LOCUS       40924_47108        8508 bp DNA     circular SYN 13-MAY-2021
DEFINITION  AAVS1 sgRNA expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8508)
  AUTHORS   Oceguera-Yanez F, Kim SI, Matsumoto T, Tan GW, Xiang L, Hatani T, 
            Kondo T, Ikeya M, Yoshida Y, Inoue H, Woltjen K
  TITLE     Engineering the AAVS1 locus for consistent and scalable transgene 
            expression in human iPSCs and their differentiated derivatives.
  JOURNAL   Methods. 2015 Dec 18. pii: S1046-2023(15)30181-X. doi: 
            10.1016/j.ymeth.2015.12.012.
  PUBMED    26707206
REFERENCE   2  (bases 1 to 8508)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8508)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Methods. 
            2015 Dec 18. pii: S1046-2023(15)30181-X. doi: 
            10.1016/j.ymeth.2015.12.012."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8508
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..241
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        443..728
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        730..1007
                     /label=chicken beta-actin promoter
     intron          1008..1235
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1247..1256
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1256..1321
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1328..1348
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1373..5473
                     /codon_start=1
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
                     /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             5474..5521
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    5555..5762
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5771..5911
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      5986..6441
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6458..6477)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     6577..6599
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6637..6655)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        6723..6827
                     /label=AmpR promoter
     CDS             6828..7685
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7859..8447
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggggccacta gggacaggat gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca
      421 gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac
      481 cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac
      541 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      601 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc
      661 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      721 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc
      781 cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg
      841 gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc
      901 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga
      961 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc
     1021 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
     1081 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
     1141 agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt
     1201 acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta
     1261 taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa
     1321 gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa
     1381 gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga
     1441 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa
     1501 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct
     1561 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga
     1621 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
     1681 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
     1741 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
     1801 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
     1861 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
     1921 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
     1981 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
     2041 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct
     2101 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
     2161 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
     2221 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
     2281 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
     2341 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
     2401 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
     2461 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
     2521 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
     2581 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
     2641 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
     2701 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
     2761 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
     2821 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa
     2881 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
     2941 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
     3001 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
     3061 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
     3121 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
     3181 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
     3241 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
     3301 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg
     3361 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
     3421 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt
     3481 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
     3541 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
     3601 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
     3661 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
     3721 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
     3781 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
     3841 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca
     3901 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg
     3961 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
     4021 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
     4081 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
     4141 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
     4201 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
     4261 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
     4321 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct
     4381 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
     4441 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
     4501 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga
     4561 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
     4621 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
     4681 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
     4741 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct
     4801 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct
     4861 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
     4921 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
     4981 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
     5041 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
     5101 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
     5161 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
     5221 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
     5281 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
     5341 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
     5401 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
     5461 gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa
     5521 gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     5581 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     5641 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     5701 ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg
     5761 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     5821 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     5881 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     5941 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     6001 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     6061 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     6121 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     6181 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     6241 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     6301 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
     6361 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     6421 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     6481 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     6541 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     6601 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     6661 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     6721 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     6781 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     6841 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     6901 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     6961 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     7021 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     7081 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     7141 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     7201 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     7261 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     7321 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     7381 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     7441 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     7501 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg
     7561 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     7621 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     7681 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     7741 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     7801 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     7861 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     7921 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     7981 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     8041 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     8101 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     8161 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     8221 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     8281 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     8341 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     8401 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     8461 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
