LOCUS       sequence-298406-        8980 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  encodes CRISPR-Cas9 system derived from plasmid pCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8980)
  AUTHORS   Ho J, Zhao M, Wojcik S, Taiaroa G, Butler M, Poulter R
  TITLE     The application of the CRISPR-Cas9 system in Pseudomonas syringae 
            pv. actinidiae.
  JOURNAL   J Med Microbiol. 2020 Mar;69(3):478-486. doi: 10.1099/jmm.0.001124. 
            Epub 2020 Jan 14.
  PUBMED    31935181
REFERENCE   2  (bases 1 to 8980)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1099/jmm.0.001124"; journalName: "J Med Microbiol"; date: 
            "2020-03"; volume: "69"; issue: "3"; pages: "478-486"
COMMENT     Sequence Label: pJH1
FEATURES             Location/Qualifiers
     source          1..8980
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(61..649)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(141..160)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(792..811)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             803..1618
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             803..1618
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(876..895)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_RNA        complement(2045..2123)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     misc_feature    6443..6574
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   6575..6610
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   6641..6676
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     primer_bind     complement(7105..7124)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        complement(join(7324..7328,7329..7345,7346..7351))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      7401..7752
                     /label=pRO1600 oriV
                     /note="broad-host-range origin of replication from 
                     Pseudomonas aeruginosa plasmid pRO1600; requires the 
                     pRO1600 Rep protein for replication (West et al., 1994)"
     CDS             7766..8599
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pRO1600 from Pseudomonas aeruginosa "
                     /label=pRO1600 Rep
                     /translation="MASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEV
                     MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
                     IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWDSIG
                     QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
                     LFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPLDLA"
ORIGIN
        1 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
       61 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
      121 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      181 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      241 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      301 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      361 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      421 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      481 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      541 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      601 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      661 gatcttttct acggggtctg acgctcagtg gaacgaaaac tctctagatc tgacgctcag
