| 质粒名称: | pLV312.3 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 119944 |
| 存储实验室: | Gerald Schwank |
|
载体骨架基本信息 |
|
| 载体名称: | px601 |
| 载体出品公司: | Feng Zhang lab |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 4316 bp |
| 完整质粒大小: | 6516 bp |
| 载体类型: | 哺乳细胞表达,AAV, CRISPR |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | C-Intein (Npu DnaE) - C-terminal (740)SaKKH-BE3，split BE3 (C-terminus) |
| 物种来源: | R. norvegicus (rat); S. aureus |
| 插入大小: | 3345 bp |
| 启动子 | CMV |
| 克隆方法: | Gibson克隆 |
| 5' 测序引物 | CGCAAATGGGCGGTAGGCGTG (CMV for) |
| 3' 测序引物 | CCTCGACTGTGCCTTCTA (bGH rev) |
|
文献引用方法 |
|
| 材料和方法部分: | pLV312.3 was a gift from Gerald Schwank (Addgene plasmid # 119944 ; http://n2t.net/addgene:119944 ; RRID:Addgene_119944) |
| 参考文献部分: | Treatment of a metabolic liver disease by in vivo genome base editing in adult mice. Villiger L, Grisch-Chan HM, Lindsay H, Ringnalda F, Pogliano CB, Allegri G, Fingerhut R, Haberle J, Matos J, Robinson MD, Thony B, Schwank G. Nat Med. 2018 Oct;24(10):1519-1525. doi: 10.1038/s41591-018-0209-1. Epub 2018 Oct 8. 10.1038/s41591-018-0209-1 [pii] PubMed 30297904 |
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 pLV312.3 6516 bp ds-DNA circular SYN 20-DEC-2018
DEFINITION Expresses C-terminal Npu DnaE Intein-SaKKH-BE3, tagRFP, and sgRNA in
mammalian cells.
ACCESSION .
VERSION .
KEYWORDS pLV312.3
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6516)
AUTHORS Villiger L, Grisch-Chan HM, Lindsay H, Ringnalda F, Pogliano CB,
Allegri G, Fingerhut R, Haberle J, Matos J, Robinson MD, Thony B,
Schwank G
TITLE Treatment of a metabolic liver disease by in vivo genome base
editing in adult mice.
JOURNAL Nat Med. 2018 Oct;24(10):1519-1525. doi: 10.1038/s41591-018-0209-1.
Epub 2018 Oct 8.
PUBMED 30297904
REFERENCE 2 (bases 1 to 6516)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Dec 20, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6516
/organism="synthetic DNA construct"
/mol_type="other DNA"
repeat_region 1..130
/label=AAV2 ITR
enhancer 178..557
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 558..761
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 712..732
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
CDS 1839..1859
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1866..1931
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 1944..2192
/codon_start=1
/product="uracil-DNA glycosylase inhibitor from a Bacillus
subtilis bacteriophage (Mol et al., 1995)"
/label=UGI
/translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
CDS 2205..2225
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 2235..2291
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 2292..3005
/codon_start=1
/product="monomeric derivative of red fluorescent protein
from Entacmaea quadricolor (Merzlyak et al., 2007)"
/label=TagRFP
/note="mammalian codon-optimized"
/translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
ARYCDLPSKLGHKLN"
primer_bind complement(3024..3041)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 3030..3237
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
promoter 3244..3484
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 3244..3264
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 3415..3434
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
misc_RNA 3515..3590
/label=Sa gRNA scaffold
/note="guide RNA scaffold for the Staphylococcus aureus
CRISPR/Cas9 system"
repeat_region 3604..3744
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 3604..3733
/label=AAV2 ITR
rep_origin 3819..4274
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3906..3925)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4116..4137
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(4291..4310)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4410..4432
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(4470..4488)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 4556..4660
/gene="bla"
/label=AmpR promoter
CDS 4661..5521
/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(4879..4898)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 5692..6280
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6181..6200
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 6434..6451
/label=L4440
/note="L4440 vector, forward primer"
ORIGIN
1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctactcg aggcgttgac
181 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
241 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
301 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
361 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
421 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
481 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
541 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
601 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
661 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
721 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta actaccggtg
781 ccaccatgat caaaattgct actaggaagt atttgggaaa gcagaacgtc tatgacatcg
841 gagtagaacg agaccacaac tttgccttga agaacggttt tatcgcaagc tcaatgcctg
901 agatcgagac agagcaggaa tacaaggaaa ttttcatcac ccctcatcag attaaacaca
961 taaaggactt caaagactat aaatactctc atagggtgga caaaaaaccc aatcgcaagc
1021 tcattaatga caccctgtac tcaacacgga aggatgataa aggtaatacc ttgattgtga
1081 ataatcttaa tggattgtat gacaaagata acgacaagct caagaagctg atcaacaagt
1141 ctccagagaa gctccttatg tatcaccacg acccacagac ttatcagaaa ttgaaactga
1201 tcatggagca atacggggat gagaagaacc cactctacaa atattatgag gaaacaggta
1261 attacctgac caagtactcc aagaaggata acggaccagt gatcaaaaag ataaagtact
