pRH030

LOCUS       pRH030                9437 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 9437)
  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 9437)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9437
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             1..4107
                     /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"
     primer_bind     complement(4257..4276)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4258..4276)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      4313..4347
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     misc_RNA        complement(4369..4444)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    4518..4539
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4554..4584
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4592..4608
                     /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)."
     primer_bind     4597..4619
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4616..4632
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4616..4632
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4648..4665)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4648..4664)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4657..4679)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8291..8836
                     /direction=RIGHT
                     /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     8737..8756
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_RNA        complement(9064..9142)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
ORIGIN
        1 atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg
       61 atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc
      121 cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa
      181 gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt
      241 tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga
      301 cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga
      361 aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa
      421 aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat
      481 atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat
      541 gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct
      601 attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga
      661 cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat
      721 ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa
      781 gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg
      841 caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt
      901 ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca
      961 atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga
     1021 caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca
     1081 ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta
     1141 gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc
     1201 aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat
     1261 gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt
     1321 gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt
     1381 cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa
     1441 gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa
     1501 aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt
     1561 tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt
     1621 tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc
     1681 gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt
     1741 tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt
     1801 attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt
     1861 ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct
     1921 cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga
     1981 cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta
     2041 gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat
     2101 agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta
     2161 catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact
     2221 gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt
     2281 attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt
     2341 atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct
     2401 gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga
     2461 gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac
     2521 attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct
     2581 gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa
     2641 aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta
     2701 acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa
     2761 ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat
     2821 actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct
     2881 aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat
     2941 taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa
     3001 tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa
     3061 atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct
     3121 aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc
     3181 cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt
     3241 gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta
     3301 cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt
     3361 gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct
     3421 tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt
     3481 aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac
     3541 tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa
     3601 tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta
     3661 caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt
     3721 cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag
     3781 cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt
     3841 attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa
     3901 ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct
     3961 cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa
     4021 gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt
     4081 gatttgagtc agctaggagg tgactgaagt atattttaga tgaagattat ttcttaatct
     4141 agagcgcttc ttgcttaccc ggcgggagcg ctttttcttt ttcttggcct tattggcccg
     4201 ggcctcgttg gccaaacgca aagaaccctc ccggcacatt tgccgagagg ggtcgaccct
     4261 atagtgagtc gtattaaaaa ggcccagtct ttcgactggg cctaagcttt agataaaaaa
     4321 cgcccggcgg caaccgagcg ttctgaacaa atccagatgg agtaaaaagc accgactcgg
     4381 tgccactttt tcaagttgat aacggactag ccttatttta acttgctatt tctagctcta
     4441 aaactgagac cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     4501 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     4561 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     4621 aacagctatg accatgatta cgaattcact ggccgtcgtt ttacaacgtc gtgactggga
     4681 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     4741 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     4801 atgaggtctc gcgtaggtac attttactca attctctaat cacggtcaag aattcgcatc
     4861 acacgcaaaa aggaaattgg aataaatgcg aaatttgaga tgttaattaa agaccttttt
     4921 gaggtctttt tttcttagat ttttggggtt atttagggga gaaaacatag gggggtacta
     4981 cgacctcccc cctaggtgtc cattgtccat tgtccaaaca aataaataaa tattgggttt
     5041 ttaatgttaa aaggttgttt tttatgttaa agtgaaaaaa acagatgttg ggaggtacag
     5101 tgatggttgt agatagaaaa gaagagaaaa aagttgctgt tactttaaga cttacaacag
     5161 aagaaaatga gatattaaat agaatcaaag aaaaatataa tattagcaaa tcagatgcaa
     5221 ccggtattct aataaaaaaa tatgcaaagg aggaatacgg tgcattttaa acaaaaaaag
     5281 atagacagca ctggcatgct gcctatctat gactaaattt tgttaaatgt attagcaccg
     5341 ttattatatc atgagcgaaa atgtaataaa agaaactgaa aacaagaaaa attcaagagg
     5401 acgtaattgg acatttgttt tatatccaga atcagcaaaa gccgagtggt tagagtattt
     5461 aaaagagtta cacattcaat ttgtagtgtc tccattacat gatagggata ctgatacaga
     5521 agataggatg aaaaaagagc attatcatat tctagtgatg tatgagggta ataaatctta
     5581 tgaacagata aaaataatta cagaagaatt gaatgcgact attccgcaga ttgcaggaag
     5641 tgtgaaaggt cttgtgagat atatgcttca cactgatgaa tcccctaatg atttttatca
     5701 aaatcattaa gttaaggtag atacacatct tgtcatatga tcaaatggtt tcgccaaaaa
     5761 tcaataatca gacaacaaaa tgtgcgaact cgatatttta cacgactctc tttaccaatt
     5821 ctgccccgaa ttacacttaa aacgactcaa cagcttaacg ttggcttgcc acgccttact
     5881 tgactgtaaa actctcactc ttaccgaact tggccgtaac ctgccaacca aagcgagaac
     5941 aaaacataac atcaaacgaa tcgaccgatt gttaggtaat cgtcacctcc acaaagagcg
     6001 actcgctgta taccgttggc atgctagctt tatctgttcg ggcaatacga tgcccattgt
