LOCUS       Exported                8100 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  CRISPR‐Cas9 tool for the genetic engineering of Bacillus phages.
ACCESSION   .
VERSION     .
KEYWORDS    pRH030
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8100)
  AUTHORS   Otte K, Kuhne NM, Furrer AD, Baena Lozada LP, Lutz VT, Schilling T, 
            Hertel R
  TITLE     A CRISPR-Cas9 tool to explore the genetics of Bacillus subtilis 
            phages.
  JOURNAL   Lett Appl Microbiol. 2020 Jul 2. doi: 10.1111/lam.13349.
  PUBMED    32615024
REFERENCE   2  (bases 1 to 8100)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8100
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     2187..2209
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2201..2218
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2202..2218
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2234..2250)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2234..2250)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2247..2269)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2258..2274
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     promoter        complement(2282..2312)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2327..2348
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_RNA        2422..2497
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2519..2553
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     primer_bind     2590..2609
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2590..2608
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(2759..6865)
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /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"
     misc_RNA        7161..7239
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     rep_origin      complement(7467..8012)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(7547..7566)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgaggtcatc gttcaaaatg gtatgcgttt tgacacatcc actatatatc cgtgtcgttc
       61 tgtccactcc tgaatcccat tccagaaatt ctctagcgat tccagaagtt tctcagagtc
      121 ggaaagttga ccagacatta cgaactggca cagatggtca taacctgaag gaagatctga
      181 ttgcttaact gcttcagtta agaccgaagc gctcgtcgta taacagatgc gatgatgcag
      241 accaatcaac atggcacctg ccattgctac ctgcacagtc aaggatggta gaaatgttgt
      301 cggtccttgc acacgaatat tacgccattt gcctgcatat tcaaacagct cttctacgat
      361 aagggcacaa atcgcatcgt ggaacgtttg ggcttctacc gatttagcag tttgatacac
      421 tttctctaag tatccacctg aatcataaat cggcaaaata gagaaaaatt gaccatgtgt
      481 aagcggccaa tctgattcca cctgagatgc ataatctagt agaatctctt cgctatcaaa
      541 attcacttcc accttccact caccggttgt ccattcatgg ctgaactctg cttcctctgt
      601 tgacatgaca cacatcatct caatatccga atagggccca tcagtctgac gaccaagaga
      661 gccataaaca ccaatagcct taacatcatc cccatattta tccaatattc gttccttaat
      721 ttcatgaaca atcttcattc tttcttctct agtcattatt attggtccat tcactattct
      781 cattcccttt tcagataatt ttagatttgc ttttctaaat aagaatattt ggagagcacc
      841 gttcttattc agctattaaa cccattatat cgggtttttg aggggatttc aactgcagac
      901 acctaaattc aaaatctatc ggtcagattt ataccgattt gattttatat attcttgaat
      961 aacatacgcc gagttatcac ataaaagcgg gaaccaatca tcaaatttaa acttcattgc
     1021 ataatccatt aaactcttaa attctacgat tccttgttca tcaataaact caatcatttc
     1081 tttaattaat ttatatctat ctgttgttgt tttctttaat aattcatcaa catctacacc
     1141 gccataaact atcatatctt ctttttgata tttaaattta ttaggatcgt ccatgtgaag
     1201 catatatctc acaagacctt tcacacttcc tgcaatctgc ggaatagtcg cattcaattc
     1261 ttctgttatt atttttatct gttcataaga tttattaccc tcatacatca ctagaatatg
     1321 ataatgctct tttttcatcc taccttctgt atcagtatcc ctatcatgta atggagacac
     1381 tacaaattga atgtgtaact cttttaaata ctctaaccac tcggcttttg ctgattctgg
     1441 atataaaaca aatgtccaat tacgtcctct tgaatttttc ttgttttcag tttcttttat
     1501 tacattttcg ctcatgatat aataacggtg ctaatacatt taacaaaatt tagtcataga
     1561 taggcagcat gccagtgctg tctatctttt tttgtttaaa atgcaccgta ttcctccttt
