LOCUS       62056_13395        8722 bp DNA     circular SYN 07-JUL-2019
DEFINITION  Cloning vector pKill3, complete sequence.
ACCESSION   MK756313
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8722)
  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 8722)
  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 8722)
  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..8722
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..58
                     /label=attPbxb1
                     /note="attPbxb1"
     rep_origin      539..761
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             809..1756
                     /label=Rep101(Ts)
                     /note="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
     protein_bind    2192..2239
                     /label=FRT
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     regulatory      2267..2348
                     /label=apFAB391
                     /note="apFAB391"
                     /regulatory_class="terminator"
     regulatory      2349..2398
                     /allele="PN25"
                     /regulatory_class="promoter"
     misc_feature    2399..2418
                     /label=spacer
                     /note="spacer"
     misc_RNA        2419..2494
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      2499..2529
                     /label=B1006U10
                     /note="B1006U10"
                     /regulatory_class="terminator"
     regulatory      2590..2612
                     /label=apFAB391
                     /note="apFAB391"
                     /regulatory_class="terminator"
     regulatory      2613..2662
                     /label=PN25
                     /note="PN25"
                     /regulatory_class="promoter"
     misc_RNA        2663..2762
                     /product="gRNA_2"
     misc_feature    2663..2682
                     /label=spacer
                     /note="spacer"
     regulatory      2763..2793
                     /label=B1006U10
                     /note="B1006U10"
                     /regulatory_class="terminator"
     regulatory      2854..2876
                     /label=apFAB391
                     /note="apFAB391"
                     /regulatory_class="terminator"
     regulatory      2877..2926
                     /allele="PN25"
                     /regulatory_class="promoter"
     misc_RNA        2927..3026
                     /product="gRNA_3"
     misc_feature    2927..2946
                     /label=spacer
                     /note="spacer"
     regulatory      3027..3057
                     /label=B1006U10
                     /note="B1006U10"
                     /regulatory_class="terminator"
     regulatory      3255..3304
                     /regulatory_class="promoter"
     CDS             3393..4184
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     CDS             4616..8719
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
ORIGIN
        1 gtcgtggttt gtctggtcaa ccaccgcggt ctcagtggtg tacggtacaa accccgaccg
       61 acagtaagac gggtaagcct gttgatgata ccgctgcctt actgggtgca ttagccagtc
      121 tgaatgacct gtcacgggat aatccgaagt ggtcagactg gaaaatcaga gggcaggaac
      181 tgctgaacag caaaaagtca gatagcacca catagcagac ccgccataaa acgccctgag
      241 aagcccgtga cgggcttttc ttgtattatg ggtagtttcc ttgcatgaat ccataaaagg
      301 cgcctgtagt gccatttacc cccattcact gccagagccg tgagcgcagc gaactgaatg
      361 tcacgaaaaa gacagcgact caggtgcctg atggtcggag acaaaaggaa tattcagcga
      421 tttgcccgag cttgcgaggg tgctacttaa gcctttaggg ttttaaggtc tgttttgtag
      481 aggagcaaac agcgtttgcg acatcctttt gtaatactgc ggaactgact aaagtagtga
      541 gttatacaca gggctgggat ctattctttt tatctttttt tattctttct ttattctata
      601 aattataacc acttgaatat aaacaaaaaa aacacacaaa ggtctagcgg aatttacaga
      661 gggtctagca gaatttacaa gttttccagc aaaggtctag cagaatttac agatacccac
      721 aactcaaagg aaaaggacta gtaattatca ttgactagcc catctcaatt ggtatagtga
      781 ttaaaatcac ctagaccaat tgagatgtat gtctgaatta gttgttttca aagcaaatga
      841 actagcgatt agtcgctatg acttaacgga gcatgaaacc aagctaattt tatgctgtgt
      901 ggcactactc aaccccacga ttgaaaaccc tacaaggaaa gaacggacgg tatcgttcac
      961 ttataaccaa tacgttcaga tgatgaacat cagtagggaa aatgcttatg gtgtattagc
     1021 taaagcaacc agagagctga tgacgagaac tgtggaaatc aggaatcctt tggttaaagg
     1081 ctttgagatt ttccagtgga caaactatgc caagttctca agcgaaaaat tagaattagt
     1141 ttttagtgaa gagatattgc cttatctttt ccagttaaaa aaattcataa aatataatct
     1201 ggaacatgtt aagtcttttg aaaacaaata ctctatgagg atttatgagt ggttattaaa
