LOCUS       Exported                7246 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses SaCas9 and a gRNA targeting mouse YAP.
ACCESSION   .
VERSION     .
KEYWORDS    Icam 2 SaCas9 YAP sgRNA2
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 May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7246
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      554..563
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             566..586
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             611..3766
                     /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             3767..3814
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3821..3901
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(3929..3946)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3935..4142
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4149..4389
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     4149..4169
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4320..4339
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4420..4495
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4509..4649
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4509..4638
                     /label=AAV2 ITR
     rep_origin      4724..5179
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4811..4830)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5021..5042
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5196..5215)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5315..5337
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5375..5393)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5461..5565
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5566..6426
                     /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(5784..5803)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6597..7185
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7086..7105
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct ggggcctcta gactcgaggt ctcccaggca tgactccaac aatgcatccc
      181 atgggatttg gggttcccca gatctggggc ttgtaggcct gactctcccc tgtgcacacg
      241 tctcatacac gcatgcgtgc acccattgcc tgccccgccc cttgcacagg gagtcagcag
      301 ggaggactgg gttatgccct gcttatcagc agcttcccag cttcctctgc ctggattctt
      361 agaggcctgg ggtcctagaa cgagctggtg cacgtggctt cccaaagatc tctcagataa
      421 tgagaggaaa tgcagtcatc agtttgcaga aggctaggga ttctgggcca tagctcagac
      481 ctgcgcccac catctccctc caggcagccc ttggctggtc cctgcgagcc cgtggagact
      541 gccagtcacc ggtgccacca tggccccaaa gaagaagcgg aaggtcggta tccacggagt
      601 cccagcagcc aagcggaact acatcctggg cctggacatc ggcatcacca gcgtgggcta
      661 cggcatcatc gactacgaga cacgggacgt gatcgatgcc ggcgtgcggc tgttcaaaga
      721 ggccaacgtg gaaaacaacg agggcaggcg gagcaagaga ggcgccagaa ggctgaagcg
      781 gcggaggcgg catagaatcc agagagtgaa gaagctgctg ttcgactaca acctgctgac
      841 cgaccacagc gagctgagcg gcatcaaccc ctacgaggcc agagtgaagg gcctgagcca
      901 gaagctgagc gaggaagagt tctctgccgc cctgctgcac ctggccaaga gaagaggcgt
      961 gcacaacgtg aacgaggtgg aagaggacac cggcaacgag ctgtccacca aagagcagat
     1021 cagccggaac agcaaggccc tggaagagaa atacgtggcc gaactgcagc tggaacggct
     1081 gaagaaagac ggcgaagtgc ggggcagcat caacagattc aagaccagcg actacgtgaa
     1141 agaagccaaa cagctgctga aggtgcagaa ggcctaccac cagctggacc agagcttcat
     1201 cgacacctac atcgacctgc tggaaacccg gcggacctac tatgagggac ctggcgaggg
     1261 cagccccttc ggctggaagg acatcaaaga atggtacgag atgctgatgg gccactgcac
     1321 ctacttcccc gaggaactgc ggagcgtgaa gtacgcctac aacgccgacc tgtacaacgc
     1381 cctgaacgac ctgaacaatc tcgtgatcac cagggacgag aacgagaagc tggaatatta
     1441 cgagaagttc cagatcatcg agaacgtgtt caagcagaag aagaagccca ccctgaagca
     1501 gatcgccaaa gaaatcctcg tgaacgaaga ggatattaag ggctacagag tgaccagcac
     1561 cggcaagccc gagttcacca acctgaaggt gtaccacgac atcaaggaca ttaccgcccg
     1621 gaaagagatt attgagaacg ccgagctgct ggatcagatt gccaagatcc tgaccatcta
     1681 ccagagcagc gaggacatcc aggaagaact gaccaatctg aactccgagc tgacccagga
     1741 agagatcgag cagatctcta atctgaaggg ctataccggc acccacaacc tgagcctgaa
     1801 ggccatcaac ctgatcctgg acgagctgtg gcacaccaac gacaaccaga tcgctatctt
     1861 caaccggctg aagctggtgc ccaagaaggt ggacctgtcc cagcagaaag agatccccac
     1921 caccctggtg gacgacttca tcctgagccc cgtcgtgaag agaagcttca tccagagcat
     1981 caaagtgatc aacgccatca tcaagaagta cggcctgccc aacgacatca ttatcgagct
     2041 ggcccgcgag aagaactcca aggacgccca gaaaatgatc aacgagatgc agaagcggaa
     2101 ccggcagacc aacgagcgga tcgaggaaat catccggacc accggcaaag agaacgccaa
     2161 gtacctgatc gagaagatca agctgcacga catgcaggaa ggcaagtgcc tgtacagcct
     2221 ggaagccatc cctctggaag atctgctgaa caaccccttc aactatgagg tggaccacat
     2281 catccccaga agcgtgtcct tcgacaacag cttcaacaac aaggtgctcg tgaagcagga
     2341 agaaaacagc aagaagggca accggacccc attccagtac ctgagcagca gcgacagcaa
     2401 gatcagctac gaaaccttca agaagcacat cctgaatctg gccaagggca agggcagaat
     2461 cagcaagacc aagaaagagt atctgctgga agaacgggac atcaacaggt tctccgtgca
     2521 gaaagacttc atcaaccgga acctggtgga taccagatac gccaccagag gcctgatgaa
