| 质粒名称: | pCMV-AsCas12f1-v5.1+T1-empty |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 204977 |
| 存储实验室: | Quanjiang Ji |
|
载体骨架基本信息 |
|
| 载体名称: | Col1E |
| 载体出品公司: | Clontech |
| 完整质粒大小: | 6816bp |
| 载体类型: | 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: | AsCas12f1-v5.1, sgRNA_T1 / Cas12U3 |
| 物种来源: | Synthetic |
| 基因突变: | N70Q/K103R/A104R/S118A/D364R |
| GenBank编号 | JN247409 |
| 启动子: | PCMV,U6 |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | Unknown (unknown if destroyed) |
| 3’克隆位点: | Unknown (unknown if destroyed) |
|
文献引用方法 |
|
| 材料和方法部分: | pCMV-AsCas12f1-v5.1+T1-empty was a gift from Quanjiang Ji (Addgene plasmid # 204977 ; http://n2t.net/addgene:204977 ; RRID:Addgene_204977) |
| 参考文献部分: | Structure and engineering of miniature Acidibacillus sulfuroxidans Cas12f1. Wu Z, Liu D, Pan D, Yu H, Shi J, Ma J, Fu W, Wang Z, Zheng Z, Qu Y, Li F, Chen W, Huang X, Shen H, Ji Q. Nature Catalysis (2023) 6, 695–709 10.1038/s41929-023-00995-4 |
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 pCMV-AsCas12f1-v5.1+T1-empty 6816 bp ds-DNA circular SYN 16-AUG-2023
DEFINITION AsCas12f1-v5.1+T1-based Human cell genome editing.
ACCESSION .
VERSION .
KEYWORDS pCMV-AsCas12f1-v5.1+T1-empty
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6816)
AUTHORS Wu, Z., Liu, D., Pan, D., Yu, H., Shi, J., Ma, J., Fu, W., Wang, Z.,
Zheng, Z., Qu, Y., Li, F., Chen, W., Huang, X., Shen, H., Ji, Q
TITLE Structure and engineering of miniature Acidibacillus sulfuroxidans
Cas12f1
JOURNAL Nature Catalysis, 2023
REFERENCE 2 (bases 1 to 6816)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 16, 2023 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..6816
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 72..312
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 72..92
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 243..262
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
misc_feature 538..655
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 704..1214
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
primer_bind 1133..1151
/label=hPGK-F
/note="Human PGK promoter, forward primer"
CDS 1236..1835
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind complement(1236..1255)
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
primer_bind 1732..1752
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
polyA_signal 1982..2103
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(1993..2012)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 2047..2066
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind complement(2351..2368)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(2522..3110)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(2602..2621)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(3281..4141)
/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 3904..3923
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(4142..4246)
/gene="bla"
/label=AmpR promoter
primer_bind 4273..4289
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 4273..4289
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
polyA_signal 4360..4584
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
primer_bind 4573..4590
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
CDS complement(4613..4630)
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS complement(4639..4659)
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS complement(5938..5958)
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
regulatory 5961..5970
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
primer_bind complement(5977..5996)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(5978..5996)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
promoter complement(6038..6241)
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind complement(6067..6087)
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
enhancer 6242..6621
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
misc_feature 6718..6809
/label=pause site
/note="RNA polymerase II transcriptional pause signal from
the human alpha-2 globin gene"
primer_bind 6758..6777
/label=RVprimer3
/note="pGL3 vector, forward primer"
ORIGIN
1 ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg
61 cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
121 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
181 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
241 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
301 tgtggaaagg acgaaacacc gattcgtcgg ttcagcgacg ataagccgag aagtgccaat
361 aaaactgtta agtggtttgg taacgctcgg taaggtagcc aaaaggctga aactccgtgc
421 acaaagaccg cacggacgct tcacatactt gtggagtgtg aaccgagacc cgagagaggg
481 tcggtctcct ttttttgaat tctcgacctc gagacaaatg gcagtattca tccacaattt
541 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
601 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt
661 ttattacagg gacagcagag atccactttg gccgcggctc gagggggttg gggttgcgcc
721 ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt ggttccggga
781 aacgcagcgg cgccgaccct gggactcgca cattcttcac gtccgttcgc agcgtcaccc
841 ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg cccctaagtc
901 gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca
961 ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga ccgcgatggg
1021 ctgtggccaa tagcggctgc tcagcagggc gcgccgagag cagcggccgg gaaggggcgg
1081 tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg
1141 cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg
1201 acctctctcc ccagggggat ccaccggagc ttaccatgac cgagtacaag cccacggtgc
1261 gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg
1321 actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc
1381 tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg
1441 acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg
1501 ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga
