LOCUS       sequence-307394-        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(66..654)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(146..165)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(797..816)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             808..1623
                     /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             808..1623
                     /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(881..900)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_RNA        complement(2050..2128)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     misc_feature    6448..6579
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   6580..6615
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   6646..6681
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     primer_bind     complement(7110..7129)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        complement(join(7329..7333,7334..7350,7351..7356))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     rep_origin      7406..7757
                     /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             7771..8604
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pRO1600 from Pseudomonas aeruginosa "
                     /label=pRO1600 Rep
                     /translation="MASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEV
                     MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
                     IIYREGEGRVELRFTEDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWDSIG
                     QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
                     LFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPLDLA"
ORIGIN
        1 ggccgcatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
       61 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
      121 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
      181 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
      241 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
      301 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
      361 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
      421 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
      481 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
      541 gttaccttcg gaaaaagagt tggtagctct tgatccggca aaaaaaccac cgctggtagc
      601 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
      661 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactctct agatctgacg
      721 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gaacaataaa actgtctgct
      781 tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctct
      841 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat
      901 aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag
      961 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga
     1021 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct
     1081 gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa
     1141 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg
     1201 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag
     1261 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgaacgtaat
     1321 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat
     1381 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta
     1441 ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc
     1501 ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat
     1561 ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc
     1621 taagaattaa ttcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     1681 ggttccgcgc acatttcccc gaaaagtgcc acctgaggcg ataagcttta atgcggtagt
     1741 ttatcacagt taaattgcta acgcagtcag gcaccgtgta tgaaatctaa caatgcgctc
     1801 atcgtcatcc tcggcaccgt caccctggat gctgtaggca taggcttggt tatgccggta
     1861 ctgccgggcc tcttgcggga ttacgaaatc atcctgtgga gcttagtagg tttagcaaga
     1921 tggcagcgcc taaatgtaga atgataaaag gattaagaga ttaatttccc taaaaatgat
     1981 aaaacaagcg ttttgaaagc gcttgttttt ttggtttgca gtcagagtag aatagaagta
     2041 tcaaaaaaag caccgactcg gtgccacttt ttcaagttga taacggacta gccttatttt
     2101 aacttgctat gctgttttga atggttccaa caagattatt ttataacttt tataacaaat
     2161 aatcaaggag aaattcaaag aaatttatca gccataaaac aatacttaat actatagaat