     6061 acttgttgac tggtctgata tccgtgagca aaaacggctt atggtattgc gagcttcagt
     6121 cgcactacac ggtcgttctg ttactcttta tgagaaagcg ttcccgcttt cagagcaatg
     6181 ttcaaagaaa gctcatgacc aatttctagc cgaccttgcg agcattctac cgagtaacac
     6241 cacaccgctc attgtcagtg atgctggctt taaagtgcca tggtataaat ccgttgagaa
     6301 gctgggttgg tactggttaa gtcgagtaag aggaaaagta caatatgcag acctaggagc
     6361 ggaaaactgg aaacctatca gcaacttaca tgatatgtca tctagtcact caaagacttt
     6421 aggctataag aggctgacta aaagcaatcc aatctcatgc caaattctat tgtataaatc
     6481 tcgctctaaa ggccgaaaaa atcagcgctc gacacggact cattgtcacc acccgtcacc
     6541 taaaatctac tcagcgtcgg caaaggagcc atggattcta gcaactaact tacctgttga
     6601 aattcgaaca cccaaacaac ttgttaatat ctattcgaag cgaatgcaga ttgaagaaac
     6661 cttccgagac ttgaaaagtc ctgcctacgg actaggccta cgccatagcc gaacgagcag
     6721 ctcagagcgt tttgatatca tgctgctaat cgccctgatg cttcaactaa catgttggct
     6781 tgcgggcgtt catgctcaga aacaaggttg ggacaagcac ttccaggcta acacagtcag
     6841 aaatcgaaac gtactctcaa cagttcgctt aggcatggaa gttttgcggc attctggcta
     6901 cacaataaca agggaagact tactcgtggc tgcaacccta ctagctcaaa atttattcac
     6961 acatggttac gctttgggga aattatgagg ggatctctca gtgcttcaca tggacgatcc
     7021 taataaattt aaatatcaaa aagaagatat gatagtttat ggcggtgtag atgttgatga
     7081 attattaaag aaaacaacaa cagatagata taaattaatt aaagaaatga ttgagtttat
     7141 tgatgaacaa ggaatcgtag aatttaagag tttaatggat tatgcaatga agtttaaatt
     7201 tgatgattgg ttcccgcttt tatgtgataa ctcggcgtat gttattcaag aatatataaa
     7261 atcaaatcgg tataaatctg accgatagat tttgaattta ggtgtctgca gttgaaatcc
     7321 cctcaaaaac ccgatataat gggtttaata gctgaataag aacggtgctc tccaaatatt
     7381 cttatttaga aaagcaaatc taaaattatc tgaaagggga atgagaatag tgaatggacc
     7441 aataataatg actagagaag aaagaatgaa gattgttcat gaaattaagg aacgaatatt
     7501 ggataaatat ggggatgatg ttaaggctat tggtgtttat ggctctcttg gtcgtcagac
     7561 tgatgggccc tattcggata ttgagatgat gtgtgtcatg tcaacagagg aagcagagtt
     7621 cagccatgaa tggacaaccg gtgagtggaa ggtggaagtg aattttgata gcgaagagat
     7681 tctactagat tatgcatctc aggtggaatc agattggccg cttacacatg gtcaattttt
     7741 ctctattttg ccgatttatg attcaggtgg atacttagag aaagtgtatc aaactgctaa
     7801 atcggtagaa gcccaaacgt tccacgatgc gatttgtgcc cttatcgtag aagagctgtt
     7861 tgaatatgca ggcaaatggc gtaatattcg tgtgcaagga ccgacaacat ttctaccatc
     7921 cttgactgtg caggtagcaa tggcaggtgc catgttgatt ggtctgcatc atcgcatctg
     7981 ttatacgacg agcgcttcgg tcttaactga agcagttaag caatcagatc ttccttcagg
     8041 ttatgaccat ctgtgccagt tcgtaatgtc tggtcaactt tccgactctg agaaacttct
     8101 ggaatcgcta gagaatttct ggaatgggat tcaggagtgg acagaacgac acggatatat
     8161 agtggatgtg tcaaaacgca taccattttg aacgatgacc tcgagctcgg ctgacttcag
     8221 gtgctacatt tgaagagata aattgcactg aaatctagaa atattttatc tgattaataa
     8281 gatgatcttc ttgagatcgt tttggtctgc gcgtaatctc ttgctctgaa aacgaaaaaa
     8341 ccgccttgca gggcggtttt tcgaaggttc tctgagctac caactctttg aaccgaggta
     8401 actggcttgg aggagcgcag tcaccaaaac ttgtcctttc agtttagcct taaccggcgc
     8461 atgacttcaa gactaactcc tctaaatcaa ttaccagtgg ctgctgccag tggtgctttt
     8521 gcatgtcttt ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcggac
     8581 tgaacggggg gttcgtgcat acagtccagc ttggagcgaa ctgcctaccc ggaactgagt
     8641 gtcaggcgtg gaatgagaca aacgcggcca taacagcgga atgacaccgg taaaccgaaa
     8701 ggcaggaaca ggagagcgca cgagggagcc gccaggggga aacgcctggt atctttatag
     8761 tcctgtcggg tttcgccacc actgatttga gcgtcagatt tcgtgatgct tgtcaggggg
     8821 gcggagccta tggaaaaacg gctttgccgc ggccctctca cttccctgtt aagtgtacag
     8881 ggcctcttgc gggattacga aatcatcctg tggagcttag taggtttagc aagatggcag
     8941 cgcctaaatg tagaatgata aaaggattaa gagattaatt tccctaaaaa tgataaaaca
     9001 agcgttttga aagcgcttgt ttttttggtt tgcagtcaga gtagaataga agtatcaaaa
     9061 aaagcaccga ctcggtgcca ctttttcaag ttgataacgg actagcctta ttttaacttg
     9121 ctatgctgtt ttgaatggtt ccaacaagat tattttataa cttttataac aaataatcaa
     9181 ggagaaattc aaagaaattt atcagccata aaacaatact taatactata gaatgataac
     9241 aaaataaact actttttaaa agaattttgt gttataatct atttattatt aagtattggg
     9301 taatattttt tgaagagata ttttgaaaaa gaaaaattaa agcatattaa actaatttcg
     9361 gaggtcatta aaactattat tgaaatcatc aaactcatta tggatttaat ttaaactttt
     9421 tattttagga ggcaaaa
//