| 质粒名称: | pCAT100 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 180512 |
| 存储实验室: | Jennifer Doudna |
|
载体骨架基本信息 |
|
| 载体名称: | pBLO62.5 |
| 载体出品公司: | -- |
| 空载体大小: | 4849 bp |
| 完整质粒大小: | 7909 bp |
| 载体修饰: | -- |
| 载体类型: | 哺乳细胞表达, CRISPR |
| 筛选标记: | -- |
| 载体抗性: | 氨苄霉素100 μg/mL |
| 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: | CasX sgRNAv2 |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子: | U6 |
| 标签/融合蛋白: | -- |
| 克隆方法: | Gibson克隆 |
| 5’克隆位点: | -- |
| 3’克隆位点: | -- |
| 5’测序引物 | GCTGTTAGAGAGATAATTGG |
| 3’测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pCAT100 was a gift from Jennifer Doudna (Addgene plasmid # 180512 ; http://n2t.net/addgene:180512 ; RRID:Addgene_180512) |
| 参考文献部分: | Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity. Tsuchida CA, Zhang S, Doost MS, Zhao Y, Wang J, O'Brien E, Fang H, Li CP, Li D, Hai ZY, Chuck J, Brotzmann J, Vartoumian A, Burstein D, Chen XW, Nogales E, Doudna JA, Liu JG. Mol Cell. 2022 Mar 17;82(6):1199-1209.e6. doi: 10.1016/j.molcel.2022.02.002. Epub 2022 Feb 25. 10.1016/j.molcel.2022.02.002 PubMed 35219382 |
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 pCAT100 7874 bp ds-DNA circular SYN 20-APR-2022
DEFINITION Plasmid expressing mammalian codon optimized engineered PlmCasX-R1,
sgRNAv2 scaffold, and restriction sites to clone in new spacers.
ACCESSION .
VERSION .
KEYWORDS pCAT100
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7874)
TITLE Engineered CasX protein and sgRNA plasmids
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 7874)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Apr 20, 2022 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7874
/organism="synthetic DNA construct"
/mol_type="other DNA"
intron 127..354
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
regulatory 366..375
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 378..398
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3363..3383
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3384..3431
/codon_start=1
/product="two tandem FLAG(R) epitope tags"
/label=2xFLAG
/translation="DYKDDDDKDYKDDDDK"
CDS 3441..3497
/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 3498..4094
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind 3991..4011
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
primer_bind complement(4128..4145)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 4134..4341
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
repeat_region 4350..4490
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 4350..4479
/label=AAV2 ITR
rep_origin 4565..5020
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4652..4671)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4862..4883
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(5037..5056)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5156..5178
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(5216..5234)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 5302..5406
/gene="bla"
/label=AmpR promoter
CDS 5407..6267
/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(5625..5644)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6438..7026
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6927..6946
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
promoter 7088..7328
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind 7088..7108
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 7259..7278
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
enhancer 7470..7755
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
ORIGIN
1 gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga
61 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg
121 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc
181 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct
241 tctcctccgg gctgtaatta gctgagcaag aggtaagggt ttaagggatg gttggttggt
301 ggggtattaa tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttgga
361 ccggtgccac catggcccca aagaagaagc ggaaggtcag catgcaagag atcaagagaa
421 tcaacaagat cagaaggaga ctggtcaagg acagcaacac aaagaaggcc ggcaagacag
481 gccccatgaa aaccctgctc gtcagagtga tgacccctga cctgagagag cggctggaaa
541 acctgagaaa gaagcccgag aacatccctc agcctatcag caacaccagc agggccaacc
601 tgaacaagct gctgaccgac tacaccgaga tgaagaaagc catcctgcac gtgtactggg
661 aagagttcca gaaagacccc gtgggcctga tgagcagagt tgctcagccc gctcctaaga
721 acatcgacca gagaaagctg atccccgtga aggacggcaa cgagagactg acctctagcg
781 gctttgcctg cagccagtgt tgccagcctc tgtacgtgta caagctggaa caagtgaacg
841 acaagggcaa gccccacacc aactacttcg gcagatgcaa cgtgtccgag cacgagaggc
901 tgatcctgct gtctcctcac aagcccgagg ccaacgatga gctggtcaca tacagcctgg
961 gcaagttcgg acagagagcc ctggacttct acagcatcca cgtgaccagg gagagcaatc
1021 accctgtgaa gcccctggaa cagatcggcg gcaatagctg tgcctctgga cctgtgggaa
1081 aagccctgag cgacgcctgt atgggagccg tggcatcctt cctgaccaag taccaggaca
1141 tcatcctgga acaccagaaa gtgatcaaga agaacgagaa aagactggcc aacctcaagg
1201 atatcgccag cgctaacggc ctggcctttc ctaagatcac cctgcctcca cagcctcaca
1261 ccaaagaggg catcgaggcc tacaacaacg tggtggccca gatcgtgatt tgggtcaacc
1321 tgaatctgtg gcagaagctg aagatcggca gggacgaagc caagccactg cagagactga
1381 agggcttccc tagcttccct ctggtggaaa gacaggccaa tgaagtggat tggtgggaca
1441 tggtctgcaa cgtgaagaag ctgatcaacg agaagaaaga ggatggcaag gttttctggc
1501 agaacctggc cggctacaag agacaagaag ccctgctgcc ttacctgagc agcgaagagg
1561 accataaaaa gcgggagggg tctcttgaaa acccgaagaa gttcgccaga taccagttcg
1621 gcgacctgct gctgcacctg gaaaagaagc acggcgagga ctggggcaaa gtgtacgatg
1681 aggcctggga gagaatcgac aagaaggtgg aaggcctgag caagcacatt aagctggaag
1741 aggaaagaag gagcgaggac gcccaatcta aagccgctct gaccgattgg ctgagagcca
1801 aggccagctt tgtgatcgag ggcctgaaag aggccgacaa ggacgagttc tgcagatgcg
1861 agctgaagct gcagaagtgg tacggcgatc tgagaggcaa gcccttcgcc attgaggccg
1921 agaacagcat cctggacatc agcggcttca gcaagcagta caactgcgcc ttcatttggc
1981 agaaagacgg cgtcaagaaa ctgaacctgt acctgatcat caattacttc aaaggcggca
2041 agctgcggtt caagaagatc aaacccgagg ccttcgaggc taacagattc tacaccgtga
2101 tcaacaaaaa gtccggcgag atcgtgccca tggaagtgaa cttcaacttc gacgacccca
