| 质粒名称: | pGL3-U6-sgRNA-PGK-puromycin |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 51133 |
| 存储实验室: | Xingxu Huang |
|
载体骨架基本信息 |
|
| 载体名称: | pGL3-Basic |
| 载体出品公司: | Promega |
| 载体抗性: | 氨苄霉素 |
| 载体类型: | 哺乳细胞表达，CRISPR |
| 筛选标记: | 嘌呤霉素 |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 启动子 | U6 |
| 克隆方法: | 限制性内切酶 |
| 5' 测序引物 | pLKO.inducible |
| 3' 测序引物 | O.PGK1b-R |
|
文献引用方法 |
|
| 材料和方法部分: | pGL3-U6-sgRNA-PGK-puromycin was a gift from Xingxu Huang (爱迪基因 plasmid # 51133) |
| 参考文献部分: | Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins A, Iyer V, Huang X, Skarnes WC. Nat Methods. 2014 Mar 2. doi: 10.1038/nmeth.2857. 10.1038/nmeth.2857 PubMed 24584192 |
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 pGL3-U6-sgRNA-PGK-puromycin 4952 bp ds-DNA circular SYN 10-JAN-2018
DEFINITION expresses sgRNA from the U6 promoter..
ACCESSION .
VERSION .
KEYWORDS pGL3-U6-sgRNA-PGK-puromycin
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4952)
AUTHORS Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins
A, Iyer V, Huang X, Skarnes WC
TITLE Efficient genome modification by CRISPR-Cas9 nickase with minimal
off-target effects.
JOURNAL Nat Methods. 2014 Mar 2. doi: 10.1038/nmeth.2857.
PUBMED 24584192
COMMENT This file is created by Vector NTI
http://www.biofeng.com/
COMMENT ORIGDB|GenBank
COMMENT VNTAUTHORNAME|biofeng.com|
COMMENT VNTNAME|pGL3-U6-sgRNA-PGK-puromycin|
FEATURES Location/Qualifiers
source 1..4952
/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_RNA 344..419
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
misc_feature 471..588
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 637..1147
/label=hPGK promoter
/note="human phosphoglycerate kinase 1 promoter"
primer_bind 1066..1084
/label=hPGK-F
/note="Human PGK promoter, forward primer"
CDS 1169..1768
/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(1169..1188)
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
primer_bind 1665..1685
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
polyA_signal 1915..2036
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(1926..1945)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 1980..1999
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind complement(2284..2301)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(2455..3043)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(2535..2554)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(3214..4074)
/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 3837..3856
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(4075..4179)
/gene="bla"
/label=AmpR promoter
rep_origin 4206..4661
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4293..4312)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4503..4524
/label=F1ori-F
/note="F1 origin, forward primer"
polyA_signal 4792..4840
/note="synthetic polyadenylation signal"
misc_feature 4854..4945
/label=pause site
/note="RNA polymerase II transcriptional pause signal from
the human alpha-2 globin gene"
primer_bind 4894..4913
/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 ggtgagaccg agagagggtc tcagttttag agctagaaat
361 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
421 ttttttaaag aattctcgac ctcgagacaa atggcagtat tcatccacaa ttttaaaaga
481 aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac
541 atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac
601 agggacagca gagatccact ttggccgcgg ctcgaggggg ttggggttgc gccttttcca
661 aggcagccct gggtttgcgc agggacgcgg ctgctctggg cgtggttccg ggaaacgcag
721 cggcgccgac cctgggactc gcacattctt cacgtccgtt cgcagcgtca cccggatctt
781 cgccgctacc cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg
841 ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc tcactagtac
901 cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc
961 caatagcggc tgctcagcag ggcgcgccga gagcagcggc cgggaagggg cggtgcggga
1021 ggcggggtgt ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg
1081 caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc
1141 tccccagggg gatccaccgg agcttaccat gaccgagtac aagcccacgg tgcgcctcgc
1201 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
1261 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga
1321 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
1381 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
1441 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
1501 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc
1561 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
1621 cgagcgcgcc ggggtgcccg ccttcctgga aacctccgcg ccccgcaacc tccccttcta
1681 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
1741 gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga
1801 aaggagcgca cgaccccatg catcggtacc tttaagacca atgacttaca aggcagctgt
1861 agatcttagc cactttctag agtcggggcg gccggccgct tcgagcagac atgataagat
1921 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
1981 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
2041 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
2101 gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga tccgtcgacc gatgcccttg
2161 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca
2221 cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctcttccgc
2281 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
2341 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
2401 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
2461 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
2521 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
2581 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
2641 gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
2701 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
2761 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
2821 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
2881 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
2941 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
3001 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
3061 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
3121 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
3181 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
3241 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
3301 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgggaccc
3361 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
3421 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
3481 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
3541 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
3601 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
3661 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
3721 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
3781 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
3841 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
3901 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
3961 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
4021 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
4081 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
4141 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
4201 acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
4261 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
4321 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
4381 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
4441 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
4501 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
4561 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
4621 atttaacgcg aattttaaca aaatattaac gtttacaatt tcccattcgc cattcaggct
4681 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agcccaagct
4741 accatgataa gtaagtaata ttaaggtacg ggaggtactt ggagcggccg caataaaata
4801 tctttatttt cattacatct gtgtgttggt tttttgtgtg aatcgatagt actaacatac
4861 gctctccatc aaaacaaaac gaaacaaaac aaactagcaa aataggctgt ccccagtgca
4921 agtgcaggtg ccagaacatt tctctatcga ta
//
