag agatccgcgg
4381 ccgctaatac gactcactat agggagagcc gccacc
//