2161 acctgattat cctgcctctg gccttcggca agagacaggg cagagagttc atctggaacg
2221 atctgctgag cctggaaacc ggctctctga agctggccaa tggcagagtg atcgagaaaa
2281 ccctgtacaa caggagaacc agacaggacg agcctgctct gtttgtggcc ctgaccttcg
2341 agagaagaga ggtgctggac agcagcaaca tcaagcccat gaacctgatc ggcatcgacc
2401 ggggcgagaa tatccctgct gtgatcgccc tgacagaccc tgaaggatgc ccactgagca
2461 gattcaagga ctccctgggc aaccctacac acatcctgag aatcggcgag agctacaaag
2521 agaagcagag gacaatccag gccgccaaag aggtggaaca gagaagagcc ggcggatact
2581 ctaggaagta cgccagcaag gccaagaatc tggccgacga catggtccga aacaccgcca
2641 gagatctgct gtactacgcc gtgacacagg acgccatgct gatcttcgag aatctgagca
2701 gaggcttcgg ccggcagggc aagagaacct ttatggccga gaggcagtac accagaatgg
2761 aagattggct cacagctaaa ctggcctacg agggactgcc cagcaagacc tacctgtcca
2821 aaacactggc ccagtatacc tccaagacct gcagcaattg cggcttcacc atcaccagcg
2881 ccgactacga cagagtgctg gaaaagctca agaaaaccgc caccggctgg atgaccacca
2941 tcaacggcaa agagctgaag gttgagggcc agatcaccta ctacaacagg tacaagaggc
3001 agaacgtcgt gaaggatctg agcgtggaac tggacagact gagcgaagag agcgtgaaca
3061 acgacatcag cagctggaca aagggcagat caggcgaggc tctgagcctg ctgaagaaga
3121 ggtttagcca cagacctgtg caagagaagt tcgtgtgcct gaactgcggc ttcgagacac
3181 acgccgatga acaggctgcc ctgaacattg ccagaagctg gctgttcctg agaagccaag
3241 agtacaagaa gtaccagacc aacaagacca ccggcaacac cgacaagagg gcctttgtgg
3301 aaacctggca gagcttctac agaaaaaagc tgaaagaagt ctggaagccc gccgtgacta
3361 gtccaaaaaa gaagagaaag gtagattaca aagatgacga tgacaaagac tacaaggatg
3421 atgatgataa gggatccggc gcaacaaact tctctctgct gaaacaagcc ggagatgtcg
3481 aagagaatcc tggaccgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg
3541 tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca
3601 ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc
3661 gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct
3721 ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg
3781 ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc
3841 accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg
3901 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg
3961 tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct
4021 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca
4081 agcccggtgc ctgaacgcgt taagaattcc tagagctcgc tgatcagcct cgactgtgcc
4141 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
4201 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
4261 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
4321 gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc
4381 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
4441 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
4501 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
4561 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
4621 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc
4681 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
4741 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
4801 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
4861 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat
4921 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa
4981 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
5041 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
5101 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
5161 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
5221 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
5281 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
5341 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
5401 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
5461 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
5521 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
5581 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
5641 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
5701 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
5761 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
5821 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
5881 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
5941 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
6001 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
6061 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
6121 ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
6181 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
6241 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
6301 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
6361 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
6421 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
6481 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
6541 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
6601 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
6661 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
6721 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
6781 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
6841 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
6901 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
6961 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
7021 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
7081 cacatgtgag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
7141 tagagagata attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
7201 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
7261 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
7321 gaaaggacga aacaccggcg cttttatctc attactttga gagccatcac cagcgactat
7381 gtcgtatggg taaagcgctt atttatcgga gaaaccgata aataagaagc atcaaagggg
7441 tcttcgagaa gacccttttt tttggtaccc gttacataac ttacggtaaa tggcccgcct
7501 ggctgaccgc ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc
7561 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
7621 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
7681 tgtgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
7741 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc
7801 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
7861 gcgggggggg gggg
//
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