LOCUS       40924_175        8507 bp DNA     circular SYN 13-MAY-2021
DEFINITION  CRISPR/Cas to target the AAVS1 locus in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8507)
  AUTHORS   Natsume T, Kiyomitsu T, Saga Y, Kanemaki MT
  TITLE     Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron 
            Tagging with Short Homology Donors.
  JOURNAL   Cell Rep. 2016 Apr 5;15(1):210-8. doi: 10.1016/j.celrep.2016.03.001.
            Epub 2016 Mar 24.
  PUBMED    27052166
REFERENCE   2  (bases 1 to 8507)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8507)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2016.03.001"; journalName: "Cell Rep"; date: 
            "2016-04-5- 5"; volume: "15"; issue: "1"; pages: "210-8"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8507
                     /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        270..345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        442..727
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1007..1234
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1246..1255
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1255..1320
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1327..1347
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1372..5472
                     /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             5473..5520
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    5554..5761
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5770..5910
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      5985..6440
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6457..6476)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     6576..6598
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6636..6654)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        6722..6826
                     /label=AmpR promoter
     CDS             6827..7684
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7858..8446
                     /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 gggccactag ggacaggatg ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag
      361 ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag
      421 acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc
      481 gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg
      541 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      601 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca
      661 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      721 taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
      781 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
      841 gggggggggg cgcgcgccrg gsggggsggg ggggggsggg ggggggggsg gggsgrggsg
      901 grgrggkgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
      961 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
     1021 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
     1081 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta
     1141 gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta
     1201 cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catggactat
     1261 aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga tgacgataag
     1321 atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag
     1381 tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag
     1441 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag
     1501 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg
     1561 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag
     1621 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc
     1681 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac
     1741 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac
     1801 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc
     1861 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg
     1921 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc
     1981 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat
     2041 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg
     2101 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg
     2161 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac
     2221 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac
     2281 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga
     2341 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct
     2401 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac
     2461 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac
     2521 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc
     2581 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg
     2641 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg
     2701 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg
     2761 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag
     2821 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac
     2881 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg
     2941 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag
     3001 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg
     3061 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa
     3121 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag
     3181 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca
     3241 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac
     3301 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg
     3361 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag
     3421 tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt
     3481 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt
     3541 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg
     3601 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc
     3661 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc
     3721 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc
     3781 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg
     3841 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag
     3901 agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg
     3961 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg
     4021 cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag
     4081 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc
     4141 cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac
     4201 gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc
     4261 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc
     4321 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg
     4381 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag
     4441 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac
     4501 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag
     4561 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg
     4621 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc
     4681 ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag
     4741 gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg
     4801 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg
     4861 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc
     4921 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc
     4981 gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac
     5041 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag
     5101 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac
     5161 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac
     5221 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag
     5281 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc
     5341 aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac
     5401 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag
     5461 ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag
     5521 taagaattcc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     5581 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     5641 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     5701 gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcagg catgctgggg
     5761 agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     5821 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     5881 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     5941 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     6001 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     6061 cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     6121 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     6181 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     6241 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     6301 tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     6361 ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     6421 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     6481 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     6541 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     6601 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     6661 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     6721 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     6781 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     6841 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     6901 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     6961 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     7021 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     7081 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     7141 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     7201 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     7261 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     7321 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     7381 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     7441 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     7501 tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc
     7561 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     7621 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     7681 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     7741 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     7801 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     7861 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     7921 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     7981 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     8041 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     8101 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     8161 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     8221 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     8281 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     8341 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     8401 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     8461 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