     2221 gataacaaaa taaactaatt tcggaggtca ttaaaactat tattgaaatc atcaaactca
     2281 ttatggattt aatttaaact ttttatttta ggaggcaaaa atggataaga aatactcaat
     2341 aggcttagat atcggcacaa atagcgtcgg atgggcggtg atcactgatg aatataaggt
     2401 tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc cacagtatca aaaaaaatct
     2461 tataggggct cttttatttg acagtggaga gacagcggaa gcgactcgtc tcaaacggac
     2521 agctcgtaga aggtatacac gtcggaagaa tcgtatttgt tatctacagg agattttttc
     2581 aaatgagatg gcgaaagtag atgatagttt ctttcatcga cttgaagagt cttttttggt
     2641 ggaagaagac aagaagcacg aacggcatcc tatttttgga aatatagtag atgaagttgc
     2701 ttatcatgag aaatatccaa ctatctatca tctgcgaaaa aaattggtag attctactga
     2761 taaagcggat ttgcgcttaa tctatttggc cttagcgcat atgatcaagt tccgcggcca
     2821 ctttttgatc gagggggatt tcaacccgga taacagcgac gtggacaaac tgttcatcca
     2881 gttggtgcag acctacaacc agttcttcga agaaaacccg atcaacgcaa gcggagtaga
     2941 tgctaaagcg atcctgtccg cacgattcag taagtcgagc cgattagaaa atctcattgc
     3001 tcagctcccc ggcgagaaga aaaatggctt attcgggaac ctcattgctt tgtcgttggg
     3061 cttgaccccg aacttcaagt cgaacttcga cttggcagaa gacgctaaat ttcagctctc
     3121 caaggacacg tacgacgatg acctggacaa tttgttggcg caaatcggcg accaatacgc
     3181 tgacttgttc ttgggagcca agaacttgtc tgacgctatc ttgctctctg acatcctcag
     3241 cgtaaacact gaaatcacta aggcgcccct atcagcttca atgattaaac gctacgacga
     3301 gcaccatcag gacttgactc ttttgaaagc gttggtccga cagcagctcc cggagaagta
     3361 caaagagatc ttcttcgacc agtccaaaaa cggatatgca ggttatattg acgggggcgc
     3421 cagccaggag gagttctaca aattcatcaa accgatcttg gaaaaaatgg acggtacgga
     3481 ggagttgttg gtgaaactaa atcgtgaaga tttgctgcgc aagcaacgga cctttgacaa
     3541 cggctctatt ccccatcaaa ttcacttggg tgagctgcac gcgatcttga gaagacagga
     3601 ggacttctac ccgttcttga aagacaaccg tgagaagatc gagaaaatct tgaccttccg
     3661 gattccttat tatgttggtc cattggcgcg tggcaatagt cgttttgcat ggatgacccg
     3721 gaagtccgaa gaaacgatca ccccatggaa tttcgaagaa gttgtcgaca aaggtgcctc
     3781 agctcaatct ttcattgagc gcatgacgaa cttcgacaaa aacctcccga acgagaaagt
     3841 cctgccgaaa cacagcttgc tctacgagta cttcacggtc tacaacgagt tgacgaaggt
     3901 caaatacgtc actgaaggaa tgcgaaaacc agcatttctt tcaggtgagc agaagaaagc
     3961 catcgtggac ttgctcttca aaacgaaccg caaagtcacc gtgaagcagt tgaaagagga
     4021 ctacttcaaa aaaatcgagt gcttcgacag cgtcgagatc tccggagttg aggacagctt
     4081 caacgcttcc ttgggcacct accacgactt gctgaaaatc atcaaagaca aagacttctt
     4141 ggacaacgag gagaacgagg acatcttgga ggacatcgtc ttgacgttga ccttgttcga
     4201 ggacagggag atgatcgagg agagactcaa aacatatgct cacctctttg atgataaggt
     4261 gatgaaacag cttaaacgtc gccgttatac tggttgggga cgtttgtctc gaaaattgat
     4321 taatggtatt agggataagc aatctggcaa aacaatatta gattttttga aatcagatgg
     4381 ttttgccaat cgcaatttta tgcagctgat ccatgatgat agtttgacat ttaaagaaga
     4441 cattcaaaaa gcgcaggtgt ctggacaagg cgatagttta catgaacata ttgcaaattt
     4501 agctggtagc cctgctatta aaaaaggtat tttacagact gtaaaagttg ttgatgaatt
     4561 ggtcaaagta atggggcggc ataagccaga aaatatcgtt attgaaatgg cacgtgaaaa
     4621 tcagacaact caaaagggcc agaaaaattc gcgagagcgt atgaaacgaa tcgaagaagg
     4681 tatcaaagaa ttaggaagtc agattcttaa agagcatcct gttgaaaata ctcaattgca
     4741 aaatgaaaag ctctatctct attatctcca aaatggaaga gacatgtatg tggaccaaga
     4801 attagatatt aatcgtttaa gtgattatga tgtcgatcac attgttccac aaagtttcct
     4861 taaagacgat tcaatagaca ataaggtctt aacgcgttct gataaaaatc gtggtaaatc
     4921 ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa aactattgga gacaacttct
     4981 aaacgccaag ttaatcactc aacgtaagtt tgataattta acgaaagctg aacgtggagg
     5041 tttgagtgaa cttgataaag ctggttttat caaacgccaa ttggttgaaa ctcgccaaat
     5101 cactaagcat gtggcacaaa ttttggatag tcgcatgaat actaaatacg atgaaaatga
     5161 taaacttatt cgagaggtta aagtgattac cttaaaatct aaattagttt ctgacttccg
     5221 aaaagatttc caattctata aagtacgtga gattaacaat taccatcatg cccatgatgc
     5281 gtatctaaat gccgtcgttg gaactgcttt gattaagaaa tatccaaaac ttgaatcgga
     5341 gtttgtctat ggtgattata aagtttatga tgttcgtaaa atgattgcta agtctgagca
     5401 agaaataggc aaagcaaccg caaaatattt cttttactct aatatcatga acttcttcaa
     5461 aacagaaatt acacttgcaa atggagagat tcgcaaacgc cctctaatcg aaactaatgg
     5521 ggaaactgga gaaattgtct gggataaagg gcgagatttt gccacagtgc gcaaagtatt
