LOCUS       sequence-288284-        7238 bp    DNA     circular SYN 14-APR-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 7238)
  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 7238)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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 sgRNA1
FEATURES             Location/Qualifiers
     source          1..7238
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      554..563
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     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..4046
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4141..4389
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4141..4161
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4312..4331
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4412..4487
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4501..4641
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4501..4630
                     /label=AAV2 ITR
     rep_origin      4716..5171
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4803..4822)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5013..5034
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5188..5207)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5307..5329
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5367..5385)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5453..5557
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5558..5626,5627..6418)
                     /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(5776..5795)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6589..7177
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7078..7097
                     /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 gcggcctcta 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 agaatagctg gggaggtacc
     4141 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
     4201 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
     4261 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
     4321 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
     4381 cgaaacaccg tctgaggcac gttggccgtc tgttttagta ctctggaaac agaatctact
     4441 aaaacaaggc aaaatgccgt gtttatctcg tcaacttgtt ggcgagattt ttgcggccgc
     4501 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     4561 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     4621 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     4681 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     4741 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
     4801 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     4861 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     4921 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     4981 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     5041 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
     5101 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     5161 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     5221 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     5281 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     5341 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     5401 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     5461 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     5521 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     5581 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     5641 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     5701 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     5761 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     5821 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     5881 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     5941 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     6001 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     6061 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     6121 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     6181 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     6241 tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg
     6301 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     6361 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     6421 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     6481 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     6541 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     6601 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     6661 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     6721 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     6781 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     6841 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     6901 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     6961 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     7021 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     7081 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     7141 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     7201 acggttcctg gccttttgct ggccttttgc tcacatgt
//
