| 质粒名称: | pLBH531_MBP-Cas14a1 expression |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 112500 |
| 存储实验室: | Jennifer Doudna |
|
载体骨架基本信息 |
|
| 载体名称: | pMBP |
| 载体类型: | 细菌表达,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: | Cas14a1 |
| 插入大小: | 1590 bp |
| 启动子: | T7 |
| 标签/融合蛋白: | MBP（插入件上的N端子） |
| 克隆方法: | Gibson克隆 |
| 5’测序引物 | GATGAAGCCCTGAAAGACGCGCAG |
| 3’测序引物 | CTAGTTATTGCTCAGCGGT |
|
文献引用方法 |
|
| 材料和方法部分: | pLBH531_MBP-Cas14a1 expression was a gift from Jennifer Doudna (Addgene plasmid # 112500 ; http://n2t.net/addgene:112500 ; RRID:Addgene_112500) |
| 参考文献部分: | Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Harrington LB, Burstein D, Chen JS, Paez-Espino D, Ma E, Witte IP, Cofsky JC, Kyrpides NC, Banfield JF, Doudna JA. Science. 2018 Oct 18. pii: science.aav4294. doi: 10.1126/science.aav4294. 10.1126/science.aav4294 PubMed 30337455 |
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 pLBH531_MBP-Cas14a1 expression 7556 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses MBP fused to Cas14a1.
ACCESSION .
VERSION .
KEYWORDS pLBH531_MBP-Cas14a1 expression
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7556)
AUTHORS Harrington LB, Burstein D, Chen JS, Paez-Espino D, Ma E, Witte IP,
Cofsky JC, Kyrpides NC, Banfield JF, Doudna JA
TITLE Programmed DNA destruction by miniature CRISPR-Cas14 enzymes.
JOURNAL Science. 2018 Oct 18. pii: science.aav4294. doi:
10.1126/science.aav4294.
PUBMED 30337455
REFERENCE 2 (bases 1 to 7556)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7556
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 58..77
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 161..180
/label=T7
/note="T7 promoter, forward primer"
promoter 161..179
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
RBS 268..290
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 310..339
/codon_start=1
/product="10xHis affinity tag"
/label=10xHis
/translation="HHHHHHHHHH"
CDS 349..1449
/codon_start=1
/gene="malE (mutated)"
/product="maltose binding protein from E. coli"
/label=MBP
/note="This version of the gene does not encode a signal
sequence, so MBP will remain in the cytosol."
/translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
WYAVRTAVINAASGRQTVDEALKDAQT"
primer_bind 1423..1446
/label=MBP-F
/note="Maltose binding protein, forward primer"
CDS 1504..1524
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQS"
primer_bind complement(3246..3264)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 3260..3307
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
promoter complement(3673..3701)
/label=tet promoter
/note="E. coli promoter for tetracycline efflux protein
gene"
primer_bind complement(3728..3746)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 3814..3918
/gene="bla"
/label=AmpR promoter
CDS 3919..4779
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(4137..4156)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 4950..5538
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5439..5458
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 5692..5709
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 5724..5863
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(5789..5808)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5949..5971
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(5965..6156)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
ORIGIN
1 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
61 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
121 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg
181 agaccacaac ggtttccctc tagtgccggc tccggagagc tctttaatta agcggccgcc
241 ctgcaggact cgagttctag aaataatttt gtttaacttt aagaaggaga tatacatatg
301 aaatcttctc accatcacca tcaccatcac catcaccatg gttcttctat gaaaatcgaa
361 gaaggtaaac tggtaatctg gattaacggc gataaaggct ataacggtct cgctgaagtc
421 ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg ttgagcatcc ggataaactg
481 gaagagaaat tcccacaggt tgcggcaact ggcgatggcc ctgacattat cttctgggca
541 cacgaccgct ttggtggcta cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa
601 gcgttccagg acaagctgta tccgtttacc tgggatgccg tacgttacaa cggcaagctg
661 attgcttacc cgatcgctgt tgaagcgtta tcgctgattt ataacaaaga tctgctgccg
721 aacccgccaa aaacctggga agagatcccg gcgctggata aagaactgaa agcgaaaggt
781 aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca cctggccgct gattgctgct
841 gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg
901 gataacgctg gcgcgaaagc gggtctgacc ttcctggttg acctgattaa aaacaaacac
961 atgaatgcag acaccgatta ctccatcgca gaagctgcct ttaataaagg cgaaacagcg
1021 atgaccatca acggcccgtg ggcatggtcc aacatcgaca ccagcaaagt gaattatggt
1081 gtaacggtac tgccgacctt caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc
1141 gcaggtatta acgccgccag tccgaacaaa gagctggcaa aagagttcct cgaaaactat
1201 ctgctgactg atgaaggtct ggaagcggtt aataaagaca aaccgctggg tgccgtagcg
1261 ctgaagtctt acgaggaaga gttggcgaaa gatccacgta ttgccgccac tatggaaaac
1321 gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg
1381 cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg tcgatgaagc cctgaaagac
1441 gcgcagacta attcgagctc gaacaacaac aacaataaca ataacaacaa cctcgggatc
1501 gaggaaaacc tgtacttcca atccaatgca atggccaaaa acaccattac caaaacactg
1561 aaactgcgta ttgtgcgtcc gtataatagc gcagaagtgg aaaaaattgt tgccgacgaa
1621 aaaaacaacc gcgaaaaaat cgcactggaa aagaacaaag acaaagtgaa agaagcctgc
1681 agcaaacatc tgaaagttgc agcatattgt accacacagg ttgaacgtaa tgcatgcctg
1741 ttttgtaaag cacgtaaact ggatgacaaa ttctaccaaa aactgcgtgg tcagtttccg
1801 gatgcagttt tttggcaaga aatcagcgaa atttttcgcc agctgcagaa acaggcagca
1861 gaaatctata atcagagcct gatcgaactg tactacgaga tttttatcaa aggcaaaggt
1921 attgcaaatg ccagcagcgt tgaacattat ctgagtgatg tttgttatac ccgtgcagca
1981 gaactgttta aaaacgcagc aattgcaagc ggtctgcgta gcaaaatcaa aagcaatttt
2041 cgtctgaaag aactgaaaaa catgaaaagt ggtctgccga ccaccaaaag cgataatttt