      721 tggaacgaaa actcacgtta agggattttg gtcatgaaca ataaaactgt ctgcttacat
      781 aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtctt gctctaggcc
      841 gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt
      901 cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt
      961 tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa
     1021 ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga
     1081 tgcatggtta ctcaccactg cgatccccgg gaaaacagca ttccaggtat tagaagaata
     1141 tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc
     1201 gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca
     1261 atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg
     1321 gcctgttgaa caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt
     1381 cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg
     1441 ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg
     1501 gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat
     1561 tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaaga
     1621 attaattcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     1681 cgcgcacatt tccccgaaaa gtgccacctg aggcgataag ctttaatgcg gtagtttatc
     1741 acagttaaat tgctaacgca gtcaggcacc gtgtatgaaa tctaacaatg cgctcatcgt
     1801 catcctcggc accgtcaccc tggatgctgt aggcataggc ttggttatgc cggtactgcc
     1861 gggcctcttg cgggattacg aaatcatcct gtggagctta gtaggtttag caagatggca
     1921 gcgcctaaat gtagaatgat aaaaggatta agagattaat ttccctaaaa atgataaaac
     1981 aagcgttttg aaagcgcttg tttttttggt ttgcagtcag agtagaatag aagtatcaaa
     2041 aaaagcaccg actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt
     2101 gctatgctgt tttgaatggt tccaacaaga ttattttata acttttataa caaataatca
     2161 aggagaaatt caaagaaatt tatcagccat aaaacaatac ttaatactat agaatgataa
     2221 caaaataaac taatttcgga ggtcattaaa actattattg aaatcatcaa actcattatg
     2281 gatttaattt aaacttttta ttttaggagg caaaaatgga taagaaatac tcaataggct
     2341 tagatatcgg cacaaatagc gtcggatggg cggtgatcac tgatgaatat aaggttccgt
     2401 ctaaaaagtt caaggttctg ggaaatacag accgccacag tatcaaaaaa aatcttatag
     2461 gggctctttt atttgacagt ggagagacag cggaagcgac tcgtctcaaa cggacagctc
     2521 gtagaaggta tacacgtcgg aagaatcgta tttgttatct acaggagatt ttttcaaatg
     2581 agatggcgaa agtagatgat agtttctttc atcgacttga agagtctttt ttggtggaag
     2641 aagacaagaa gcacgaacgg catcctattt ttggaaatat agtagatgaa gttgcttatc
     2701 atgagaaata tccaactatc tatcatctgc gaaaaaaatt ggtagattct actgataaag
     2761 cggatttgcg cttaatctat ttggccttag cgcatatgat caagttccgc ggccactttt
     2821 tgatcgaggg ggatttcaac ccggataaca gcgacgtgga caaactgttc atccagttgg
     2881 tgcagaccta caaccagttc ttcgaagaaa acccgatcaa cgcaagcgga gtagatgcta
     2941 aagcgatcct gtccgcacga ttcagtaagt cgagccgatt agaaaatctc attgctcagc
     3001 tccccggcga gaagaaaaat ggcttattcg ggaacctcat tgctttgtcg ttgggcttga
     3061 ccccgaactt caagtcgaac ttcgacttgg cagaagacgc taaatttcag ctctccaagg
     3121 acacgtacga cgatgacctg gacaatttgt tggcgcaaat cggcgaccaa tacgctgact
     3181 tgttcttggg agccaagaac ttgtctgacg ctatcttgct ctctgacatc ctcagcgtaa
     3241 acactgaaat cactaaggcg cccctatcag cttcaatgat taaacgctac gacgagcacc
     3301 atcaggactt gactcttttg aaagcgttgg tccgacagca gctcccggag aagtacaaag
     3361 agatcttctt cgaccagtcc aaaaacggat atgcaggtta tattgacggg ggcgccagcc
     3421 aggaggagtt ctacaaattc atcaaaccga tcttggaaaa aatggacggt acggaggagt
