| 质粒名称: | pCAG-Cas12i-HiFi-T2A-EGFP |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 188269 |
| 存储实验室: | Wei Li |
|
载体骨架基本信息 |
|
| 载体名称: | pUC19 |
| 完整质粒大小: | 8473 bp |
| 载体类型: | 哺乳细胞表达、CRISPR |
| 载体抗性: | 氨苄霉素 |
| 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: | Cas12i-HiFi |
| 物种来源: | Synthetic |
| 基因突变: | 无 |
| 启动子: | CAG |
| 克隆方法: | Gibson Cloning |
|
文献引用方法 |
|
| 材料和方法部分: |
pCAG-Cas12i-HiFi-T2A-EGFP was a gift from Wei Li (Addgene plasmid # 188269 ; http://n2t.net/addgene:188269 ; RRID:Addgene_188269)
|
| 参考文献部分: | Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing. Chen Y, Hu Y, Wang X, Luo S, Yang N, Chen Y, Li Z, Zhou Q, Li W. Innovation (Camb). 2022 May 26;3(4):100264. doi: 10.1016/j.xinn.2022.100264. eCollection 2022 Jul 12. 10.1016/j.xinn.2022.100264 PubMed 35693153 |
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 pCAG-Cas12i-HiFi-T2A-EGFP 8473 bp ds-DNA circular SYN 03-OCT-2022
DEFINITION Mammalian expression, Genome editing.
ACCESSION .
VERSION .
KEYWORDS pCAG-Cas12i-HiFi-T2A-EGFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8473)
AUTHORS Chen Y, Hu Y, Wang X, Luo S, Yang N, Chen Y, Li Z, Zhou Q, Li W
TITLE Synergistic engineering of CRISPR-Cas nucleases enables robust
mammalian genome editing.
JOURNAL Innovation (Camb). 2022 May 26;3(4):100264. doi:
10.1016/j.xinn.2022.100264. eCollection 2022 Jul 12.
PUBMED 35693153
REFERENCE 2 (bases 1 to 8473)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Oct 3, 2022 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..8473
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 8..387
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 389..664
/label=chicken beta-actin promoter
intron 665..1681
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind 1605..1627
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 1689..1708
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
promoter 1742..1760
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1772..1792
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4967..4987
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4994..5083
/codon_start=1
/product="three tandem HA epitope tags"
/label=3xHA
/translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
CDS 5105..5158
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 5159..5878
/codon_start=1
/product="the original enhanced GFP (Yang et al., 1996)"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 5159..5875
/codon_start=1
/product="enhanced GFP"
/label=EGFP
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
primer_bind complement(5204..5225)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(5465..5484)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 5812..5833
/label=EGFP-C
/note="EGFP, forward primer"
polyA_signal 5921..6042
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(5958..5977)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 6012..6031
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(6085..6101)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(6085..6101)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(6098..6120)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 6109..6125
/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(6133..6163)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6178..6199
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(6316..6333)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(6487..7075)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(6567..6586)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(7246..8106)
/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 7869..7888
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(8107..8211)
/gene="bla"
/label=AmpR promoter
primer_bind complement(8290..8309)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
ORIGIN
1 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
61 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
121 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
181 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
361 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc
421 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt
481 gcagcgatgg gggcgggggg gggggggggg cgcgccaggc ggggcggggc ggggcgaggg
541 gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
601 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
661 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
721 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
781 tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg
841 tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc
901 gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga
961 gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc
1021 cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg
1081 tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct
1141 gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg
1201 gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg
1261 ggcggggccg cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagcgc
1321 cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag
1381 ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg
1441 caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg
1501 ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg
1561 tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg
1621 tgtgaccggc ggctctagcg cctctgctaa ccatgttcat gccttcttct ttttcctaca
1681 gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag ctagtgaatt
1741 ctaatacgac tcactatagg ccgccaccat gcccaagaag aagaggaagg ttcccgggat
1801 gagcagcgcc atcaagagct acaagagcgt gctgcgcccc aacgagcgca agaaccagct
1861 gctgaagagc accatccagt gcctggagga cggcagcgcc ttcttcttca agatgctgca
1921 gggcctgttc ggcggcatca cccccgagat cgtgcgcttc agcaccgagc aggagaagca
1981 gcagcaggac atcgccctgt ggtgcgccgt gaactggttc cgccccgtga gccaggacag
2041 cctgacccac accatcgcca gcgacaacct ggtggagaag ttcgaggagt actacggcgg
2101 caccgccagc gacgccatca agcagtactt cagcgccagc atcggcgaga gctactactg
2161 gaacgactgc cgccagcagt actacgacct gtgccgcgag ctgggcgtgg aggtgagcga
2221 cctgacccac gacctggaga tcctgtgccg cgagaagtgc ctggccgtgg ccaccgagag
2281 caaccagtac aacagcatca tcagcgtgct gttcggcacc ggcagaaagg aggaccgcag
2341 cgtgaagctg cgcatcacca agaagatcct ggaggccatc agcaacctga aggagatccc
2401 caagaacgtg gcccccatcc aggagatcat cctgaacgtg gccaaggcca ccaaggagac
2461 cttccgccag gtgtacgccg gcaacctggg cgcccccagc accctggaga gattcatcgc
