LOCUS       Exported                6187 bp ds-DNA     circular SYN 18-OCT-2017
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 6187)
  AUTHORS   Trial User
  TITLE     Direct Submission
  JOURNAL   Exported Nov 1, 2017 from SnapGene 4.0.5
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6187
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     terminator      184..227
                     /label=bacterial terminator
                     /note="/ApEinfo_fwdcolor=#c6c9d1
                     /ApEinfo_revcolor=#c6c9d1"
     misc_feature    230
                     /label=BioBrick prefix
                     /note="/ApEinfo_fwdcolor=#84b0dc
                     /ApEinfo_revcolor=#84b0dc"
     misc_feature    231..249
                     /label=BioBrick prefix CDS
                     /label=BioBrick prefix
                     /note="/Biobrick prefix for coding sequences
                     /ApEinfo_fwdcolor=#84b0dc
                     /ApEinfo_revcolor=#84b0dc"
     primer_bind     245..279
                     /label=G35
     CDS             251..4357
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             278..280
                     /label=D10A
                     /label=dCas9
                     /note="/ApEinfo_fwdcolor=#ff9ccd
                     /ApEinfo_revcolor=#ff9ccd"
     primer_bind     complement(1559..1598)
                     /label=G34
     primer_bind     1589..1619
                     /label=G33
     misc_feature    1594
                     /label=T->A
     CDS             2768..2770
                     /label=H840A
                     /label=dCas9
                     /note="/ApEinfo_fwdcolor=#ff9ccd
                     /ApEinfo_revcolor=#ff9ccd"
     misc_feature    4371..4389
                     /label=BioBrick suffix
                     /note="/ApEinfo_fwdcolor=#84b0dc
                     /ApEinfo_revcolor=#84b0dc"
     terminator      4390..4447
                     /label=his operon terminator
                     /note="/ApEinfo_fwdcolor=#c6c9d1
                     /ApEinfo_revcolor=#c6c9d1"
     misc_feature    complement(4629..5243)
                     /label=rep (pMB1)
                     /note="/ApEinfo_fwdcolor=#f8d3a9
                     /ApEinfo_revcolor=#f8d3a9"
     terminator      5413..5507
                     /label=lambda t0 terminator
                     /note="/ApEinfo_fwdcolor=#c6c9d1
                     /ApEinfo_revcolor=#c6c9d1"
     CDS             complement(5528..6187)
                     /label=CmR
                     /note="/ApEinfo_fwdcolor=#b7e6d7
                     /ApEinfo_revcolor=#b7e6d7"
ORIGIN
        1 tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg gtagtgatct
       61 tatttcatta tggtgaaagt tggaacctct tacgtgcccg atcaactcga gtgccacctg
      121 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
      181 agaatttcag ataaaaaaaa tccttagctt tcgctaagga tgatttctgg aattcgcggc
      241 cgcttctaga atggataaga aatactcaat aggcttagct atcggcacaa atagcgtcgg
      301 atgggcggtg atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa
      361 tacagaccgc cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga
      421 gacagcggaa gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa
      481 tcgtatttgt tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt
      541 ctttcatcga cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc
      601 tatttttgga aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca
      661 tctgcgaaaa aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc
      721 cttagcgcat atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga
      781 taatagtgat gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga
      841 agaaaaccct attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag
      901 taaatcaaga cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt
      961 atttgggaat ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga
     1021 tttggcagaa gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa
     1081 tttattggcg caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc
     1141 agatgctatt ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct
     1201 atcagcttca atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc
     1261 tttagttcga caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa
     1321 cggatatgca ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa
     1381 accaatttta gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga
     1441 tttgctgcgc aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg
     1501 tgagctgcat gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg
     1561 tgagaagatt gaaaaaatct tgacttttcg aataccttat tatgttggtc cattggcgcg
     1621 tggcaatagt cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa
     1681 ttttgaagaa gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa
     1741 ctttgataaa aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta
     1801 ttttacggtt tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc
     1861 agcatttctt tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg
     1921 aaaagtaacc gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag
     1981 tgttgaaatt tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt
     2041 gctaaaaatt attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga
     2101 ggatattgtt ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa
     2161 aacatatgct cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac
     2221 tggttgggga cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa
     2281 aacaatatta gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat
     2341 ccatgatgat agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg
     2401 cgatagttta catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat
     2461 tttacagact gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga
     2521 aaatatcgtt attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc
     2581 gcgagagcgt atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa
     2641 agagcatcct gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca
     2701 aaatggaaga gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga
     2761 tgtcgatgcc attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt
     2821 aacgcgttct gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa
     2881 aaagatgaaa aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt
     2941 tgataattta acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat
     3001 caaacgccaa ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag
     3061 tcgcatgaat actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac
     3121 cttaaaatct aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga
     3181 gattaacaat taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt
     3241 gattaagaaa tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga
     3301 tgttcgtaaa atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt
     3361 cttttactct aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat
     3421 tcgcaaacgc cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg
     3481 gcgagatttt gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa
     3541 aacagaagta cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga
     3601 caagcttatt gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc
     3661 aacggtagct tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt
     3721 aaaatccgtt aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa
     3781 tccgattgac tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa
     3841 actacctaaa tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc
     3901 cggagaatta caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata
     3961 tttagctagt cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt
     4021 gtttgtggag cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc
     4081 taagcgtgtt attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca
     4141 tagagacaaa ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa
     4201 tcttggagct cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac
     4261 gtctacaaaa gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga
     4321 aacacgcatt gatttgagtc agctaggagg tgactaagtc gacctcgaga ctagtagcgg
     4381 ccgctgcagt ccggcaaaaa agggcaaggt gtcaccaccc tgcccttttt ctttaaaacc
     4441 gaaaagatta cttcgcgtta tgcaggcttc ctcgctcact gactcgctgc gctcggtcgt
     4501 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4561 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4621 aaaggccgcg ttgctggcgt ttttccacag gctccgcccc cctgacgagc atcacaaaaa
     4681 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4741 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4801 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4861 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4921 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4981 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5041 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5101 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5161 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5221 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5281 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5341 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5401 ctcgaggctt ggattctcac caataaaaaa cgcccggcgg caaccgagcg ttctgaacaa
     5461 atccagatgg agttctgagg tcattactgg atctatcaac aggagtccaa gcgagctcga
     5521 tatcaaatta cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc
     5581 tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca
     5641 ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag aagttgtcca
     5701 tattggccac gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa
     5761 acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat
     5821 cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg
     5881 aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca
     5941 ccagctcacc gtctttcatt gccatacgaa attccggatg agcattcatc aggcgggcaa
     6001 gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg
     6061 ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct
     6121 caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt
     6181 tctccat
//