     2581 cctgctgcgg agctacttca gagtgaacaa cctggacgtg aaagtgaagt ccatcaatgg
     2641 cggcttcacc agctttctgc ggcggaagtg gaagtttaag aaagagcgga acaaggggta
     2701 caagcaccac gccgaggacg ccctgatcat tgccaacgcc gatttcatct tcaaagagtg
     2761 gaagaaactg gacaaggcca aaaaagtgat ggaaaaccag atgttcgagg aaaagcaggc
     2821 cgagagcatg cccgagatcg aaaccgagca ggagtacaaa gagatcttca tcacccccca
     2881 ccagatcaag cacattaagg acttcaagga ctacaagtac agccaccggg tggacaagaa
     2941 gcctaataga gagctgatta acgacaccct gtactccacc cggaaggacg acaagggcaa
     3001 caccctgatc gtgaacaatc tgaacggcct gtacgacaag gacaatgaca agctgaaaaa
     3061 gctgatcaac aagagccccg aaaagctgct gatgtaccac cacgaccccc agacctacca
     3121 gaaactgaag ctgattatgg aacagtacgg cgacgagaag aatcccctgt acaagtacta
     3181 cgaggaaacc gggaactacc tgaccaagta ctccaaaaag gacaacggcc ccgtgatcaa
     3241 gaagattaag tattacggca acaaactgaa cgcccatctg gacatcaccg acgactaccc
     3301 caacagcaga aacaaggtcg tgaagctgtc cctgaagccc tacagattcg acgtgtacct
     3361 ggacaatggc gtgtacaagt tcgtgaccgt gaagaatctg gatgtgatca aaaaagaaaa
     3421 ctactacgaa gtgaatagca agtgctatga ggaagctaag aagctgaaga agatcagcaa
     3481 ccaggccgag tttatcgcct ccttctacaa caacgatctg atcaagatca acggcgagct
     3541 gtatagagtg atcggcgtga acaacgacct gctgaaccgg atcgaagtga acatgatcga
     3601 catcacctac cgcgagtacc tggaaaacat gaacgacaag aggcccccca ggatcattaa
     3661 gacaatcgcc tccaagaccc agagcattaa gaagtacagc acagacattc tgggcaacct
     3721 gtatgaagtg aaatctaaga agcaccctca gatcatcaaa aagggcaaaa ggccggcggc
     3781 cacgaaaaag gccggccagg caaaaaagaa aaagggatcc tacccatacg atgttccaga
     3841 ttacgcttac ccatacgatg ttccagatta cgcttaccca tacgatgttc cagattacgc
     3901 ttaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     3961 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     4021 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     4081 ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg
     4141 gaggtaccga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     4201 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     4261 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     4321 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     4381 ggaaaggacg aaacaccgtg cacgacctgg tggccggccg ttttagtact ctggaaacag
     4441 aatctactaa aacaaggcaa aatgccgtgt ttatctcgtc aacttgttgg cgagattttt
     4501 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     4561 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     4621 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     4681 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     4741 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4801 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4861 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4921 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     4981 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     5041 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     5101 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     5161 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     5221 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     5281 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     5341 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     5401 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     5461 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5521 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5581 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5641 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5701 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5761 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     5821 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5881 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5941 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     6001 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     6061 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     6121 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     6181 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6241 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca
     6301 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6361 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6421 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6481 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6541 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6601 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6661 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6721 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     6781 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6841 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6901 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6961 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     7021 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     7081 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     7141 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     7201 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