1321 atggcaacaa acttaatgcg catttggaca taactgacga ttaccccaat tctcgaaaca
1381 aggttgtgaa gctctccctg aagccttata gatttgacgt gtacctggat aatggggttt
1441 ataaattcgt caccgtgaaa aatctggacg tgatcaaaaa ggagaactat tatgaagtaa
1501 actcaaagtg ctatgaggag gcgaagaagc tgaagaagat ctccaatcag gccgagttca
1561 tcgcttcctt ctataagaac gatctcatca agatcaatgg agagctttat cgcgtcattg
1621 gtgtgaacaa tgacttgctg aacaggatcg aagtcaatat gatagacatt acctaccggg
1681 agtatctcga aaacatgaat gataaacggc cgcctcacat catcaagaca atcgcatcta
1741 aaactcagtc aataaaaaag tactctaccg atatcctggg gaatctctat gaagtgaagt
1801 caaagaagca cccacaaatc attaaaaaag gtggatcccc caagaagaag aggaaagtct
1861 cgagcgacta caaagaccat gacggtgatt ataaagatca tgacatcgat tacaaggatg
1921 acgatgacaa gtctggtggt tctactaatc tgtcagatat tattgaaaag gagaccggta
1981 agcaactggt tatccaggaa tccatcctca tgctcccaga ggaggtggaa gaagtcattg
2041 ggaacaagcc ggaaagcgat atactcgtgc acaccgccta cgacgagagc accgacgaga
2101 atgtcatgct tctgactagc gacgcccctg aatacaagcc ttgggctctg gtcatacagg
2161 atagcaacgg tgagaacaag attaagatgc tctctggtgg ttctcccaag aagaagagga
2221 aagtcggaag cggcgcaaca aacttctctc tgctgaaaca agccggagat gtcgaagaga
2281 atcctggacc gatggtgtct aagggcgaag agctgattaa ggagaacatg cacatgaagc
2341 tgtacatgga gggcaccgtg aacaaccacc acttcaagtg cacatccgag ggcgaaggca
2401 agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc cctctcccct
2461 tcgccttcga catcctggct accagcttca tgtacggcag cagaaccttc atcaaccaca
2521 cccagggcat ccccgacttc tttaagcagt ccttccctga gggcttcaca tgggagagag
2581 tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg
2641 gctgcctcat ctacaacgtc aagatcagag gggtgaactt cccatccaac ggccctgtga
2701 tgcagaagaa aacactcggc tgggaggcca acaccgagat gctgtacccc gctgacggcg
2761 gcctggaagg cagaagcgac atggccctga agctcgtggg cgggggccac ctgatctgca
2821 acttcaagac cacatacaga tccaagaaac ccgctaagaa cctcaagatg cccggcgtct
2881 actatgtgga ccacagactg gaaagaatca aggaggccga caaagagacc tacgtcgagc
2941 agcacgaggt ggctgtggcc agatactgcg acctccctag caaactgggg cacaaactta
3001 attaactaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
3061 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
3121 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
3181 ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggaggt
3241 accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
3301 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
3361 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
3421 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
3481 ggacgaaaca ccgccttccg agtctcccac tgcagtttta gtactctgga aacagaatct
3541 actaaaacaa ggcaaaatgc cgtgtttatc tcgtcaactt gttggcgaga tttttgcggc
3601 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
3661 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
3721 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt
3781 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
3841 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc
3901 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
3961 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
4021 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
4081 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
4141 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt
4201 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
4261 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
4321 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
4381 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
4441 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
4501 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
4561 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
4621 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
4681 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
4741 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
4801 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
4861 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
4921 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
4981 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
5041 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
5101 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
5161 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
5221 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
5281 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
5341 gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact
5401 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
5461 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
5521 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
5581 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
5641 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
5701 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
5761 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
5821 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
5881 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
5941 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
6001 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
6061 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
6121 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
6181 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
6241 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
6301 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
6361 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
6421 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
6481 gcctctcccc gcgcgttggc cgattcatta atgcag
//
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