| 质粒名称: | pLBH559_Tet-HisCas14a1Locus |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 112502 |
| 存储实验室: | Jennifer Doudna |
|
载体骨架基本信息 |
|
| 载体名称: | pTet |
| 载体抗性: | 氯霉素 |
| 载体类型: | 细菌表达，CRISPR |
| 高拷贝/低拷贝: | 低拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | HisCas14a1 |
| 插入大小: | 2181 bp |
| 启动子 | Tet |
| 克隆方法: | Gibson克隆 |
| 5' 测序引物 | gtgccgatcaacgtAtcattttcg |
| 3' 测序引物 | acgcagaaaggcccacccgaag |
|
文献引用方法 |
|
| 材料和方法部分: | pLBH559_Tet-HisCas14a1Locus was a gift from Jennifer Doudna (Addgene plasmid # 112502 ; http://n2t.net/addgene:112502 ; RRID:Addgene_112502) |
| 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 pLBH559_Tet-HisCas14a1Locus 4670 bp ds-DNA circular SYN 26-OCT-2018
DEFINITION Expresses 10xHis-Cas14a1 locus (including crRNA and tracrRNA) under
control of a tetracycline inducible promoter.
ACCESSION .
VERSION .
KEYWORDS pLBH559_Tet-HisCas14a1Locus
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4670)
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 4670)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Oct 26, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..4670
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 16..71
/gene="tetR"
/label=tetR/tetA promoters
/note="overlapping promoters for bacterial tetR and tetA"
protein_bind 52..70
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
RBS 89..100
/note="strong bacterial ribosome binding site (Elowitz and
Leibler, 2000)"
CDS 116..133
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 134..154
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQS"
terminator 2289..2316
/label=T7Te terminator
/note="phage T7 early transcription terminator"
primer_bind complement(2344..2361)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(2478..3023)
/direction=LEFT
/label=p15A ori
/note="Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin.
"
primer_bind complement(2558..2577)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
terminator 3137..3231
/label=lambda t0 terminator
/note="transcription terminator from phage lambda"
CDS complement(3252..3911)
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
primer_bind 3826..3845
/label=CAT-R
/note="Chloramphenicol resistance gene, reverse primer"
promoter complement(3912..4014)
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
CDS complement(4044..4667)
/codon_start=1
/gene="tetR from transposon Tn10"
/product="tetracycline repressor TetR"
/label=TetR
/note="TetR binds to the tetracycline operator tetO to
inhibit transcription. This inhibition can be relieved by
adding tetracycline or doxycycline."
/translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGSKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
primer_bind 4575..4594
/label=Tet-R
/note="Tetracycline resistance gene, reverse primer"
ORIGIN
1 taattcctaa tttttgttga cactctatcg ttgatagagt tattttacca ctccctatca
61 gtgatagaga aaagaattca aaagatctaa agaggagaaa ggatctatga aatctcacca
121 tcaccatcac catgaaaacc tgtacttcca atccaatatt ggaagtggaa tggccaaaaa
181 caccattacc aaaacactga aactgcgtat tgtgcgtccg tataatagcg cagaagtgga
241 aaaaattgtt gccgacgaaa aaaacaaccg cgaaaaaatc gcactggaaa agaacaaaga
301 caaagtgaaa gaagcctgca gcaaacatct gaaagttgca gcatattgta ccacacaggt
361 tgaacgtaat gcatgcctgt tttgtaaagc acgtaaactg gatgacaaat tctaccaaaa
421 actgcgtggt cagtttccgg atgcagtttt ttggcaagaa atcagcgaaa tttttcgcca
481 gctgcagaaa caggcagcag aaatctataa tcagagcctg atcgaactgt actacgagat
541 ttttatcaaa ggcaaaggta ttgcaaatgc cagcagcgtt gaacattatc tgagtgatgt
601 ttgttatacc cgtgcagcag aactgtttaa aaacgcagca attgcaagcg gtctgcgtag
661 caaaatcaaa agcaattttc gtctgaaaga actgaaaaac atgaaaagtg gtctgccgac
721 caccaaaagc gataattttc cgattccgct ggttaaacag aaaggtggtc agtataccgg
781 ttttgaaatt agcaatcata atagcgactt catcatcaag attccgtttg gtcgttggca
841 ggtcaaaaaa gagattgata aatatcgtcc gtgggagaaa tttgactttg aacaggttca
901 gaaaagcccg aaaccgatta gcctgctgct gagcacccag cgtcgtaaac gtaataaagg
961 ttggagcaaa gatgaaggca ccgaagccga aatcaaaaaa gttatgaatg gcgattatca
1021 gaccagctac attgaagtta aacgtggcag caaaatcggt gaaaaaagcg catggatgct
1081 gaatctgagc attgatgttc cgaaaattga taaaggtgtg gatccgagca ttattggtgg