     5581 gtccatgccc caagtcaata ttgtcaagaa aacagaagta cagacaggcg gattctccaa
     5641 ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt gctcgtaaaa aagactggga
     5701 tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct tattcagtcc tagtggttgc
     5761 taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt aaagagttac tagggatcac
     5821 aattatggaa agaagttcct ttgaaaaaaa tccgattgac tttttagaag ctaaaggata
     5881 taaggaagtt aaaaaagact taatcattaa actacctaaa tatagtcttt ttgagttaga
     5941 aaacggtcgt aaacggatgc tggctagtgc cggagaatta caaaaaggaa atgagctggc
     6001 tctgccaagc aaatatgtga attttttata tttagctagt cattatgaaa agttgaaggg
     6061 tagtccagaa gataacgaac aaaaacaatt gtttgtggag cagcataagc attatttaga
     6121 tgagattatt gagcaaatca gtgaattttc taagcgtgtt attttagcag atgccaattt
     6181 agataaagtt cttagtgcat ataacaaaca tagagacaaa ccaatacgtg aacaagcaga
     6241 aaatattatt catttattta cgttgacgaa tcttggagct cccgctgctt ttaaatattt
     6301 tgatacaaca attgatcgta aacgatatac gtctacaaaa gaagttttag atgccactct
     6361 tatccatcaa tccatcactg gtctttatga aacacgcatt gatttgagtc agctaggagg
     6421 tgactgaagt atattttaga tgaagattat ttcttaataa ctaaaaatat ggtataatac
     6481 tcttaataaa tgcagtaata caggggcttt tcaagactga agtctagctg agacaaatag
     6541 tgcgattacg aaatttttta gacaaaaata gtctacgagg ttttagagct atgctgtttt
     6601 gaatggtccc aaaactgaga ccagtctcgg aagctcaaag gtctcgtttt agagctatgc
     6661 tgttttgaat ggtcccaaaa cttcagcaca ctgagacttg ttgagttcca tgttttagag
     6721 ctatgctgtt ttgaatggac tccattcaac attgccgatg ataacttgag aaagagggtt
     6781 aataccagca gtcggatacc ttcctattct ttctgttaaa gcgttttcat gttataatag
     6841 gcaaaagaag agtagtgtga tcgtccattc cgacagcatc gccagtcact atggcgtgct
     6901 gctagcgcta tatgcgttga tgcaatttct atgcgcaccc gttctcggag cactgtccga
     6961 ccgctttggc cgccgcccag tcctgctcgc ttcgctactt ggagccacta tcgactacgc
     7021 gatcatggcg accacacccg tcctgtggat cctctacgcc ggacgcatcg tggccggcat
     7081 caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga
     7141 tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc
     7201 cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt
     7261 gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga
     7321 gcgtcgacgt ttacgaaccg aacaggctta tgtcaattcg cctctcaggc gccgctggtg
     7381 ccgctggttg gacgccaagg gtgaatccgc ctcgataccc tgactactcg cttcctgcgc
     7441 cctctcaggc ggcgataggg gactggtaaa acggggattg cccagacgcc tcccccgccc
     7501 cttcaggggc acaaatgcgg ccccaacggg gccacgtagt ggtgcgtttt ttgcgtttcc
     7561 acccttttct tccttttccc ttttaaacct tttaggacgt ctacaggcca cgtaatccgt
     7621 ggcctgtaga gtttaaaaag ggacggattt gttgccatta agggacggat ttgttgttaa
     7681 gaagggacgg atttgttgtt gtaaagggac ggatttgttg tattgtggga cgcagataca
     7741 gtgtcccctt atacacaagg aatgtcgaac gtggcctcac ccccaatggt ttacaaaagc
     7801 aatgccctgg tcgaggccgc gtatcgcctc agtgttcagg aacagcggat cgttctggcc
     7861 tgtattagcc aggtgaagag gagcgagcct gtcaccgatg aagtgatgta ttcagtgacg
     7921 gcggaggaca tagcgacgat ggcgggtgtc cctatcgaat cttcctacaa ccagctcaaa
     7981 gaagcggccc tgcgcctgaa acggcgggaa gtccggttaa cccaagagcc caatggcaag
     8041 gggaaaagac cgagtgtgat gattaccggc tgggtgcaaa caatcatcta ccgggagggt
     8101 gagggccgtg tagaactcag gttcaccgaa gacatgctgc cgtacctgac ggaactcacc
     8161 aaacagttca ccaaatacgc cttggctgac gtggccaaga tggacagcac ccacgcgatc
     8221 aggctttacg agctgctcat gcaatgggac agcatcggcc agcgcgaaat agaaattgac
     8281 cagctgcgaa agtggtttca actggaaggc cggtatccct cgatcaagga cttcaagttg
     8341 cgagtgcttg atccagccgt gacgcagatc aacgagcaca gcccgctaca ggtggagtgg
     8401 gcgcagcgaa agaccgggcg caaggtcaca catctgttgt tcagttttgg accgaagaag
     8461 cccgccaagg cggtgggtaa ggccccagcg aagcgcaagg ccgggaagat ttcagatgct
     8521 gagatcgcga aacaggctcg ccctggtgag acatgggaag cggcccgcgc tcgactaacc
     8581 cagatgccgc tggatctggc ctagaggccg tggccaccac ggcccggcct gcctttcagg
     8641 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg cgtcgaccga
     8701 tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg
     8761 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
     8821 tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc
     8881 ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt cccgccacca
     8941 aacgtttcgg cgagaagcag gccattatcg ccggcatggc
//
