LOCUS       40924_13590        8215 bp DNA     circular SYN 13-MAY-2021
DEFINITION  For in vivo expression of Cas9 or generation of Cas9 mRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8215)
  AUTHORS   Gagnon JA, Valen E, Thyme SB, Huang P, Ahkmetova L, Pauli A, 
            Montague TG, Zimmerman S, Richter C, Schier AF
  TITLE     Efficient mutagenesis by Cas9 protein-mediated oligonucleotide 
            insertion and large-scale assessment of single-guide RNAs.
  JOURNAL   PLoS One. 2014 May 29;9(5):e98186. doi: 
            10.1371/journal.pone.0098186. eCollection 2014.
  PUBMED    24873830
REFERENCE   2  (bases 1 to 8215)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8215)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2014-05-29"; pages: "
            10.1371/journal.pone.0098186. eCollection 2014"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8215
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        242..260
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    373..507
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        512..529
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(540..560)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(573..4676)
                     /codon_start=1
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     promoter        complement(4721..4739)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     promoter        complement(4774..5752)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus
                     major immediate early transcription unit IE94"
     rep_origin      5829..6284
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        6310..6414
                     /label=AmpR promoter
     CDS             6415..7272
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7446..8034
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8188..8205
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgctccc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg
      241 caattaaccc tcactaaagg gaacaaaagc tgggtaccgg gcccaatgca ttggcgccgc
      301 ggccgcgaat taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca
      361 attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
      421 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
      481 atcaatgtat cttatcatgt ctggatctac gtaatacgac tcactatagt tctagattac
      541 accttcctct tcttcttggg gtcagccctg ctgtctccac cgagctgaga gaggtcgatt
      601 cttgtttcat agagccccgt aattgactga tgaatcagtg tggcgtccag gacctccttt
      661 gtagaggtgt accgctttct gtctatggtg gtgtcgaagt acttgaaggc tgcaggcgcg
      721 cccaagttgg tcagagtaaa caagtggata atgttttctg cctgctccct gatgggctta
      781 tccctgtgct tattgtaagc agaaagcacc ttatcgaggt tagcgtcggc gaggatcact
      841 cttttggaga attcgcttat ttgctcgatg atctcatcaa ggtagtgttt gtgttgttcc
      901 acgaacagct gcttctgctc attatcttcg ggagaccctt tgagcttttc atagtggctg
      961 gccagataca agaaattaac gtatttagag ggcagtgcca gctcgttacc tttctgcagc
     1021 tcgcccgcac tagcgagcat tcgtttccgg ccgttttcaa gctcaaagag agagtacttg
     1081 ggaagcttaa tgatgaggtc ttttttgacc tctttatatc ctttcgcctc gagaaagtcg
     1141 atggggtttt tttcgaagct tgatcgctcc atgattgtga tgcccagcag ttccttgacg
     1201 cttttgagtt ttttagactt ccctttctcc actttggcca caaccagtac actgtaagcg
     1261 actgtaggag aatcgaatcc gccgtatttc ttggggtccc aatctttttt gcgtgcgatc
     1321 agcttgtcgc tgttcctttt cgggaggata ctttccttgg agaagcctcc ggtctgtact
     1381 tcggtctttt taacgatgtt cacctgcggc atggacagga ccttccggac tgtcgcgaaa
     1441 tccctaccct tgtcccacac gatttctcct gtttctccgt ttgtttcgat aagtggtcgc
     1501 ttccgaatct ctccattggc cagtgtaatc tcggtcttga aaaaattcat aatattgctg
     1561 taaaagaagt acttagcggt ggccttgcct atttcctgct cagactttgc gatcattttc
     1621 ctaacatcgt acactttata gtctccgtaa acaaattcag attcaagctt gggatatttt
     1681 ttgataagtg cagtgcctac cactgcattc aggtaggcat catgcgcatg gtggtaattg
