| 出品公司: | 爱迪基因 |
|---|---|
| 载体名称: | pgRNA-humanized |
| 质粒类型: | CRISPR/Cas9载体；哺乳动物表达载体；慢病毒载体；双启动子载体 |
| 高拷贝/低拷贝: | 高拷贝 |
| 插入位点: | 限制性内切酶，多克隆位点 |
| 启动子: | U6 |
| 载体大小: | 8308 bp |
| 5' 测序引物及序列: | -- |
| 3' 测序引物及序列: | -- |
| 载体标签: | -- |
| 载体抗性: | 氨苄青霉素 |
| 筛选标记: | puromycin、mCherry |
| 克隆菌株: | NEB Stable |
| 宿主细胞（系）: | 哺乳动物 |
| 备注: | |
| 产品目录号: | #44248 |
| 稳定性: | 稳表达 |
| 组成型/诱导型: | 组成型 |
| 病毒/非病毒: | 慢病毒 |
LOCUS Exported 8308 bp ds-DNA circular SYN 06-JAN-2016
DEFINITION Lentiviral vector for expressing a guide RNA (gRNA) together with a
puromycin resistance marker and mCherry.
ACCESSION .
VERSION .
KEYWORDS pgRNA-humanized
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8308)
AUTHORS Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim
WA.
TITLE Repurposing CRISPR as an RNA-guided platform for sequence-specific
control of gene expression.
JOURNAL Cell 2013;152:1173-83.
PUBMED 23452860
REFERENCE 2 (bases 1 to 8308)
AUTHORS Qi Lab / Addgene #44248
TITLE Direct Submission
JOURNAL Exported Wednesday, July 27, 2016 from SnapGene Viewer 3.1.4
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8308
/organism="synthetic DNA construct"
/lab_host="Mammalian Cells"
/mol_type="other DNA"
protein_bind 209..242
/bound_moiety="Cre recombinase"
/note="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
promoter 243..327
/note="U6 promoter"
/note="RNA polymerase III promoter for mouse U6 snRNA"
misc_RNA 349..424
/note="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
terminator 425..430
/note="polIII terminator"
/note="RNA polymerase III transcription terminator"
enhancer 549..852
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 853..1056
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 1090..1686
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/note="PuroR"
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
CDS 1696..1749
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/note="T2A"
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 1762..2472
/codon_start=1
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/note="mCherry"
/note="mammalian codon-optimized"
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK"
protein_bind 2492..2525
/bound_moiety="Cre recombinase"
/note="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_feature 2581..3169
/note="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 3698..3878
/note="LTR (truncated)"
/note="truncated long terminal repeat (LTR) from HIV-1"
rep_origin complement(3940..4528)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4699..5559)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(5560..5664)
/gene="bla"
/note="AmpR promoter"
LTR 6536..6716
/note="LTR (truncated)"
/note="truncated long terminal repeat (LTR) from HIV-1"
misc_feature 6763..6888
/note="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 7387..7620
/note="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 8147..8264
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
ORIGIN
1 gggcccgctc tagagatccg acgcgccatc tctaggcccg cgccggcccc ctcgcacaga
61 cttgtgggag aagctcggct actcccctgc cccggttaat ttgcatataa tatttcctag
121 taactataga ggcttaatgt gcgataaaag acagataatc tgttcttttt aatactagct
181 acattttaca tgataggctt ggatttctat aacttcgtat agcatacatt atacgaagtt
241 ataaacagca caaaaggaaa ctcaccctaa ctgtaaagta attgtgtgtt ttgagactat
301 aagtatccct tggagaacca ccttgttgga ccaggatggg caccacccgt tttagagcta
361 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
421 gtgctttttt tctcgagtac taggatccat taggcggccg cgtggataac cgtattaccg
481 ccatgcatta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
541 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
601 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
661 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
721 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
781 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
841 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
901 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
961 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
1021 aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc
1081 tagcccacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
1141 cgggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
1201 gacccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
1261 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
1321 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
1381 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
1441 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
1501 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
1561 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
1621 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
1681 ggtgccggat cgggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat
1741 cctggcccac cggtcgccac catggtgagc aagggcgagg aggataacat ggccatcatc
1801 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag
1861 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg
1921 accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc
1981 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc
2041 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
2101 caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac
2161 ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag
2221 cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag
2281 gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag
2341 ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac
2401 accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag
2461 ctgtacaagt aagaattcgt cgagggacct aataacttcg tatagcatac attatacgaa
2521 gttatacatg tttaagggtt ccggttccac taggtacaat tcgatatcaa gcttatcgat
2581 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
2641 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
2701 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
2761 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
2821 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
2881 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
2941 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
3001 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
3061 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
3121 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt
3181 cgacctcgat cgagacctag aaaaacatgg agcaatcaca agtagcaata cagcagctac
3241 caatgctgat tgtgcctggc tagaagcaca agaggaggag gaggtgggtt ttccagtcac
3301 acctcaggta cctttaagac caatgactta caaggcagct gtagatctta gccacttttt
3361 aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag atatccttga
3421 tctgtggatc taccacacac aaggctactt ccctgattgg cagaactaca caccagggcc
3481 agggatcaga tatccactga cctttggatg gtgctacaag ctagtaccag ttgagcaaga
3541 gaaggtagaa gaagccaatg aaggagagaa cacccgcttg ttacaccctg tgagcctgca
3601 tgggatggat gacccggaga gagaagtatt agagtggagg tttgacagcc gcctagcatt
3661 tcatcacatg gcccgagagc tgcatccgga ctgtactggg tctctctggt tagaccagat
3721 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
3781 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
3841 cctcagaccc ttttagtcag tgtggaaaat ctctagcagc atgtgagcaa aaggccagca
3901 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
3961 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
4021 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
4081 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
4141 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
4201 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
4261 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
4321 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
4381 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
4441 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
4501 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
4561 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
4621 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
4681 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
4741 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
4801 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
4861 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
4921 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
4981 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
5041 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
5101 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
5161 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
5221 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
5281 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
5341 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
5401 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
5461 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
5521 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
5581 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
5641 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgcttaagc
5701 ggtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
5761 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
5821 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
5881 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
5941 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
6001 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
6061 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
6121 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
6181 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
6241 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
6301 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
6361 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
6421 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
6481 ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg cgttttgcct gtactgggtc
6541 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
6601 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
6661 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
6721 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
6781 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
6841 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
6901 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
6961 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
7021 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
7081 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
7141 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
7201 aaaagtaaga ccaccgcaca gcaagcggcc ggccgcgctg atcttcagac ctggaggagg
7261 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
7321 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
7381 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
7441 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
7501 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
7561 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
7621 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
7681 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
7741 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
7801 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
7861 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
7921 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
7981 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat
8041 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatcggc
8101 actgcgtgcg ccaattctgc agacaaatgg cagtattcat ccacaatttt aaaagaaaag
8161 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
8221 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
8281 acagcagaga tccagtttgg ttagtacc
//