2101 ccgattccgc tggttaaaca gaaaggtggt cagtataccg gttttgaaat tagcaatcat
2161 aatagcgact tcatcatcaa gattccgttt ggtcgttggc aggtcaaaaa agagattgat
2221 aaatatcgtc cgtgggagaa atttgacttt gaacaggttc agaaaagccc gaaaccgatt
2281 agcctgctgc tgagcaccca gcgtcgtaaa cgtaataaag gttggagcaa agatgaaggc
2341 accgaagccg aaatcaaaaa agttatgaat ggcgattatc agaccagcta cattgaagtt
2401 aaacgtggca gcaaaatctg tgaaaaaagc gcatggatgc tgaatctgag cattgatgtt
2461 ccgaaaattg ataaaggtgt ggatccgagc attattggtg gtattgatgt tggtgttaaa
2521 tcaccgctgg tttgcgcaat taacaatgca tttagccgtt atagcatcag cgataacgac
2581 ctgtttcact tcaacaagaa aatgtttgca cgtcgtcgta tcctgctgaa aaaaaaccgt
2641 cataaacgtg caggtcatgg tgcaaaaaac aaactgaaac cgatcaccat tctgaccgaa
2701 aaaagtgaac gttttcgcaa aaagctgatt gaacgttggg catgtgaaat cgcggatttc
2761 ttcattaaaa acaaagttgg caccgtgcag atggaaaatc tggaaagcat gaaacgtaaa
2821 gaggacagct attttaacat tcgcctgcgt ggcttttggc cgtatgcaga aatgcagaac
2881 aaaatcgaat tcaaactgaa gcagtatggc atcgaaattc gtaaagttgc accgaataat
2941 accagcaaaa cctgtagcaa atgtggccat ctgaacaact atttcaactt cgagtaccgc
3001 aagaaaaaca aattcccgca ctttaaatgc gaaaaatgca acttcaaaga aaacgccgat
3061 tataatgcag ccctgaatat ttcaaacccg aaactgaaaa gcaccaaaga ggaaccgtaa
3121 taacattgga agtggataac ggatccgcga tcgcggcgcg ccacctggtg gccggccggt
3181 accacgcgtg cgcgctgatc cggctgctaa caaagcccga aaggaagctg agttggctgc
3241 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
3301 ttttttgctg aaaggaggaa ctatatccgg atatccacag gacgggtgtg gtcgccatga
3361 tcgcgtagtc gatagtggct ccaagtagcg aagcgagcag gactgggcgg cggccaaagc
3421 ggtcggacag tgctccgaga acgggtgcgc atagaaattg catcaacgca tatagcgcta
3481 gcagcacgcc atagtgactg gcgatgctgt cggaatggac gatatcccgc aagaggcccg
3541 gcagtaccgg cataaccaag cctatgccta cagcatccag ggtgacggtg ccgaggatga
3601 cgatgagcgc attgttagat ttcatacacg gtgcctgact gcgttagcaa tttaactgtg
3661 ataaactacc gcattaaagc ttatcgatga taagctgtca aacatgagaa ttcttgaaga
3721 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
3781 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
3841 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
3901 cattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
3961 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
4021 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
4081 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
4141 tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac
4201 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
4261 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
4321 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
4381 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
4441 gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc
4501 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
4561 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
4621 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
4681 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
4741 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
4801 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
4861 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
4921 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
4981 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
5041 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
5101 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
5161 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
5221 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
5281 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
5341 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
5401 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
5461 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
5521 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
5581 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
5641 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
5701 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
5761 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
5821 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
5881 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
5941 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
6001 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
6061 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
6121 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
6181 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
6241 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
6301 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
6361 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
6421 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
6481 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
6541 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
6601 gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc gccgcgtgcg
6661 gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt gcgcattcac
6721 agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt tagcgaggtg
6781 ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg
6841 aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca tgtgctcgcc
6901 gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag gctggtaaga
6961 gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct ggacagcatg
7021 gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat ggggaaggcc
7081 atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc cgccatgccg
7141 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
7201 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
7261 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
7321 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
7381 gagctgactg ggttgaaggc tctcaagggc atcggtcgac gctctccctt atgcgactcc
7441 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat
7501 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccat
//
