| 质粒名称: | pKDsgRNA-p15 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 62656 |
| 存储实验室: | Kristala Prather |
|
载体骨架基本信息 |
|
| 载体名称: | pKD46 |
| 载体出品公司: | -- |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 载体抗性: | 大观霉素 |
| 生长温度: | 30℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 低拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | exo, beta, gam, sgRNA |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子: | -- |
| 标签/融合蛋白: | -- |
| 克隆方法: | -- |
| 5’克隆位点: | -- |
| 3’克隆位点: | -- |
| 5’测序引物 | -- |
| 3’测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pKDsgRNA-p15 was a gift from Kristala Prather (Addgene plasmid # 62656 ; http://n2t.net/addgene:62656 ; RRID:Addgene_62656) |
| 参考文献部分: | The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli. Reisch CR, Prather KL. Sci Rep. 2015 Oct 14;5:15096. doi: 10.1038/srep15096. 10.1038/srep15096 PubMed 26463009 |
买家导航
pKDsgRNAp15载体基本信息
pKDsgRNAp15载体图谱质粒图谱和多克隆位点信息
pKDsgRNAp15载体简介
pKDsgRNAp15载体序列
LOCUS pKDsgRNA-p15 6928 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Arabinose inducible lambda red and anhydrotetracycline inducible
sgRNA expression that targets pCas9 plasmid.
ACCESSION .
VERSION .
KEYWORDS pKDsgRNA-p15
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6928)
AUTHORS Reisch CR, Prather KL
TITLE The no-SCAR (Scarless Cas9 Assisted Recombineering) system for
genome editing in Escherichia coli.
JOURNAL Sci Rep. 2015 Oct 14;5:15096. doi: 10.1038/srep15096.
PUBMED 26463009
REFERENCE 2 (bases 1 to 6928)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6928
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(11..889)
/codon_start=1
/gene="araC"
/product="L-arabinose regulatory protein"
/label=araC
/translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
EKVNDVAVKLS"
promoter 916..1200
/gene="araBAD"
/label=araBAD promoter
/note="promoter of the L-arabinose operon of E. coli; the
araC regulatory gene is transcribed in the opposite
direction (Guzman et al., 1995)"
primer_bind 1123..1142
/label=pBAD Forward
/note="For vectors with E. coli araBAD promoter, forward
primer"
RBS 1227..1235
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
CDS 1244..1660
/codon_start=1
/gene="gam"
/product="inhibitor of the host RecBCD nuclease in the
lambda Red system"
/label=Gam
/note="protects the double-stranded DNA substrate for
recombination"
/translation="MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNAYY
IQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKSITR
AFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV"
CDS 1666..2451
/codon_start=1
/gene="bet"
/product="single-stranded DNA binding recombinase in the
lambda Red system"
/label=Beta
/note="mediates pairing of a single-stranded DNA with a
complementary target"
/translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI
ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC
TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA
FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP
LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA"
CDS 2448..3128
/codon_start=1
/gene="exo"
/product="5' to 3' double-stranded DNA exonuclease in the
lambda Red system"
/label=Exo
/note="creates 3' single-stranded DNA tails"