     3481 tgttggtgaa actaaatcgt gaagatttgc tgcgcaagca acggaccttt gacaacggct
     3541 ctattcccca tcaaattcac ttgggtgagc tgcacgcgat cttgagaaga caggaggact
     3601 tctacccgtt cttgaaagac aaccgtgaga agatcgagaa aatcttgacc ttccggattc
     3661 cttattatgt tggtccattg gcgcgtggca atagtcgttt tgcatggatg acccggaagt
     3721 ccgaagaaac gatcacccca tggaatttcg aagaagttgt cgacaaaggt gcctcagctc
     3781 aatctttcat tgagcgcatg acgaacttcg acaaaaacct cccgaacgag aaagtcctgc
     3841 cgaaacacag cttgctctac gagtacttca cggtctacaa cgagttgacg aaggtcaaat
     3901 acgtcactga aggaatgcga aaaccagcat ttctttcagg tgagcagaag aaagccatcg
     3961 tggacttgct cttcaaaacg aaccgcaaag tcaccgtgaa gcagttgaaa gaggactact
     4021 tcaaaaaaat cgagtgcttc gacagcgtcg agatctccgg agttgaggac agcttcaacg
     4081 cttccttggg cacctaccac gacttgctga aaatcatcaa agacaaagac ttcttggaca
     4141 acgaggagaa cgaggacatc ttggaggaca tcgtcttgac gttgaccttg ttcgaggaca
     4201 gggagatgat cgaggagaga ctcaaaacat atgctcacct ctttgatgat aaggtgatga
     4261 aacagcttaa acgtcgccgt tatactggtt ggggacgttt gtctcgaaaa ttgattaatg
     4321 gtattaggga taagcaatct ggcaaaacaa tattagattt tttgaaatca gatggttttg
     4381 ccaatcgcaa ttttatgcag ctgatccatg atgatagttt gacatttaaa gaagacattc
     4441 aaaaagcgca ggtgtctgga caaggcgata gtttacatga acatattgca aatttagctg
     4501 gtagccctgc tattaaaaaa ggtattttac agactgtaaa agttgttgat gaattggtca
     4561 aagtaatggg gcggcataag ccagaaaata tcgttattga aatggcacgt gaaaatcaga
     4621 caactcaaaa gggccagaaa aattcgcgag agcgtatgaa acgaatcgaa gaaggtatca
     4681 aagaattagg aagtcagatt cttaaagagc atcctgttga aaatactcaa ttgcaaaatg
     4741 aaaagctcta tctctattat ctccaaaatg gaagagacat gtatgtggac caagaattag
     4801 atattaatcg tttaagtgat tatgatgtcg atcacattgt tccacaaagt ttccttaaag
     4861 acgattcaat agacaataag gtcttaacgc gttctgataa aaatcgtggt aaatcggata
     4921 acgttccaag tgaagaagta gtcaaaaaga tgaaaaacta ttggagacaa cttctaaacg
     4981 ccaagttaat cactcaacgt aagtttgata atttaacgaa agctgaacgt ggaggtttga
     5041 gtgaacttga taaagctggt tttatcaaac gccaattggt tgaaactcgc caaatcacta
     5101 agcatgtggc acaaattttg gatagtcgca tgaatactaa atacgatgaa aatgataaac
     5161 ttattcgaga ggttaaagtg attaccttaa aatctaaatt agtttctgac ttccgaaaag
     5221 atttccaatt ctataaagta cgtgagatta acaattacca tcatgcccat gatgcgtatc
     5281 taaatgccgt cgttggaact gctttgatta agaaatatcc aaaacttgaa tcggagtttg
     5341 tctatggtga ttataaagtt tatgatgttc gtaaaatgat tgctaagtct gagcaagaaa
     5401 taggcaaagc aaccgcaaaa tatttctttt actctaatat catgaacttc ttcaaaacag
     5461 aaattacact tgcaaatgga gagattcgca aacgccctct aatcgaaact aatggggaaa
     5521 ctggagaaat tgtctgggat aaagggcgag attttgccac agtgcgcaaa gtattgtcca
     5581 tgccccaagt caatattgtc aagaaaacag aagtacagac aggcggattc tccaaggagt
     5641 caattttacc aaaaagaaat tcggacaagc ttattgctcg taaaaaagac tgggatccaa
     5701 aaaaatatgg tggttttgat agtccaacgg tagcttattc agtcctagtg gttgctaagg
     5761 tggaaaaagg gaaatcgaag aagttaaaat ccgttaaaga gttactaggg atcacaatta
     5821 tggaaagaag ttcctttgaa aaaaatccga ttgacttttt agaagctaaa ggatataagg
     5881 aagttaaaaa agacttaatc attaaactac ctaaatatag tctttttgag ttagaaaacg
     5941 gtcgtaaacg gatgctggct agtgccggag aattacaaaa aggaaatgag ctggctctgc
     6001 caagcaaata tgtgaatttt ttatatttag ctagtcatta tgaaaagttg aagggtagtc
     6061 cagaagataa cgaacaaaaa caattgtttg tggagcagca taagcattat ttagatgaga
     6121 ttattgagca aatcagtgaa ttttctaagc gtgttatttt agcagatgcc aatttagata
     6181 aagttcttag tgcatataac aaacatagag acaaaccaat acgtgaacaa gcagaaaata
