LOCUS pCMV-2NLS-Cas9-6XMS2 8217 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expression of 6X MS2 stem loops fused SpCas9 mRNA for packaging of
SpCas9 mRNA within lentiviral particles. .
ACCESSION .
VERSION .
KEYWORDS pCMV-2NLS-Cas9-6XMS2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8217)
AUTHORS Ling S, Yang S, Hu X, Yin D, Dai Y, Qian X, Wang D, Pan X, Hong J,
Sun X, Yang H, Paludan SR, Cai Y
TITLE Lentiviral delivery of co-packaged Cas9 mRNA and a Vegfa-targeting
guide RNA prevents wet age-related macular degeneration in mice.
JOURNAL Nat Biomed Eng. 2021 Jan 4. pii: 10.1038/s41551-020-00656-y. doi:
10.1038/s41551-020-00656-y.
PUBMED 33398131
REFERENCE 2 (bases 1 to 8217)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8217
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 151..170
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 364..386
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 378..395
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 379..395
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
enhancer 422..725
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 726..929
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 880..900
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 926..950
/label=LNCX
/note="Human CMV promoter, forward primer"
intron 1067..1163
/label=SV40 intron
/note="modified SV40 intron with splice donor and acceptor
sites"
regulatory 1223..1232
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1235..1255
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1280..5380
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 5381..5428
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
misc_RNA 5450..5468
/label=MS2 stem loop
/note="stem loop that binds the bacteriophage MS2 coat
protein"
misc_RNA 5489..5507
/label=MS2 stem loop
/note="stem loop that binds the bacteriophage MS2 coat
protein"
misc_RNA 5559..5577
/label=MS2 stem loop
/note="stem loop that binds the bacteriophage MS2 coat
protein"
misc_RNA 5598..5616
/label=MS2 stem loop
/note="stem loop that binds the bacteriophage MS2 coat
protein"
misc_RNA 5668..5686
/label=MS2 stem loop
/note="stem loop that binds the bacteriophage MS2 coat
protein"
misc_RNA 5707..5725
/label=MS2 stem loop
/note="stem loop that binds the bacteriophage MS2 coat
protein"
polyA_signal 5751..5885
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(5775..5794)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 5829..5848
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind complement(5996..6012)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5996..6012)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(6009..6031)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 6020..6036
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(6044..6074)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6089..6110
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(6227..6244)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(6398..6986)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(6478..6497)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(7157..8017)
/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 7780..7799
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(8018..8122)
/gene="bla"
/label=AmpR promoter
primer_bind 8190..8208
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
ORIGIN
1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctccca gagacggtca
61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagcttgca tgcctgcagg
421 tcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
481 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
721 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
781 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
841 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
901 tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac
961 gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccggactc
1021 tagaggatcc ggtactcgag gaactgaaaa accagaaagt taactggtaa gtttagtctt
1081 tttgtctttt atttcaggtc ccggatccgg tggtggtgca aatcaaagaa ctgctcctca
1141 gtggatgttg cctttacttc taggcctgta cggaagtgtt acttctgctc taaaagctgc
1201 ggaattgtac ccgcggacgc gtgccaccat ggccccaaag aagaagcgga aggtcggtat
1261 ccacggagtc ccagcagccg acaagaagta cagcatcggc ctggacatcg gcaccaactc
1321 tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct
1381 gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag
1441 cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg
1501 gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga
1561 cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg
1621 gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat
1681 ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta
1741 tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa
1801 ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct
1861 gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag
1921 actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa
1981 tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa
2041 cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct
2101 ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa
2161 cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc
2221 ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct
2281 gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag
2341 caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt
2401 catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag
2461 agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca
2521 cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga
2581 caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct
2641 ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc
2701 ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat
2761 gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta
2821 cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag
2881 aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac
2941 caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt
3001 cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca
3061 cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat
3121 tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg
3181 gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag
3241 atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc
3301 cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca
3361 gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg
3421 ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa
3481 gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa
3541 gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa
3601 gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat
3661 cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta
3721 cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga
3781 ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa
3841 ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt
3901 cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag
3961 aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg
4021 cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct
4081 ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt
4141 gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt
4201 gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac
4261 cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt
4321 gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa
4381 gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg
4441 cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga
4501 taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt
4561 gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa
4621 cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga
4681 cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa
4741 gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga
4801 gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat
4861 catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc
4921 ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt
4981 cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa
5041 acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga
5101 gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa
5161 caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct
5221 gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag
5281 gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct
5341 gtacgagaca cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa
5401 aaaggccggc caggcaaaaa agaaaaagta aaccggtctt gcagcacata catgaggatc
5461 acccatgtct gcaggtcgac tctagaaaac atgaggatca cccatgtctg cagtattccc
5521 gggttcatta gatcctaagg tacctaattg cctagaaaac atgaggatca cccatgtctg
5581 caggtcgact ctagaaaaca tgaggatcac ccatgtctgc agtattcccg ggttcattag
5641 atcctaaggt acctaattgc ctagaaaaca tgaggatcac ccatgtctgc aggtcgactc
5701 tagaaaacat gaggatcacc catgtgtgca ccttaagctt ggcccgcggg gatccagaca
5761 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
5821 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
5881 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg
5941 ttttttcgga tcctctagag tcgacctgca ggcatgcaag cttggcgtaa tcatggtcat
6001 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
6061 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
6121 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
6181 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
6241 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
6301 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
6361 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
6421 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
6481 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
6541 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
6601 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
6661 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
6721 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
6781 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
6841 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
6901 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
6961 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
7021 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
7081 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
7141 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
7201 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
7261 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
7321 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
7381 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
7441 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
7501 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
7561 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
7621 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
7681 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
7741 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
7801 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
7861 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
7921 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
7981 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
8041 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
8101 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
8161 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtc
//
