LOCUS       62056_13505        6216 bp DNA     circular SYN 07-JUL-2019
DEFINITION  Cloning vector pKN02, complete sequence.
ACCESSION   MK756314
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6216)
  AUTHORS   Neil K, Allard N, Jordan D, Rodrigue S.
  TITLE     Assembly of large mobilizable genetic cargo by double recombinase 
            operated insertion of DNA (DROID)
  JOURNAL   Plasmid (2019) In press
  PUBMED    31247227
REFERENCE   2  (bases 1 to 6216)
  AUTHORS   Neil K, Rodrigue S.
  TITLE     Direct Submission
  JOURNAL   Submitted (04-APR-2019) Biology, University of Sherbrooke, 2500 
            Boulevard de l'Universite, Sherbrooke, Quebec J1K 2R1, Canada
REFERENCE   3  (bases 1 to 6216)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plasmid 
            (2019) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (04-APR-2019) Biology, University of Sherbrooke, 2500 Boulevard de 
            l'Universite, Sherbrooke, Quebec J1K 2R1, Canada"
COMMENT     ##Assembly-Data-START##
            Assembly Method       :: ApE v. 2018-05-04
            Sequencing Technology :: Putative Sequence
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..6216
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             53..910
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     misc_RNA        1070..1145
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     oriT            1351..1459
                     /label=oriT
                     /note="incP origin of transfer"
     rep_origin      1478..1866
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     CDS             2110..6213
                     /codon_start=1
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
                     /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"
ORIGIN
        1 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
       61 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
      121 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
      181 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
      241 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga
      301 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
      361 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
      421 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
      481 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
      541 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc
      601 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
      661 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
      721 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
      781 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
      841 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
      901 taagcattgg taacaggtcg gtcagtttca cctgatttac gtaaaaaccc gcttcggcgg
      961 gtttttgctt ttggaggggc agaaagatga atgactgtct cataaaaaat ttatttgctt
     1021 tcaggaaaat ttttctgtat aatagattct cgcaagatgt ggcgtgttag ttttagagct
     1081 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     1141 ggtgctttta aaaaaaaaac cccgccctgt caggggcggg gttttttttt acaagtctca