/translation="MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPR
SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
EQWR"
terminator 3129..3373
/label=-lambda tL3 terminator
/note="transcription terminator tL3 from phage lambda"
CDS complement(3496..4446)
/codon_start=1
/gene="rep101 encoding an A56V mutation"
/product="temperature-sensitive version of the RepA protein
needed for replication with the pSC101 origin (Armstrong et
al., 1984)"
/label=Rep101(Ts)
/translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
rep_origin 4494..4716
/label=pSC101 ori
/note="low-copy replication origin that requires the Rep101
protein"
protein_bind 5282..5300
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 5307..5325
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
misc_RNA 5356..5431
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
CDS 5452..5481
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
CDS 5497..5514
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(5570..5587)
/label=pBAD Reverse
/note="For vectors with E. coli araBAD promoter, reverse
primer"
terminator 5740..5786
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
CDS complement(5925..6716)
/codon_start=1
/gene="aadA"
/product="aminoglycoside adenylyltransferase (Murphy,
1985)"
/label=SmR
/note="confers resistance to spectinomycin and
streptomycin"
/translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
promoter complement(6717..6808)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 catcgattta ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac
61 ggaactcgct cgggctggcc ccggtgcatt ttttaaatac ccgcgagaag tagagttgat
121 cgtcaaaacc aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca
181 gcttcgcctg gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct
241 ggcggaaaag atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga
301 tatcaaaatt gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat
361 tatccatcgg tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct
421 caagcagatt tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga
481 tttgcccaaa caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg
541 tattggcaaa tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt
601 aaacccactg gtgataccat tcgcgagcct ccggatgacg accgtagtga tgaatctctc
661 ctggcgggaa cagcaaaata tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca
721 ccaccccctg accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt
781 cgataaaaaa atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg
841 cattaaacga gtatcccggc agcaggggat cattttgcgc ttcagccata cttttcatac
901 tcccgccatt cagagaagaa accaattgtc catattgcat cagacattgc cgtcactgcg
961 tcttttactg gctcttctcg ctaaccaaac cggtaacccc gcttattaaa agcattctgt
1021 aacaaagcgg gaccaaagcc atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca
1081 gaaaagtcca cattgattat ttgcacggcg tcacactttg ctatgccata gcatttttat
1141 ccataagatt agcggatcct acctgacgct ttttatcgca actctctact gtttctccat
1201 acccgttttt ttgggaattc gagctctaag gaggttataa aaaatggata ttaatactga
1261 aactgagatc aagcaaaagc attcactaac cccctttcct gttttcctaa tcagcccggc
1321 atttcgcggg cgatattttc acagctattt caggagttca gccatgaacg cttattacat
1381 tcaggatcgt cttgaggctc agagctgggc gcgtcactac cagcagctcg cccgtgaaga