     1741 ttgatctctc tcaccttata aaactgaaag tcctttctga aatctgagac cagcttagac
     1801 ttcagagtaa taactttcac ctctcgaatc agtttgtcat tttcatcgta cttggtgttc
     1861 atgcgtgaat cgagaatttg ggccacgtgc ttggtgatct ggcgtgtctc aacaagctgc
     1921 cttttgatga agccggcttt atccaactca gacaggccac ctcgttcagc cttagtcaga
     1981 ttatcgaact tccgttgtgt gatcagtttg gcgttcagca gctgccgcca ataatttttc
     2041 attttcttga caacttcttc tgaggggacg ttatcactct tccctctatt tttatcggat
     2101 cttgtcaaca ctttattatc aatagaatca tctttgagaa aagactgggg cacgatatga
     2161 tccacgtcgt agtcggagag ccgattgatg tccagttcct gatccacgta catgtccctg
     2221 ccgttctgca ggtagtacag gtagagcttc tcattctgaa gctgggtgtt ttcaactggg
     2281 tgttccttaa ggatttggga ccccagttct tttataccct cttcaatcct cttcatcctt
     2341 tccctactgt tcttctgtcc cttctgggta gtttggttct ctcgggccat ctcgataacg
     2401 atattctcgg gcttatgcct tcccattact ttgacgagtt catccacgac cttaacggtc
     2461 tgcagtattc cctttttgat agctgggcta cctgcaagat tagcgatgtg ctcgtgaaga
     2521 ctgtccccct ggccagaaac ttgtgctttc tggatgtcct ccttaaaggt gagagagtca
     2581 tcatggatca actgcatgaa gttccggttg gcaaatccat cggacttaag aaaatccagg
     2641 attgtctttc cactctgctt gtctcggatc ccattgatca gttttcttga cagccgcccc
     2701 catcctgtat atcggcgcct cttgagctgt ttcatgactt tgtcgtcgaa gagatgagcg
     2761 taagttttca agcgttcttc aatcatctcc ctatcttcaa acaacgtaag ggtgaggaca
     2821 atgtcctcaa gaatgtcctc gttctcctca ttgtccagga agtccttgtc tttaatgatt
     2881 ttcaggagat cgtgatacgt tcccagggat gcgttgaagc gatcctccac tccgctgatt
     2941 tcaacagagt cgaaacattc aatctttttg aaatagtctt ctttgagctg tttcacggta
     3001 actttccggt tcgtcttgaa gaggaggtcc acgatagctt tcttctgctc tccagacagg
     3061 aatgctggct ttctcatccc ttctgtgacg tatttgacct tggtgagctc gttataaact
     3121 gtgaagtact cgtacagcag agagtgttta ggaagcacct tttcgttagg cagattttta
     3181 tcaaagttag tcatcctttc gatgaaggac tgggcagagg cccccttatc cacgacttcc
     3241 tcgaagttcc agggagtgat ggtctcttct gatttgcgag tcatccacgc gaatctggaa
     3301 tttccccggg cgagggggcc tacatagtag ggtatccgaa atgtgaggat tttctcaatc
     3361 ttttccctgt tatctttcaa aaaggggtag aaatcctctt gccgcctgag gatagcgtgc
     3421 agttcgccca ggtgaatctg gtgggggatg cttccattgt cgaaagtgcg ctgtttgcgc
     3481 aacagatctt ctctgttaag ctttaccagc agctcctcgg tgccgtccat tttttccaag
     3541 atgggcttaa taaatttgta aaattcctcc tggcttgctc cgccgtcaat gtatccggcg
     3601 tagccatttt tagactgatc gaagaaaatt tccttgtact tctcaggcag ttgctgtctg
     3661 acaagggcct tcagcaaagt caagtcttgg tggtgctcat catagcgctt gatcatacta
     3721 gcgctcagcg gagctttggt gatctccgtg ttcactcgca gaatatcact cagcagaatg
     3781 gcgtctgaca ggttctttgc cgccaaaaaa aggtctgcgt actggtcgcc gatctgggcc
     3841 agcagattgt cgagatcatc atcgtaggtg tctttgctca gttgaagctt ggcatcttcg
     3901 gccaggtcga agttagattt aaagttgggg gtcagcccga gtgacagggc gataagatta
     3961 ccaaacaggc cgttcttctt ctccccaggg agctgtgcga tgaggttttc gagccgccgg
     4021 gatttggaca gcctagcgct caggattgct ttggcgtcaa ctccggatgc gttgatcggg
     4081 ttctcttcga aaagctgatt gtaagtctga accagttgga taaagagttt gtcgacatcg
     4141 ctgttgtctg ggttcaggtc cccctcgatg aggaagtgtc cccgaaattt gatcatatgc
     4201 gccagcgcga gatagatcaa ccgcaagtca gccttatcag tactgtctac aagcttcttc
     4261 ctcagatgat atatggttgg gtacttttca tggtacgcca cctcgtccac gatattgcca
     4321 aagattgggt ggcgctcgtg ctttttatcc tcctccacca aaaaggactc ctccagccta
     4381 tggaagaaag agtcatccac cttagccatc tcattactaa agatctcctg caggtagcag
     4441 atccgattct ttctgcgggt atatctgcgc cgtgctgttc ttttgagccg cgtggcttcg
     4501 gccgtctccc cggagtcgaa caggagggcg ccaatgaggt tcttctttat gctgtggcga
     4561 tcggtattgc ccagaacttt gaattttttg ctcggcacct tgtactcgtc cgtaatgacg
     4621 gcccagccga cgctgtttgt gccgatatcg agcccaatgg agtacttctt gtccatggtg
     4681 gcatcgatgg gatcctgcaa aaagaacaag tagcttgtat tctatagtgt cacctaaatc
