LOCUS       sequence-197781-        7246 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SaCas9 and a gRNA targeting mouse YAP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7246)
  AUTHORS   Wang L, Luo JY, Li B, Tian XY, Chen LJ, Huang Y, Liu J, Deng D, Lau 
            CW, Wan S, Ai D, Mak KK, Tong KK, Kwan KM, Wang N, Chiu JJ, Zhu Y, 
            Huang Y
  TITLE     Integrin-YAP/TAZ-JNK cascade mediates atheroprotective effect of 
            unidirectional shear flow.
  JOURNAL   Nature. 2016 Dec 7. doi: 10.1038/nature20602.
  PUBMED    27926730
REFERENCE   2  (bases 1 to 7246)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2016 Dec 7. doi: 10.1038/nature20602."
COMMENT     Sequence Label: Icam 2 SaCas9 YAP sgRNA3
FEATURES             Location/Qualifiers
     source          1..7246
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   3..132
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      556..565
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             568..588
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             613..3768
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             3769..3816
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3823..3903
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(3931..3948)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3937..4144
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4151..4399
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4151..4171
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4322..4341
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4422..4497
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4511..4651
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4511..4640
                     /label=AAV2 ITR
     rep_origin      4726..5181
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4813..4832)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5023..5044
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5198..5217)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5317..5339
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5377..5395)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5463..5567
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5568..5636,5637..6428)
                     /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     complement(5786..5805)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6599..7187
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7088..7107
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtcctgcagg cagctgcgcg ctcgctcgct cactgaggcc gcccgggcgt cgggcgacct
       61 ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca
      121 ctaggggttc ctgcggcctc tagactcgag gtctcccagg catgactcca acaatgcatc
      181 ccatgggatt tggggttccc cagatctggg gcttgtaggc ctgactctcc cctgtgcaca
      241 cgtctcatac acgcatgcgt gcacccattg cctgccccgc cccttgcaca gggagtcagc
      301 agggaggact gggttatgcc ctgcttatca gcagcttccc agcttcctct gcctggattc
      361 ttagaggcct ggggtcctag aacgagctgg tgcacgtggc ttcccaaaga tctctcagat
      421 aatgagagga aatgcagtca tcagtttgca gaaggctagg gattctgggc catagctcag
      481 acctgcgccc accatctccc tccaggcagc ccttggctgg tccctgcgag cccgtggaga
      541 ctgccagtca ccggtgccac catggcccca aagaagaagc ggaaggtcgg tatccacgga
      601 gtcccagcag ccaagcggaa ctacatcctg ggcctggaca tcggcatcac cagcgtgggc
      661 tacggcatca tcgactacga gacacgggac gtgatcgatg ccggcgtgcg gctgttcaaa
      721 gaggccaacg tggaaaacaa cgagggcagg cggagcaaga gaggcgccag aaggctgaag
      781 cggcggaggc ggcatagaat ccagagagtg aagaagctgc tgttcgacta caacctgctg
      841 accgaccaca gcgagctgag cggcatcaac ccctacgagg ccagagtgaa gggcctgagc
      901 cagaagctga gcgaggaaga gttctctgcc gccctgctgc acctggccaa gagaagaggc
      961 gtgcacaacg tgaacgaggt ggaagaggac accggcaacg agctgtccac caaagagcag
     1021 atcagccgga acagcaaggc cctggaagag aaatacgtgg ccgaactgca gctggaacgg
     1081 ctgaagaaag acggcgaagt gcggggcagc atcaacagat tcaagaccag cgactacgtg
     1141 aaagaagcca aacagctgct gaaggtgcag aaggcctacc accagctgga ccagagcttc
     1201 atcgacacct acatcgacct gctggaaacc cggcggacct actatgaggg acctggcgag
     1261 ggcagcccct tcggctggaa ggacatcaaa gaatggtacg agatgctgat gggccactgc
     1321 acctacttcc ccgaggaact gcggagcgtg aagtacgcct acaacgccga cctgtacaac
     1381 gccctgaacg acctgaacaa tctcgtgatc accagggacg agaacgagaa gctggaatat
     1441 tacgagaagt tccagatcat cgagaacgtg ttcaagcaga agaagaagcc caccctgaag
     1501 cagatcgcca aagaaatcct cgtgaacgaa gaggatatta agggctacag agtgaccagc
     1561 accggcaagc ccgagttcac caacctgaag gtgtaccacg acatcaagga cattaccgcc
     1621 cggaaagaga ttattgagaa cgccgagctg ctggatcaga ttgccaagat cctgaccatc
     1681 taccagagca gcgaggacat ccaggaagaa ctgaccaatc tgaactccga gctgacccag
     1741 gaagagatcg agcagatctc taatctgaag ggctataccg gcacccacaa cctgagcctg
     1801 aaggccatca acctgatcct ggacgagctg tggcacacca acgacaacca gatcgctatc
     1861 ttcaaccggc tgaagctggt gcccaagaag gtggacctgt cccagcagaa agagatcccc
     1921 accaccctgg tggacgactt catcctgagc cccgtcgtga agagaagctt catccagagc
     1981 atcaaagtga tcaacgccat catcaagaag tacggcctgc ccaacgacat cattatcgag
     2041 ctggcccgcg agaagaactc caaggacgcc cagaaaatga tcaacgagat gcagaagcgg
     2101 aaccggcaga ccaacgagcg gatcgaggaa atcatccgga ccaccggcaa agagaacgcc
     2161 aagtacctga tcgagaagat caagctgcac gacatgcagg aaggcaagtg cctgtacagc
     2221 ctggaagcca tccctctgga agatctgctg aacaacccct tcaactatga ggtggaccac
     2281 atcatcccca gaagcgtgtc cttcgacaac agcttcaaca acaaggtgct cgtgaagcag