     1201 gtgtgctgaa ctagtctttt ccgctgcata accctgcttc ggggtcatta tagcgatttt
     1261 ttcggtatat ccatcctttt tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga
     1321 ctttccttgg tgtatccaac ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc
     1381 gagcgggtgt tccttcttca ctgtccctta ttcgcacctg gcggtgctca acgggaatcc
     1441 tgctctgcga ggctggccgt aggccggccg gcgcgccgat ctgaagatca gcagttcaac
     1501 ctgttgatag tacgtactaa gctctcatgt ttcacgtact aagctctcat gtttaacgta
     1561 ctaagctctc atgtttaacg aactaaaccc tcatggctaa cgtactaagc tctcatggct
     1621 aacgtactaa gctctcatgt ttcacgtact aagctctcat gtttgaacaa taaaattaat
     1681 ataaatcagc aacttaaata gcctctaagg ttttaagttt tataagaaaa aaaagaatat
     1741 ataaggcttt taaagccttt aaggtttaac ggttgtggac aacaagccag ggatgtaacg
     1801 cactgagaag cccttagagc ctctcaaagc aattttgagt gacacaggaa cacttaacgg
     1861 ctgacatggt ttatcagcca taaaacaata cttaatacta tagaatgata acaaaataaa
     1921 ctacttttta aaagaatttt gtgttataat ctatttatta ttaagtattg ggtaatattt
     1981 tttgaagaga tattttgaaa aagaaaaatt aaagcatatt aaactaattt cggaggtcat
     2041 taaaactatt attgaaatca tcaaactcat tatggattta atttaaactt tttattttag
     2101 gaggcaaaaa tggataagaa atactcaata ggcttagata tcggcacaaa tagcgtcgga
     2161 tgggcggtga tcactgatga atataaggtt ccgtctaaaa agttcaaggt tctgggaaat
     2221 acagaccgcc acagtatcaa aaaaaatctt ataggggctc ttttatttga cagtggagag
     2281 acagcggaag cgactcgtct caaacggaca gctcgtagaa ggtatacacg tcggaagaat
     2341 cgtatttgtt atctacagga gattttttca aatgagatgg cgaaagtaga tgatagtttc
     2401 tttcatcgac ttgaagagtc ttttttggtg gaagaagaca agaagcatga acgtcatcct
     2461 atttttggaa atatagtaga tgaagttgct tatcatgaga aatatccaac tatctatcat
     2521 ctgcgaaaaa aattggtaga ttctactgat aaagcggatt tgcgcttaat ctatttggcc
     2581 ttagcgcata tgattaagtt tcgtggtcat tttttgattg agggagattt aaatcctgat
     2641 aatagtgatg tggacaaact atttatccag ttggtacaaa cctacaatca attatttgaa
     2701 gaaaacccta ttaacgcaag tggagtagat gctaaagcga ttctttctgc acgattgagt
     2761 aaatcaagac gattagaaaa tctcattgct cagctccccg gtgagaagaa aaatggctta
     2821 tttgggaatc tcattgcttt gtcattgggt ttgaccccta attttaaatc aaattttgat
     2881 ttggcagaag atgctaaatt acagctttca aaagatactt acgatgatga tttagataat
     2941 ttattggcgc aaattggaga tcaatatgct gatttgtttt tggcagctaa gaatttatca
     3001 gatgctattt tactttcaga tatcctaaga gtaaatactg aaataactaa ggctccccta
     3061 tcagcttcaa tgattaaacg ctacgatgaa catcatcaag acttgactct tttaaaagct
     3121 ttagttcgac aacaacttcc agaaaagtat aaagaaatct tttttgatca atcaaaaaac
     3181 ggatatgcag gttatattga tgggggagct agccaagaag aattttataa atttatcaaa
     3241 ccaattttag aaaaaatgga tggtactgag gaattattgg tgaaactaaa tcgtgaagat
     3301 ttgctgcgca agcaacggac ctttgacaac ggctctattc cccatcaaat tcacttgggt
     3361 gagctgcatg ctattttgag aagacaagaa gacttttatc catttttaaa agacaatcgt
     3421 gagaagattg aaaaaatctt gacttttcga attccttatt atgttggtcc attggcgcgt
     3481 ggcaatagtc gttttgcatg gatgactcgg aagtctgaag aaacaattac cccatggaat
     3541 tttgaagaag ttgtcgataa aggtgcttca gctcaatcat ttattgaacg catgacaaac
     3601 tttgataaaa atcttccaaa tgaaaaagta ctaccaaaac atagtttgct ttatgagtat
     3661 tttacggttt ataacgaatt gacaaaggtc aaatatgtta ctgaaggaat gcgaaaacca