     4741 aagcttgctc cgacgtcccc aggcagaatg gcggttccct aaacgagcat tgcttatata
     4801 gacctcccat taggcacgcc taccgcccat ttacgtcaat ggaacgccca tttgcgtcat
     4861 tgcccctccc cattgacgtc aatggggatg tacttggcag ccatcgcggg ccatttaccg
     4921 ccattgacgt caatgggagt actgccaatg taccctggcg tacttccaat agtaatgtac
     4981 ttgccaagtt actattaata gatattgatg tactgccaag tgggccattt accgtcattg
     5041 acgtcaatag ggggcgtgag aacggatatg aatgggcaat gagccatccc attgacgtca
     5101 atggtgggtg gtcctattga cgtcaatggg cattgagcca ggcgggccat ttaccgtaat
     5161 tgacgtcaat gggggaggcg ccatatacgt caataggacc gcccatatga cgtcaatagg
     5221 aaagaccata tatagagacc attgacgtca atgggggagt ggctatgggc ggtattagga
     5281 agccccatat atggtatatg ggaccgccca ttgggagggg ctatctacgt caataggaaa
     5341 acccatatat ggaatactat atggcatagg gccaatacat agtattgaac ctggccaata
     5401 gccatattgg catagggcca tattggatat tgcctatata ttgatcctgg catatagcca
     5461 atatggccgc cattattggc accatgccaa ttcaatatgg cggacttggc acagggccaa
     5521 gacccctcct caaacttggc accgtgccaa gtagacccct ccccagttgg cacagtgcca
     5581 ggtccgccat attgaattgg catggtgcca agtccgccat attgaattgg ctggggtcca
     5641 gatccaccat attgaattgg cacgagtcca tatacgccat attgaattgg cacaggtcca
     5701 gatccaccat attgaattgg catgagtcca tatacgccat attgaattgg cggtcgactg
     5761 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     5821 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     5881 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     5941 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     6001 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     6061 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     6121 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     6181 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     6241 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     6301 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     6361 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     6421 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     6481 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     6541 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     6601 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     6661 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     6721 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     6781 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6841 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6901 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6961 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     7021 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     7081 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     7141 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     7201 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     7261 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     7321 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     7381 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     7441 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     7501 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     7561 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     7621 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     7681 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     7741 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     7801 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     7861 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7921 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7981 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     8041 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     8101 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     8161 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaag
//