2521 caaggacggc cagaaggagt tcgacctgaa gaagctgcag accgacctga agaaggtgat
2581 ccgcggcaag agcaaggagc gcgactggtg ctgccaggag gagctgcgca gctacgtgga
2641 gcagaacacc atccagtacg acctgtgggc ctggggcgag atgttcaaca aggcccacac
2701 cgccctgaag atcaagagca cccgcaacta caacttcgcc aagcagcgcc tggagcagtt
2761 caagagaatc cagagcctga acaacctgct ggtggtgaag aagctgaacg acttcttcga
2821 cagcgagttc ttcagcggcg aggagaccta caccatctgc gtgcaccacc tgggcggcaa
2881 gagactgagc aagctgtaca aggcctggga ggacgacccc gccgaccccg agaacgccat
2941 cgtggtgctg tgcgacgacc tgaagaacaa cttcaaggcc gagcccatcc gcaacatcct
3001 gcgctacatc ttcaccatcc gccaggagtg cagcgcccag gacatcctgg ccgccgccaa
3061 gtacaaccag cagctggacc gctacaagag ccagaaggcc aaccccagcg tgctgggcaa
3121 ccagggcttc acctggagaa acgccgtgat cctgcccgag aaggcccagc gcaacgaccg
3181 ccccaacagc ctggacctgc gcatctggct gtacctgaag ctgcgccacc ccgacggccg
3241 ctggaagaag caccacatcc ccttctacga cacccgcttc ttccaggaga tctacgccgc
3301 cggcaacagc cccgtggaca cctgccagtt ccgcaccccc cgcttcggct accacctgcc
3361 caagctgacc gaccagaccg ccatccgcgt gaacaagaag cacgtgaagg ccgccaagac
3421 cgaggcccgc atccgcctgg ccatccagca gggcaccctg cccgtgagca acctgaagat
3481 caccagaatc agcgccacca tcaacagcaa gggccaggtg cgcatccccg tgaagttcga
3541 cgtgggccgc cagaagggca ccctgcagat cggcgaccgc ttctgcggct acgaccagaa
3601 ccagaccgcc agccacgcct acagcctgtg ggaggtggtg aaggagggcc agtaccacaa
3661 ggagctgggc tgcttcgtgc gcttcatcag cagcggcgac atcgtgagca tcaccgagaa
3721 ccgcggcaac cagttcgacc agctgagcta cgagggcctg gcctaccccc agtacgccga
3781 ctggcgcaag aaggccagca agttcgtgag cctgtggcag atcaccaaga agaacaagaa
3841 gaaggagatc gtgaccgtgg aggccaagga gaagttcgac gccatctgca agtaccagcc
3901 ccgcctgtac aagttcaaca aggagtacgc ctacctgctg cgcgacatcg tgcgcggcaa
3961 gagcctggtg gagctgcagc agatccgcca ggagatcttc cgcttcatcg agcaggactg
4021 cggcgtgacc cgcctgggca gcctgagcct gagcaccctg gagaccgtga aggccgtgaa
4081 gggcatcatc tacagctact tcagcaccgc cctgaacgcc agcaagaaca accccatcag
4141 cgacgagcag cgcaaggagt tcgaccccga gctgttcgcc ctgctggaga agctggagct
4201 gatccgcacc cgcaagaaga agcagaaggt ggagcgcatc gccaacagcc tgatccagac
4261 ctgcctggag aacaacatca agttcatccg cggcgagggc gacctgagca ccaccaacaa
4321 cgccaccaag aagaaggcca acagccgcag catggactgg ctggcccgcg gcgtgttcaa
4381 caagatccgc cagctggccc ccatgcacaa catcaccctg ttcggctgcg gcagcctgta
4441 caccagccac caggaccccc tggtgcaccg caaccccgac aaggccatga agtgccgctg
4501 ggccgccatc cccgtgaagg acatcggcga ctgggtgctg cgcaagctga gccagaacct
4561 gcgcgccaag aacatcggca ccggcgagta ctaccaccag ggcgtgaagg agttcctgag
4621 ccactacgag ctgcaggacc tggaggagga gctgctgaag tggcgcagcg accgcaagag
4681 caacatcccc tgctgggtgc tgcagaaccg cctggccgag aagctgggca acaaggaggc
4741 cgtggtgtac atccccgtgc gcggcggccg catctacttc gccacccaca aggtggccac
4801 cggcgccgtg agcatcgtgt tcgaccagaa gcaggtgtgg gtgtgcaacg ccgaccacgt
4861 ggccgccgcc aacatcgccc tgaccgtgaa gggcatcggc gagcagagca gcgacgagga
4921 gaaccccgac ggcagccgca tcaagctgca gctgaccagc gctagcccaa agaagaagag
4981 gaaagtctct agataccctt atgatgttcc agattatgcc ggatacccat acgatgtccc
5041 tgactatgca ggctcctacc cttatgacgt cccagactac gccggatcca ggtccggcgg
5101 cggagagggc agaggaagtc ttctaacatg cggtgacgtg gaggagaatc ccggcccaat
5161 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
5221 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
5281 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
5341 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
5401 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
5461 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
5521 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
5581 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
5641 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
5701 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
5761 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
5821 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaact
5881 gcagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
5941 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
6001 gcattctagt tgtggtttgt ccaaactcat caatgtatct tattaattaa cgtcgacctc
6061 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
6121 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctagg gtgcctaatg
6181 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
6241 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
6301 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
6361 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
6421 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
6481 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
6541 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
6601 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
6661 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
6721 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
6781 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
6841 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
6901 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
6961 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
7021 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
7081 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
7141 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
7201 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
7261 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
7321 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
7381 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
7441 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
7501 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
7561 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
7621 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
7681 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
7741 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
7801 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
7861 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
7921 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
7981 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
8041 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
8101 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
8161 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
8221 ccgaaaagtg ccacctgacg tcgacggatc gggagatcga tctcccgatc ccctagggtc
8281 gactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
8341 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
8401 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt
8461 acgggccaga tat
//