     2341 gaagaaaaca gcaagaaggg caaccggacc ccattccagt acctgagcag cagcgacagc
     2401 aagatcagct acgaaacctt caagaagcac atcctgaatc tggccaaggg caagggcaga
     2461 atcagcaaga ccaagaaaga gtatctgctg gaagaacggg acatcaacag gttctccgtg
     2521 cagaaagact tcatcaaccg gaacctggtg gataccagat acgccaccag aggcctgatg
     2581 aacctgctgc ggagctactt cagagtgaac aacctggacg tgaaagtgaa gtccatcaat
     2641 ggcggcttca ccagctttct gcggcggaag tggaagttta agaaagagcg gaacaagggg
     2701 tacaagcacc acgccgagga cgccctgatc attgccaacg ccgatttcat cttcaaagag
     2761 tggaagaaac tggacaaggc caaaaaagtg atggaaaacc agatgttcga ggaaaagcag
     2821 gccgagagca tgcccgagat cgaaaccgag caggagtaca aagagatctt catcaccccc
     2881 caccagatca agcacattaa ggacttcaag gactacaagt acagccaccg ggtggacaag
     2941 aagcctaata gagagctgat taacgacacc ctgtactcca cccggaagga cgacaagggc
     3001 aacaccctga tcgtgaacaa tctgaacggc ctgtacgaca aggacaatga caagctgaaa
     3061 aagctgatca acaagagccc cgaaaagctg ctgatgtacc accacgaccc ccagacctac
     3121 cagaaactga agctgattat ggaacagtac ggcgacgaga agaatcccct gtacaagtac
     3181 tacgaggaaa ccgggaacta cctgaccaag tactccaaaa aggacaacgg ccccgtgatc
     3241 aagaagatta agtattacgg caacaaactg aacgcccatc tggacatcac cgacgactac
     3301 cccaacagca gaaacaaggt cgtgaagctg tccctgaagc cctacagatt cgacgtgtac
     3361 ctggacaatg gcgtgtacaa gttcgtgacc gtgaagaatc tggatgtgat caaaaaagaa
     3421 aactactacg aagtgaatag caagtgctat gaggaagcta agaagctgaa gaagatcagc
     3481 aaccaggccg agtttatcgc ctccttctac aacaacgatc tgatcaagat caacggcgag
     3541 ctgtatagag tgatcggcgt gaacaacgac ctgctgaacc ggatcgaagt gaacatgatc
     3601 gacatcacct accgcgagta cctggaaaac atgaacgaca agaggccccc caggatcatt
     3661 aagacaatcg cctccaagac ccagagcatt aagaagtaca gcacagacat tctgggcaac
     3721 ctgtatgaag tgaaatctaa gaagcaccct cagatcatca aaaagggcaa aaggccggcg
     3781 gccacgaaaa aggccggcca ggcaaaaaag aaaaagggat cctacccata cgatgttcca
     3841 gattacgctt acccatacga tgttccagat tacgcttacc catacgatgt tccagattac
     3901 gcttaagaat tcctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat
     3961 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     4021 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     4081 ggggtggggt ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg
     4141 gggaggtacc gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
     4201 tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
     4261 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
     4321 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
     4381 gtggaaagga cgaaacaccg ccagagacaa cgccactggc tgttttagta ctctggaaac
     4441 agaatctact aaaacaaggc aaaatgccgt gtttatctcg tcaacttgtt ggcgagattt
     4501 ttgcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     4561 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     4621 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     4681 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     4741 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     4801 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     4861 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     4921 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     4981 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     5041 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
     5101 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     5161 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     5221 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     5281 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     5341 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     5401 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     5461 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5521 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     5581 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5641 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5701 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5761 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5821 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5881 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5941 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     6001 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     6061 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     6121 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     6181 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     6241 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg
     6301 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6361 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6421 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6481 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6541 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6601 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6661 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6721 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     6781 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6841 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6901 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6961 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     7021 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     7081 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     7141 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     7201 cggccttttt acggttcctg gccttttgct ggccttttgc tcacat
//