     6241 ttattcattt atttacgttg acgaatcttg gagctcccgc tgcttttaaa tattttgata
     6301 caacaattga tcgtaaacga tatacgtcta caaaagaagt tttagatgcc actcttatcc
     6361 atcaatccat cactggtctt tatgaaacac gcattgattt gagtcagcta ggaggtgact
     6421 gaagtatatt ttagatgaag attatttctt aataactaaa aatatggtat aatactctta
     6481 ataaatgcag taatacaggg gcttttcaag actgaagtct agctgagaca aatagtgcga
     6541 ttacgaaatt ttttagacaa aaatagtcta cgaggtttta gagctatgct gttttgaatg
     6601 gtcccaaaac tgagaccagt ctcggaagct caaaggtctc gttttagagc tatgctgttt
     6661 tgaatggtcc caaaacttca gcacactgag acttgttgag ttccatgttt tagagctatg
     6721 ctgttttgaa tggactccat tcaacattgc cgatgataac ttgagaaaga gggttaatac
     6781 cagcagtcgg ataccttcct attctttctg ttaaagcgtt ttcatgttat aataggcaaa
     6841 agaagagtag tgtgatcgtc cattccgaca gcatcgccag tcactatggc gtgctgctag
     6901 cgctatatgc gttgatgcaa tttctatgcg cacccgttct cggagcactg tccgaccgct
     6961 ttggccgccg cccagtcctg ctcgcttcgc tacttggagc cactatcgac tacgcgatca
     7021 tggcgaccac acccgtcctg tggatcctct acgccggacg catcgtggcc ggcatcaccg
     7081 gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg
     7141 ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg
     7201 ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca
     7261 acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc
     7321 gacgtttacg aaccgaacag gcttatgtca attcgcctct caggcgccgc tggtgccgct
     7381 ggttggacgc caagggtgaa tccgcctcga taccctgatt actcgcttcc tgcgccctct
     7441 caggcggcga taggggactg gtaaaacggg gattgcccag acgcctcccc cgccccttca
     7501 ggggcacaaa tgcggcccca acggggccac gtagtggtgc gttttttgcg tttccaccct
     7561 tttcttcctt ttccctttta aaccttttag gacgtctaca ggccacgtaa tccgtggcct
     7621 gtagagttta aaaagggacg gatttgttgc cattaaggga cggatttgtt gttaagaagg
     7681 gacggatttg ttgttgtaaa gggacggatt tgttgtattg tgggacgcag atacagtgtc
     7741 cccttataca caaggaatgt cgaacgtggc ctcaccccca atggtttaca aaagcaatgc
     7801 cctggtcgag gccgcgtatc gcctcagtgt tcaggaacag cggatcgttc tggcctgtat
     7861 tagccaggtg aagaggagcg agcctgtcac cgatgaagtg atgtattcag tgacggcgga
     7921 ggacatagcg acgatggcgg gtgtccctat cgaatcttcc tacaaccagc tcaaagaagc
     7981 ggccctgcgc ctgaaacggc gggaagtccg gttaacccaa gagcccaatg gcaaggggaa
     8041 aagaccgagt gtgatgatta ccggctgggt gcaaacaatc atctaccggg agggtgaggg
     8101 ccgtgtagaa ctcaggttca ccaaagacat gctgccgtac ctgacggaac tcaccaaaca
     8161 gttcaccaaa tacgccttgg ctgacgtggc caagatggac agcacccacg cgatcaggct
     8221 ttacgagctg ctcatgcaat gggacagcat cggccagcgc gaaatagaaa ttgaccagct
     8281 gcgaaagtgg tttcaactgg aaggccggta tccctcgatc aaggacttca agttgcgagt
     8341 gcttgatcca gccgtgacgc agatcaacga gcacagcccg ctacaggtgg agtgggcgca
     8401 gcgaaagacc gggcgcaagg tcacacatct gttgttcagt tttggaccga agaagcccgc
     8461 caaggcggtg ggtaaggccc cagcgaagcg caaggccggg aagatttcag atgctgagat
     8521 cgcgaaacag gctcgccctg gtgagacatg ggaagcggcc cgcgctcgac taacccagat
     8581 gccgctggat ctggcctaga ggccgtggcc accacggccc ggcctgcctt tcaggctgcg
     8641 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgcgtcg accgatgccc
     8701 ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc
     8761 gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg
     8821 gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg
     8881 gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt
     8941 ttcggcgaga agcaggccat tatcgccggc atggcggccg
//