     3721 gcatttcttt caggtgaaca gaagaaagcc attgttgatt tactcttcaa aacaaatcga
     3781 aaagtaaccg ttaagcaatt aaaagaagat tatttcaaaa aaatagaatg ttttgatagt
     3841 gttgaaattt caggagttga agatagattt aatgcttcat taggtaccta ccatgatttg
     3901 ctaaaaatta ttaaagataa agattttttg gataatgaag aaaatgaaga tatcttagag
     3961 gatattgttt taacattgac cttatttgaa gatagggaga tgattgagga aagacttaaa
     4021 acatatgctc acctctttga tgataaggtg atgaaacagc ttaaacgtcg ccgttatact
     4081 ggttggggac gtttgtctcg aaaattgatt aatggtatta gggataagca atctggcaaa
     4141 acaatattag attttttgaa atcagatggt tttgccaatc gcaattttat gcagctgatc
     4201 catgatgata gtttgacatt taaagaagac attcaaaaag cacaagtgtc tggacaaggc
     4261 gatagtttac atgaacatat tgcaaattta gctggtagcc ctgctattaa aaaaggtatt
     4321 ttacagactg taaaagttgt tgatgaattg gtcaaagtaa tggggcggca taagccagaa
     4381 aatatcgtta ttgaaatggc acgtgaaaat cagacaactc aaaagggcca gaaaaattcg
     4441 cgagagcgta tgaaacgaat cgaagaaggt atcaaagaat taggaagtca gattcttaaa
     4501 gagcatcctg ttgaaaatac tcaattgcaa aatgaaaagc tctatctcta ttatctccaa
     4561 aatggaagag acatgtatgt ggaccaagaa ttagatatta atcgtttaag tgattatgat
     4621 gtcgatcaca ttgttccaca aagtttcctt aaagacgatt caatagacaa taaggtctta
     4681 acgcgttctg ataaaaatcg tggtaaatcg gataacgttc caagtgaaga agtagtcaaa
     4741 aagatgaaaa actattggag acaacttcta aacgccaagt taatcactca acgtaagttt
     4801 gataatttaa cgaaagctga acgtggaggt ttgagtgaac ttgataaagc tggttttatc
     4861 aaacgccaat tggttgaaac tcgccaaatc actaagcatg tggcacaaat tttggatagt
     4921 cgcatgaata ctaaatacga tgaaaatgat aaacttattc gagaggttaa agtgattacc
     4981 ttaaaatcta aattagtttc tgacttccga aaagatttcc aattctataa agtacgtgag
     5041 attaacaatt accatcatgc ccatgatgcg tatctaaatg ccgtcgttgg aactgctttg
     5101 attaagaaat atccaaaact tgaatcggag tttgtctatg gtgattataa agtttatgat
     5161 gttcgtaaaa tgattgctaa gtctgagcaa gaaataggca aagcaaccgc aaaatatttc
     5221 ttttactcta atatcatgaa cttcttcaaa acagaaatta cacttgcaaa tggagagatt
     5281 cgcaaacgcc ctctaatcga aactaatggg gaaactggag aaattgtctg ggataaaggg
     5341 cgagattttg ccacagtgcg caaagtattg tccatgcccc aagtcaatat tgtcaagaaa
     5401 acagaagtac agacaggcgg attctccaag gagtcaattt taccaaaaag aaattcggac
     5461 aagcttattg ctcgtaaaaa agactgggat ccaaaaaaat atggtggttt tgatagtcca
     5521 acggtagctt attcagtcct agtggttgct aaggtggaaa aagggaaatc gaagaagtta
     5581 aaatccgtta aagagttact agggatcaca attatggaaa gaagttcctt tgaaaaaaat
     5641 ccgattgact ttttagaagc taaaggatat aaggaagtta aaaaagactt aatcattaaa
     5701 ctacctaaat atagtctttt tgagttagaa aacggtcgta aacggatgct ggctagtgcc
     5761 ggagaattac aaaaaggaaa tgagctggct ctgccaagca aatatgtgaa ttttttatat
     5821 ttagctagtc attatgaaaa gttgaagggt agtccagaag ataacgaaca aaaacaattg
     5881 tttgtggagc agcataagca ttatttagat gagattattg agcaaatcag tgaattttct
     5941 aagcgtgtta ttttagcaga tgccaattta gataaagttc ttagtgcata taacaaacat
     6001 agagacaaac caatacgtga acaagcagaa aatattattc atttatttac gttgacgaat
     6061 cttggagctc ccgctgcttt taaatatttt gatacaacaa ttgatcgtaa acgatatacg
     6121 tctacaaaag aagttttaga tgccactctt atccatcaat ccatcactgg tctttatgaa
     6181 acacgcattg atttgagtca gctaggaggt gactga
//