1441 gaaagaggca gaactggcag acgacatgga aaaaggcctg ccccagcacc tgtttgaatc
1501 gctatgcatc gatcatttgc aacgccacgg ggccagcaaa aaatccatta cccgtgcgtt
1561 tgatgacgat gttgagtttc aggagcgcat ggcagaacac atccggtaca tggttgaaac
1621 cattgctcac caccaggttg atattgattc agaggtataa aacgaatgag tactgcactc
1681 gcaacgctgg ctgggaagct ggctgaacgt gtcggcatgg attctgtcga cccacaggaa
1741 ctgatcacca ctcttcgcca gacggcattt aaaggtgatg ccagcgatgc gcagttcatc
1801 gcattactga tcgttgccaa ccagtacggc cttaatccgt ggacgaaaga aatttacgcc
1861 tttcctgata agcagaatgg catcgttccg gtggtgggcg ttgatggctg gtcccgcatc
1921 atcaatgaaa accagcagtt tgatggcatg gactttgagc aggacaatga atcctgtaca
1981 tgccggattt accgcaagga ccgtaatcat ccgatctgcg ttaccgaatg gatggatgaa
2041 tgccgccgcg aaccattcaa aactcgcgaa ggcagagaaa tcacggggcc gtggcagtcg
2101 catcccaaac ggatgttacg tcataaagcc atgattcagt gtgcccgtct ggccttcgga
2161 tttgctggta tctatgacaa ggatgaagcc gagcgcattg tcgaaaatac tgcatacact
2221 gcagaacgtc agccggaacg cgacatcact ccggttaacg atgaaaccat gcaggagatt
2281 aacactctgc tgatcgccct ggataaaaca tgggatgacg acttattgcc gctctgttcc
2341 cagatatttc gccgcgacat tcgtgcatcg tcagaactga cacaggccga agcagtaaaa
2401 gctcttggat tcctgaaaca gaaagccgca gagcagaagg tggcagcatg acaccggaca
2461 ttatcctgca gcgtaccggg atcgatgtga gagctgtcga acagggggat gatgcgtggc
2521 acaaattacg gctcggcgtc atcaccgctt cagaagttca caacgtgata gcaaaacccc
2581 gctccggaaa gaagtggcct gacatgaaaa tgtcctactt ccacaccctg cttgctgagg
2641 tttgcaccgg tgtggctccg gaagttaacg ctaaagcact ggcctgggga aaacagtacg
2701 agaacgacgc cagaaccctg tttgaattca cttccggcgt gaatgttact gaatccccga
2761 tcatctatcg cgacgaaagt atgcgtaccg cctgctctcc cgatggttta tgcagtgacg
2821 gcaacggcct tgaactgaaa tgcccgttta cctcccggga tttcatgaag ttccggctcg
2881 gtggtttcga ggccataaag tcagcttaca tggcccaggt gcagtacagc atgtgggtga
2941 cgcgaaaaaa tgcctggtac tttgccaact atgacccgcg tatgaagcgt gaaggcctgc
3001 attatgtcgt gattgagcgg gatgaaaagt acatggcgag ttttgacgag atcgtgccgg
3061 agttcatcga aaaaatggac gaggcactgg ctgaaattgg ttttgtattt ggggagcaat
3121 ggcgatgacg catcctcacg ataatatccg ggtaggcgca atcactttcg tctactccgt
3181 tacaaagcga ggctgggtat ttcccggcct ttctgttatc cgaaatccac tgaaagcaca
3241 gcggctggct gaggagataa ataataaacg aggggctgta tgcacaaagc atcttctgtt
3301 gagttaagaa cgagtatcga tatggcacat agccttgctc aaattggaat caggtttgtg
3361 ccaataccag tagaaacaga cgaagaatcc atgggtatgg acagttttcc ctttgatatg
3421 taacggtgaa cagttgttct acttttgttt gttagtcttg atgcttcact gatagataca
3481 agagccataa gaacctcaga tccttccgta tttagccagt atgttctcta gtgtggttcg
3541 ttgtttttgc gtgagccatg agaacgaacc attgagatca tacttacttt gcatgtcact
3601 caaaaatttt gcctcaaaac tggtgagctg aatttttgca gttaaagcat cgtgtagtgt
3661 ttttcttagt ccgttatgta ggtaggaatc tgatgtaatg gttgttggta ttttgtcacc
3721 attcattttt atctggttgt tctcaagttc ggttacgaga tccatttgtc tatctagttc
3781 aacttggaaa atcaacgtat cagtcgggcg gcctcgctta tcaaccacca atttcatatt
3841 gctgtaagtg tttaaatctt tacttattgg tttcaaaacc cattggttaa gccttttaaa
3901 ctcatggtag ttattttcaa gcattaacat gaacttaaat tcatcaaggc taatctctat
3961 atttgccttg tgagttttct tttgtgttag ttcttttaat aaccactcat aaatcctcat
4021 agagtatttg ttttcaaaag acttaacatg ttccagatta tattttatga atttttttaa
4081 ctggaaaaga taaggcaata tctcttcact aaaaactaat tctaattttt cgcttgagaa
4141 cttggcatag tttgtccact ggaaaatctc aaagccttta accaaaggat tcctgatttc