     1621 gcatattttt ttattagaat accggttgca tctgatttgc taatattata tttttctttg
     1681 attctattta atatctcatt ttcttctgtt gtaagtctta aagtaacagc aacttttttc
     1741 tcttcttttc tatctacaac catcactgta cctcccaaca tctgtttttt tcactttaac
     1801 ataaaaaaca accttttaac attaaaaacc caatatttat ttatttgttt ggacaatgga
     1861 caatggacac ctagggggga ggtcgtagta cccccctatg ttttctcccc taaataaccc
     1921 caaaaatcta agaaaaaaag acctcaaaaa ggtctttaat taacatctca aatttcgcat
     1981 ttattccaat ttcctttttg cgtgtgatgc gaattcttga ccgtgattag agaattgagt
     2041 aaaatgtacc tacgcgagac ctcattcgcc attcaggctg cgcaactgtt gggaagggcg
     2101 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
     2161 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga
     2221 attcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     2281 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     2341 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     2401 tgcattaatg aatcggtctc agttttagag ctagaaatag caagttaaaa taaggctagt
     2461 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttactccatc tggatttgtt
     2521 cagaacgctc ggttgccgcc gggcgttttt tatctaaagc ttaggcccag tcgaaagact
     2581 gggccttttt aatacgactc actatagggt cgacccctct cggcaaatgt gccgggaggg
     2641 ttctttgcgt ttggccaacg aggcccgggc caataaggcc aagaaaaaga aaaagcgctc
     2701 ccgccgggta agcaagaagc gctctagatt aagaaataat cttcatctaa aatatacttc
     2761 agtcacctcc tagctgactc aaatcaatgc gtgtttcata aagaccagtg atggattgat
     2821 ggataagagt ggcatctaaa acttcttttg tagacgtata tcgtttacga tcaattgttg
     2881 tatcaaaata tttaaaagca gcgggagctc caagattcgt caacgtaaat aaatgaataa
     2941 tattttctgc ttgttcacgt attggtttgt ctctatgttt gttatatgca ctaagaactt
     3001 tatctaaatt ggcatctgct aaaataacac gcttagaaaa ttcactgatt tgctcaataa
     3061 tctcatctaa ataatgctta tgctgctcca caaacaattg tttttgttcg ttatcttctg
     3121 gactaccctt caacttttca taatgactag ctaaatataa aaaattcaca tatttgcttg
     3181 gcagagccag ctcatttcct ttttgtaatt ctccggcact agccagcatc cgtttacgac
     3241 cgttttctaa ctcaaaaaga ctatatttag gtagtttaat gattaagtct tttttaactt
     3301 ccttatatcc tttagcttct aaaaagtcaa tcggattttt ttcaaaggaa cttctttcca
     3361 taattgtgat ccctagtaac tctttaacgg attttaactt cttcgatttc cctttttcca
     3421 ccttagcaac cactaggact gaataagcta ccgttggact atcaaaacca ccatattttt
     3481 ttggatccca gtctttttta cgagcaataa gcttgtccga atttcttttt ggtaaaattg
     3541 actccttgga gaatccgcct gtctgtactt ctgttttctt gacaatattg acttggggca
     3601 tggacaatac tttgcgcact gtggcaaaat ctcgcccttt atcccagaca atttctccag
     3661 tttccccatt agtttcgatt agagggcgtt tgcgaatctc tccatttgca agtgtaattt
     3721 ctgttttgaa gaagttcatg atattagagt aaaagaaata ttttgcggtt gctttgccta
     3781 tttcttgctc agacttagca atcattttac gaacatcata aactttataa tcaccataga
     3841 caaactccga ttcaagtttt ggatatttct taatcaaagc agttccaacg acggcattta
     3901 gatacgcatc atgggcatga tggtaattgt taatctcacg tactttatag aattggaaat
     3961 cttttcggaa gtcagaaact aatttagatt ttaaggtaat cactttaacc tctcgaataa
     4021 gtttatcatt ttcatcgtat ttagtattca tgcgactatc caaaatttgt gccacatgct
     4081 tagtgatttg gcgagtttca accaattggc gtttgataaa accagcttta tcaagttcac
     4141 tcaaacctcc acgttcagct ttcgttaaat tatcaaactt acgttgagtg attaacttgg
     4201 cgtttagaag ttgtctccaa tagtttttca tctttttgac tacttcttca cttggaacgt
     4261 tatccgattt accacgattt ttatcagaac gcgttaagac cttattgtct attgaatcgt
     4321 ctttaaggaa actttgtgga acaatgtgat cgacatcata atcacttaaa cgattaatat
     4381 ctaattcttg gtccacatac atgtctcttc cattttggag ataatagaga tagagctttt
     4441 cattttgcaa ttgagtattt tcaacaggat gctctttaag aatctgactt cctaattctt
     4501 tgataccttc ttcgattcgt ttcatacgct ctcgcgaatt tttctggccc ttttgagttg
     4561 tctgattttc acgtgccatt tcaataacga tattttctgg cttatgccgc cccattactt
     4621 tgaccaattc atcaacaact tttacagtct gtaaaatacc ttttttaata gcagggctac
     4681 cagctaaatt tgcaatatgt tcatgtaaac tatcgccttg tccagacact tgtgcttttt
     4741 gaatgtcttc tttaaatgtc aaactatcat catggatcag ctgcataaaa ttgcgattgg
     4801 caaaaccatc tgatttcaaa aaatctaata ttgttttgcc agattgctta tccctaatac
