LOCUS       40924_46988        8698 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses Cas9 nuclease and the PITCh-gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8698)
  AUTHORS   Sakuma T, Nakade S, Sakane Y, Suzuki KT, Yamamoto T
  TITLE     MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the 
            PITCh systems.
  JOURNAL   Nat Protoc. 2016 Jan;11(1):118-33. doi: 10.1038/nprot.2015.140. Epub
            2015 Dec 17.
  PUBMED    26678082
REFERENCE   2  (bases 1 to 8698)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8698)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nprot.2015.140"; journalName: "Nat Protoc"; date: 
            "2016-01"; volume: "11"; issue: "1"; pages: "118-33"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..293
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        295..571
                     /label=chicken beta-actin promoter
     intron          572..799
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      811..820
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             820..885
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             892..912
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             937..5037
                     /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             5038..5085
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    5119..5326
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5335..5475
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      5550..6005
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6022..6041)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     6141..6163
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6201..6219)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     CDS             6626..7414
                     /codon_start=1
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      7600..8188
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        8266..8506
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        8535..8610
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 tgagacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
       61 gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      121 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      181 ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat
      241 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg
      301 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt
      361 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg gggggcgcgc
      421 gccrggsggg gsggggsggg ggggggggsg gggsggggsg rggcggagag gtgcggcggc
      481 agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg
      541 gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt
      601 gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc
      661 acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagctga gcaagaggta
      721 agggtttaag ggatggttgg ttggtggggt attaatgttt aattacctgg agcacctgcc
      781 tgaaatcact ttttttcagg ttggaccggt gccaccatgg actataagga ccacgacgga
      841 gactacaagg atcatgatat tgattacaaa gacgatgacg ataagatggc cccaaagaag
      901 aagcggaagg tcggtatcca cggagtccca gcagccgaca agaagtacag catcggcctg
      961 gacatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc
     1021 aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga
     1081 gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga
     1141 agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag
     1201 atggccaagg tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag
     1261 gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac
     1321 gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc
     1381 gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg
     1441 atcgagggcg acctgaaccc cgacaacagc gacgttgaca agctgttcat ccagctggtg
     1501 cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag
     1561 gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg
     1621 cccggcgaga agaagaatgg cctgttcgga aacctgattg ccctgagcct gggcctgacc
     1681 cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac
     1741 acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg
     1801 tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac
     1861 accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac
     1921 caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag
     1981 attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag
     2041 gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg
     2101 ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc
     2161 atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt
     2221 tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc
     2281 tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc
     2341 gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag
     2401 agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc
     2461 aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac
     2521 gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg
     2581 gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc
     2641 aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc
     2701 tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat
     2761 gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga
     2821 gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag
     2881 cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc
     2941 atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc
     3001 aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag
     3061 aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc
     3121 agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa
     3181 gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc
     3241 acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa
     3301 gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag
     3361 aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac
     3421 atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctgaaggac
     3481 gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac
     3541 gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc
     3601 aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc
     3661 gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag
     3721 cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg
     3781 atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat
     3841 ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg
     3901 aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg
     3961 tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc
     4021 ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag
     4081 attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc
     4141 ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg
     4201 ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct
     4261 atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag
     4321 aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg
     4381 gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg
     4441 gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa
     4501 gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc
     4561 cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc
     4621 tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc
     4681 gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc
     4741 atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa
     4801 gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc
     4861 atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc
     4921 accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac
     4981 cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacaaa
     5041 aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaagtaaga attcctagag
     5101 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     5161 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     5221 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     5281 acagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggagcgg ccgcaggaac
     5341 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     5401 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     5461 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     5521 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     5581 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc
     5641 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     5701 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     5761 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     5821 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     5881 ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt
     5941 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     6001 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     6061 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     6121 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     6181 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     6241 aataatggtt tcttagacgt caggtggcac ttccagccag gacagaaatg cctcgacttc
     6301 gctgctaccc aaggttgccg ggtgacgcac accgtggaaa cggatgaagg cacgaaccca
     6361 gtggacataa gcctgttcgg ttcgtaagct gtaatgcaag tagcgtatgc gctcacgcaa
     6421 ctggtccaga accttgaccg aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc
     6481 ttgttatgac tgtttttttg gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg
     6541 ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc agcagggcag
     6601 tcgccctaaa acaaagttaa acattatgag ggaagcggtg atcgccgaag tatcgactca
     6661 actatcagag gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca
     6721 tttgtacggc tccgcagtgg atggcggcct gaagccacac agtgatattg atttgctggt
     6781 tacggtgacc gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg accttttgga
     6841 aacttcggct tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt
     6901 gcacgacgac atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg
     6961 gcagcgcaat gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc
     7021 tatcttgctg acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga
     7081 actctttgat ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct
     7141 atggaactcg ccgcccgact gggctggcga tgagcgaaat gtagtgctta cgttgtcccg
     7201 catttggtac agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc
     7261 aatggagcgc ctgccggccc agtatcagcc cgtcatactt gaagctagac aggcttatct
     7321 tggacaagaa gaagatcgct tggcctcgcg cgcagatcag ttggaagaat ttgtccacta
     7381 cgtgaaaggc gagatcacca aggtagtcgg caaataaaag cattggtaac tgtcagacca
     7441 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     7501 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     7561 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     7621 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     7681 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     7741 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     7801 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     7861 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     7921 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     7981 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     8041 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     8101 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     8161 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     8221 ggccttttgc tggccttttg ctcaggtctc actctagagg catgtgaggg cctatttccc
     8281 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
     8341 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     8401 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     8461 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgcatcg
     8521 tacgcgtacg tgttgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
     8581 caacttgaaa aagtggcacc gagtcggtgc ttttttgttt tagagctaga aatagcaagt
     8641 taaaataagg ctagtccgtt tttagcgcgt gcgccaattc tgcagacaaa tggctcta
//