1561 tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg
1621 gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg
1681 aggcggccga gcgcgccggg gtgcccgcct tcctggaaac ctccgcgccc cgcaacctcc
1741 ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc
1801 gcacctggtg catgacccgc aagcccggtg cctgacgccc gccccacgac ccgcagcgcc
1861 cgaccgaaag gagcgcacga ccccatgcat cggtaccttt aagaccaatg acttacaagg
1921 cagctgtaga tcttagccac tttctagagt cggggcggcc ggccgcttcg agcagacatg
1981 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
2041 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
2101 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt
2161 ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg ataaggatcc gtcgaccgat
2221 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt
2281 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct
2341 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
2401 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
2461 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
2521 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
2581 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
2641 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
2701 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
2761 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
2821 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
2881 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
2941 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
3001 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
3061 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
3121 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
3181 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
3241 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
3301 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
3361 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
3421 gggacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
3481 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
3541 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
3601 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
3661 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
3721 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
3781 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
3841 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
3901 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
3961 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
4021 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
4081 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
4141 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
4201 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
4261 aagtgccacc tgcaggaaac agctatgacc atgattacgc caagctctag ctagaggtcg
4321 acggtatcga gccccagctg gttctttccg cctcagaagc catagagccc accgcatccc
4381 cagcatgcct gctattgtct tcccaatcct cccccttgct gtcctgcccc accccacccc
4441 ccagaataga atgacaccta ctcagacaat gcgatgcaat ttcctcattt tattaggaaa
4501 ggacagtggg agtggcacct tccagggtca aggaaggcac gggggagggg caaacaacag
4561 atggctggca actagaaggc acagtcgagg ctgatcagcg ggtttaaact caatggtgat
4621 ggtgatgatg accggttaga ctttcctctt cttcttggga gaaccaccag acttaatgct
4681 ctcggcgatg atcttgtcga tgttggggat ggcgatattc cgggcggcgt tgtagtcggc
4741 gttggcctcg tagccgcagg cccggcactt aaagatggcc tggccgattc tgttgccgga
4801 gtcgatgttg ccgcactcgg agcatctctg gctggtgtac tggggtctga tcttgatcac
4861 tttgatgccg gcctcctcgg ccttgtagat gatcttctgc tgcaggtcgt agtaggtcca
4921 gttctgcagg aatctgctgc cgatgtctct gatgtttgtc agatcctcca tctggattgt
4981 gccgcagccc tccttgatgg ccatgtccac gatgtatctg ctgtaccggt gattggtggt
5041 gtctctgaaa ttggcgatct tatccctcag ctgctcaatg ggcttgattc tcttgtctct
5101 gccgtggccg cccctggcgc cgccggcgta cttgccctgt ctcagcatgg agatgcgccg
5161 gctctccacc tgcctcctga agttctcaat ctcgccgccc tccagcttgt acctggcggg
5221 ggtgtgctgg aaggccatgt acacggccac ggccacgccc aggtcaatgc ccatgatctt
5281 gttcaggtcc agcactctgg tctgtggctc gaagtcgtag ctgatgttca ggtaccactt
5341 gttcttccgg tcgtcgtgga taatctggct ggcgctcacc tggtactcgc cggacaggat
5401 tctgtccatg atggtcttgc cggcgcccct cacgatgatg atcacggaga tctttctctt
5461 cacgttcatc tcctgcttgg cggggttgct cagcagagac aggctggcga tgtagtcgcc
5521 gtgattcatc ctgttcacgg agatgttctc cttgatcagg tccaggggga tgtccctctt
5581 gtaggcgggg atagacatgt cgccgcgcag gatctccttc tggtaccttc tgaacctgtc
5641 tgtggctctc ttgatggtct ggctcagatt tccagagttc agtctgtagg cctttgtctt
5701 gatggtgtgg taggcgtagc cgtgcacctg ggtgtagccc aggatgtcct tgctcttggg
5761 gtactcgccg tggttatcct tgtagtcgct gctgaagccc atccactccc aggccagctg
5821 tgtggcctta ttcagggcga atctggtttc ctgctgcagc tgtctcagga tggtgccgaa
5881 ctccttccag tccaggtcca gaggcttcac gatctcgtat ctgtacacct tgatcatgac
5941 tttcctcttc ttcttggggg ccatggtggc ggctctccct atagtgagtc gtattagcgg
6001 ccgcggatct ctagcggatc tgacggttca ctaaaccagc tctgcttata tagacctccc
6061 accgtacacg cctaccgccc atttgcgtca atggggcgga gttgttacga cattttggaa
6121 agtcccgttg attttggtgc caaaacaaac tcccattgac gtcaatgggg tggagacttg
6181 gaaatccccg tgagtcaaac cgctatccac gcccattgat gtactgccaa aaccgcatca
6241 ccatggtaat agcgatgact aatacgtaga tgtactgcca agtaggaaag tcccataagg
6301 tcatgtactg ggcataatgc caggcgggcc atttaccgtc attgacgtca atagggggcg
6361 tacttggcat atgatacact tgatgtactg ccaagtgggc agtttaccgt aaatactcca
6421 cccattgacg tcaatggaaa gtccctattg gcgttactat gggaacatac gtcattattg
6481 acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg gccatttacc gtaagttatg
6541 taacgcggaa ctccatatat gggctatgaa ctaatgaccc cgtaattgat tactattaat
6601 aactagtcaa taatcaatgt caacgcgtat atctggcccg tacatcgcga agcagcgcaa
6661 aacgcctaac cctaagcaga ttcttcatgc aattgtcggt caagaatcga tagtactaac
6721 atacgctctc catcaaaaca aaacgaaaca aaacaaacta gcaaaatagg ctgtccccag
6781 tgcaagtgca ggtgccagaa catttctcta tcgata
//
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