     4861 cattaatcaa ttttcgagac aaacgtcccc aaccagtata acggcgacgt ttaagctgtt
     4921 tcatcacctt atcatcaaag aggtgagcat atgttttaag tctttcctca atcatctccc
     4981 tatcttcaaa taaggtcaat gttaaaacaa tatcctctaa gatatcttca ttttcttcat
     5041 tatccaaaaa atctttatct ttaataattt ttagcaaatc atggtaggta cctaatgaag
     5101 cattaaatct atcttcaact cctgaaattt caacactatc aaaacattct atttttttga
     5161 aataatcttc ttttaattgc ttaacggtta cttttcgatt tgttttgaag agtaaatcaa
     5221 caatggcttt cttctgttca cctgaaagaa atgctggttt tcgcattcct tcagtaacat
     5281 atttgacctt tgtcaattcg ttataaaccg taaaatactc ataaagcaaa ctatgttttg
     5341 gtagtacttt ttcatttgga agatttttat caaagtttgt catgcgttca ataaatgatt
     5401 gagctgaagc acctttatcg acaacttctt caaaattcca tggggtaatt gtttcttcag
     5461 acttccgagt catccatgca aaacgactat tgccacgcgc caatggacca acataataag
     5521 gaattcgaaa agtcaagatt ttttcaatct tctcacgatt gtcttttaaa aatggataaa
     5581 agtcttcttg tcttctcaaa atagcatgca gctcacccaa gtgaatttga tggggaatag
     5641 agccgttgtc aaaggtccgt tgcttgcgca gcaaatcttc acgatttagt ttcaccaata
     5701 attcctcagt accatccatt ttttctaaaa ttggtttgat aaatttataa aattcttctt
     5761 ggctagctcc cccatcaata taacctgcat atccgttttt tgattgatca aaaaagattt
     5821 ctttatactt ttctggaagt tgttgtcgaa ctaaagcttt taaaagagtc aagtcttgat
     5881 gatgttcatc gtagcgttta atcattgaag ctgatagggg agccttagtt atttcagtat
     5941 ttactcttag gatatctgaa agtaaaatag catctgataa attcttagct gccaaaaaca
     6001 aatcagcata ttgatctcca atttgcgcca ataaattatc taaatcatca tcgtaagtat
     6061 cttttgaaag ctgtaattta gcatcttctg ccaaatcaaa atttgattta aaattagggg
     6121 tcaaacccaa tgacaaagca atgagattcc caaataagcc atttttcttc tcaccgggga
     6181 gctgagcaat gagattttct aatcgtcttg atttactcaa tcgtgcagaa agaatcgctt
     6241 tagcatctac tccacttgcg ttaatagggt tttcttcaaa taattgattg taggtttgta
     6301 ccaactggat aaatagtttg tccacatcac tattatcagg atttaaatct ccctcaatca
     6361 aaaaatgacc acgaaactta atcatatgcg ctaaggccaa atagattaag cgcaaatccg
     6421 ctttatcagt agaatctacc aatttttttc gcagatgata gatagttgga tatttctcat
     6481 gataagcaac ttcatctact atatttccaa aaataggatg acgttcatgc ttcttgtctt
     6541 cttccaccaa aaaagactct tcaagtcgat gaaagaaact atcatctact ttcgccatct
     6601 catttgaaaa aatctcctgt agataacaaa tacgattctt ccgacgtgta taccttctac
     6661 gagctgtccg tttgagacga gtcgcttccg ctgtctctcc actgtcaaat aaaagagccc
     6721 ctataagatt ttttttgata ctgtggcggt ctgtatttcc cagaaccttg aactttttag
     6781 acggaacctt atattcatca gtgatcaccg cccatccgac gctatttgtg ccgatatcta
     6841 agcctattga gtatttctta tccatttttg cctcctaaaa taaaaagttt aaattaaatc
     6901 cataatgagt ttgatgattt caataatagt tttaatgacc tccgaaatta gtttaatatg
     6961 ctttaatttt tctttttcaa aatatctctt caaaaaatat tacccaatac ttaataataa
     7021 atagattata acacaaaatt cttttaaaaa gtagtttatt ttgttatcat tctatagtat
     7081 taagtattgt tttatggctg ataaatttct ttgaatttct ccttgattat ttgttataaa
     7141 agttataaaa taatcttgtt ggaaccattc aaaacagcat agcaagttaa aataaggcta
     7201 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttgata cttctattct
     7261 actctgactg caaaccaaaa aaacaagcgc tttcaaaacg cttgttttat catttttagg
     7321 gaaattaatc tcttaatcct tttatcattc tacatttagg cgctgccatc ttgctaaacc
     7381 tactaagctc cacaggatga tttcgtaatc ccgcaagagg ccctgtacac ttaacaggga
     7441 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac
     7501 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg
     7561 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc
     7621 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc
     7681 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt
     7741 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc
     7801 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa
     7861 ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt
     7921 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc
     7981 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaat cagataaaat
     8041 atttctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agcccgagct
//