1141 tattgatgtt ggtgttaaat caccgctggt ttgcgcaatt aacaatgcat ttagccgtta
1201 tagcatcagc gataacgacc tgtttcactt caacaagaaa atgtttgcac gtcgtcgtat
1261 cctgctgaaa aaaaaccgtc ataaacgtgc aggtcatggt gcaaaaaaca aactgaaacc
1321 gatcaccatt ctgaccgaaa aaagtgaacg ttttcgcaaa aagctgattg aacgttgggc
1381 atgtgaaatc gcggatttct tcattaaaaa caaagttggc accgtgcaga tggaaaatct
1441 ggaaagcatg aaacgtaaag aggacagcta ttttaacatt cgcctgcgtg gcttttggcc
1501 gtatgcagaa atgcagaaca aaatcgaatt caaactgaag cagtatggca tcgaaattcg
1561 taaagttgca ccgaataata ccagcaaaac ctgtagcaaa tgtggccatc tgaacaacta
1621 tttcaacttc gagtaccgca agaaaaacaa attcccgcac tttaaatgcg aaaaatgcaa
1681 cttcaaagaa aacgccgatt ataatgcagc cctgaatatt tcaaacccga aactgaaaag
1741 caccaaagag gaaccgtaaa tatttatact ttattatcct tcattgacaa aaatgagaat
1801 gttatcccag ataacatttg atgtacacag attcacactt cactgataaa gtggagaacc
1861 gcttcaccaa aagctgtccc ttaggggatt agaacttgag tgaaggtggg ctgcttgcat
1921 cagcctaatg tcgagaagtg ctttcttcgg aaagtaaccc tcgaaacaaa ttcatttttc
1981 ctctccaatt ctgcacaaaa aaaggtgagt ccttataaac cggcgtgcag aacgccggct
2041 cacctttttt cttcattcga ttttatgctt aaaagccgta aaaacgcgga attcggcgcc
2101 gttgcagaac ccgaatagac gaatgaagga atgcaactac cttacaccgc ttgcgaaagt
2161 tgtcagaaga taatcttgca gaacccgaat agacgaatga aggaatgcaa tcttgacaga
2221 gcccgattgc gttatctcca ggagaaacat ataaaagcat caaccgctga tcggactaga
2281 gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacctaggga tatattccgc
2341 ttcctcgctc actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta
2401 cgaacggggc ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg
2461 gccgcggcaa agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga
2521 cgctcaaatc agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
2581 ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg
2641 ttatggccgc gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc
2701 caagctggac tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa
2761 ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg
2821 taattgattt agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga
2881 caagttttgg tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc
2941 agagaacctt cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta
3001 cgcgcagacc aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga
3061 tttcagtgca atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt
3121 tgttactagt gcttggattc tcaccaataa aaaacgcccg gcggcaaccg agcgttctga
3181 acaaatccag atggagttct gaggtcatta ctggatctat caacaggagt ccaagcgagc
3241 tcgatatcaa attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc
3301 attctgccga catggaagcc atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc
3361 agcaccttgt cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg
3421 tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg
3481 aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc
3541 acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc
3601 gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat
3661 atcaccagct caccgtcttt cattgccata cgaaattccg gatgagcatt catcaggcgg
3721 gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa
3781 aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat
3841 gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt
3901 tttttctcca ttttagcttc cttagctcct gaaaatctcg ataactcaaa aaatacgccc
3961 ggtagtgatc ttatttcatt atggtgaaag ttggaacctc ttacgtgccg atcaacgtct
4021 cattttcgcc agatatcgac gtcttaagac ccactttcac atttaagttg tttttctaat
4081 ccgcatatga tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat
4141 aattcgatag cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct
4201 tctttagcga cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca
4261 gcgctgagtg catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat
4321 tgattttcga gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgat
4381 ccatcgcgat gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct
4441 tgccagcttt ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg
4501 gctaaggcgt cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct
4561 acacctagct tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc
4621 agctctaatg cgctgttaat cactttactt ttatctaatc tagacatcat
//