4201 cacagttctc gtcatcagct ctctggttgc tttagctaat acaccataag cattttccct
4261 actgatgttc atcatctgaa cgtattggtt ataagtgaac gataccgtcc gttctttcct
4321 tgtagggttt tcaatcgtgg ggttgagtag tgccacacag cataaaatta gcttggtttc
4381 atgctccgtt aagtcatagc gactaatcgc tagttcattt gctttgaaaa caactaattc
4441 agacatacat ctcaattggt ctaggtgatt ttaatcacta taccaattga gatgggctag
4501 tcaatgataa ttactagtcc ttttcctttg agttgtgggt atctgtaaat tctgctagac
4561 ctttgctgga aaacttgtaa attctgctag accctctgta aattccgcta gacctttgtg
4621 tgtttttttt gtttatattc aagtggttat aatttataga ataaagaaag aataaaaaaa
4681 gataaaaaga atagatccca gccctgtgta taactcacta ctttagtcag ttccgcagta
4741 ttacaaaagg atgtcgcaaa cgctgtttgc tcctctacaa aacagacctt aaaaccctaa
4801 aggcttaagt agcaccctcg caagctcggt tgcggccgca atcgggcaaa tcgctgaata
4861 ttccttttgt ctccgaccat caggcacctg agtcgctgtc tttttcgtga cattcagttc
4921 gctgcgctca cggctctggc agtgaatggg ggtaaatggc actacaggcg ccttttatgg
4981 attcatgcaa ggaaactacc cataatacaa gaaaagcccg tcacgggctt ctcagggcgt
5041 tttatggcgg gtctgctatg tggtgctatc tgactttttg ctgttcagca gttcctgccc
5101 tctgattttc cagtctgacc acttcggatt atcccgtgac aggtcattca gactggctaa
5161 tgcacccagt aaggcagcgg tatcatcaac ggggtctgac gctcagtgga acgaaaactc
5221 acgttaaggg attttggtca tgagattatc agctttcgct aaggatgatt tctggaattc
5281 ttccctatca gtgatagaga ttgacatccc tatcagtgat agagatactg agcacctatc
5341 gtcttgagtc caaccgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
5401 tcaacttgaa aaagtggcac cgagtcggtg ctttttttga agcttgggcc cgaacaaaaa
5461 ctcatctcag aagaggatct gaatagcgcc gtcgaccatc atcatcatca tcattgagtt
5521 taaacggtct ccagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga
5581 ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg
5641 tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg
5701 tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag
5761 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaactggat ccttactcga
5821 gtctagactg caggcggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
5881 caatctaaag tatatatgag taaacttggt ctgctgacag gacattattt gccgactacc
5941 ttggtgatct cgcctttcac gtagtggaca aattcttcca actgatctgc gcgcgaggcc
6001 aagcgatctt cttcttgtcc aagataagcc tgtctagctt caagtatgac gggctgatac
6061 tgggccggca ggcgctccat tgcccagtcg gcagcgacat ccttcggcgc gattttgccg
6121 gttactgcgc tgtaccaaat gcgggacaac gtaagcacta catttcgctc atcgccagcc
6181 cagtcgggcg gcgagttcca tagcgttaag gtttcattta gcgcctcaaa tagatcctgt
6241 tcaggaaccg gatcaaagag ttcctccgcc gctggaccta ccaaggcaac gctatgttct
6301 cttgcttttg tcagcaagat agccagatca atgtcgatcg tggctggctc gaagatacct
6361 gcaagaatgt cattgcgctg ccattctcca aattgcagtt cgcgcttagc tggataacgc
6421 cacggaatga tgtcgtcgtg cacaacaatg gtgacttcta cagcgcggag aatctcgctc
6481 tctccagggg aagccgaagt ttccaaaagg tcgttgatca aagctcgccg cgttgtttca
6541 tcaagcctta cggtcaccgt aaccagcaaa tcaatatcac tgtgtggctt caggccgcca
6601 tccactgcgg agccgtacaa atgtacggcc agcaacgtcg gttcgagatg gcgctcgatg
6661 acgccaacta cctctgatag ttgagtcgat acttcggcga tcaccgcttc cctcatactc
6721 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
6781 tttgaatgta tttagaaaaa taaacaaata gctagctcac tcggtcgcta ccagggttat
6841 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
6901 cgcacatttc cccgaaaagt gccacctg
//