     1261 agaactaaca caaaagaaaa ctcacaaggc aaatatagag attagccttg atgaatttaa
     1321 gttcatgtta atgcttgaaa ataactacca tgagtttaaa aggcttaacc aatgggtttt
     1381 gaaaccaata agtaaagatt taaacactta cagcaatatg aaattggtgg ttgataagcg
     1441 aggccgcccg actgatacgt tgattttcca agttgaacta gatagacaaa tggatctcgt
     1501 aaccgaactt gagaacaacc agataaaaat gaatggtgac aaaataccaa caaccattac
     1561 atcagattcc tacctacata acggactaag aaaaacacta cacgatgctt taactgcaaa
     1621 aattcagctc accagttttg aggcaaaatt tttgagtgac atgcaaagta agtatgatct
     1681 caatggttcg ttctcatggc tcacgcaaaa acaacgaacc acactagaga acatactggc
     1741 taaatacgga aggatctgag gttcttatgg ctcttgtatc tatcagtgaa gcatcaagac
     1801 taacaaacaa aagtagaaca actgttcacc gttacatatc aaagggaaaa ctgtccatat
     1861 gcacagatga aaacggtgta aaaaagatag atacatcaga gcttttacga gtttttggtg
     1921 catttaaagc tgttcaccat gaacagatcg acaatgtaac agatgaacag catgtaacac
     1981 ctaatagaac aggtgaaacc agtaaaacaa agcaactaga acatgaaatt gaacacctga
     2041 gacaacttgt tacagctcaa cagtcacaca tagacagcct gaaacaggcg atgctgctta
     2101 tcgaatcaaa gctgccgaca acacgggagc cagtgacgcc tcccgtgggg aaaaaatcat
     2161 ggcaattctg gaagaaatag cgctttcagc cgaagttcct attccgaagt tcctattctc
     2221 tagaaagtat aggaacttcg tgcactgatt aagcattggt aacaggtcgg tcagtttcac
     2281 ctgatttacg taaaaacccg cttcggcggg tttttgcttt tggaggggca gaaagatgaa
     2341 tgactgtctc ataaaaaatt tatttgcttt caggaaaatt tttctgtata atagattctc
     2401 gcaagatgtg gcgtgttagt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
     2461 ttatcaactt gaaaaagtgg caccgagtcg gtgcttttaa aaaaaaaacc ccgcgcgggg
     2521 ttttttttta caagtctcag tgtgctgaaa atgcgtacgt ccggcgaaaa tctcctgaat
     2581 gtcccgaaag ggcagaaaga tgaatgactg tctcataaaa aatttatttg ctttcaggaa
     2641 aatttttctg tataatagat tctttcgtct cagccaatcc ctgttttaga gctagaaata
     2701 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
     2761 ttaaaaaaaa aaccccgcgc ggggtttttt tttacaagtc tcagtgtgct gaactactcg
     2821 gcaaatgctt catcctctct tggtcactcg gttgggcaga aagatgaatg actgtctcat
     2881 aaaaaattta tttgctttca ggaaaatttt tctgtataat agattctgat ttaaacgtgg
     2941 ccaatagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     3001 aaaagtggca ccgagtcggt gcttttaaaa aaaaaacccc gcgcggggtt tttttttaca
     3061 agtctcagtg tgctgaaccc gggctgtagt tcgtcaaatg atggccactt ttccgacggt
     3121 gctgaccccg gatgaatgtc agctactggg ctatctggac aagggaaaac gcaagcgcaa
     3181 agagaaagca ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat
     3241 ggacagcaag cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct
     3301 gcaaagtaaa ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagat
     3361 ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     3421 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     3481 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     3541 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     3601 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     3661 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     3721 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     3781 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     3841 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     3901 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
     3961 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     4021 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     4081 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     4141 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     4201 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     4261 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     4321 ccagcgcggg gatctcatgc tggagttctt cgcccacccc agcttcaaaa gcgcttttat
     4381 cagccataaa acaatactta atactataga atgataacaa aataaactac tttttaaaag
     4441 aattttgtgt tataatctat ttattattaa gtattgggta atattttttg aagagatatt
     4501 ttgaaaaaga aaaattaaag catattaaac taatttcgga ggtcattaaa actattattg
     4561 aaatcatcaa actcattatg gatttaattt aaacttttta ttttaggagg caaaaatgga
     4621 taagaaatac tcaataggct tagatatcgg cacaaatagc gtcggatggg cggtgatcac
     4681 tgatgaatat aaggttccgt ctaaaaagtt caaggttctg ggaaatacag accgccacag
     4741 tatcaaaaaa aatcttatag gggctctttt atttgacagt ggagagacag cggaagcgac
     4801 tcgtctcaaa cggacagctc gtagaaggta tacacgtcgg aagaatcgta tttgttatct
     4861 acaggagatt ttttcaaatg agatggcgaa agtagatgat agtttctttc atcgacttga
     4921 agagtctttt ttggtggaag aagacaagaa gcatgaacgt catcctattt ttggaaatat
     4981 agtagatgaa gttgcttatc atgagaaata tccaactatc tatcatctgc gaaaaaaatt
     5041 ggtagattct actgataaag cggatttgcg cttaatctat ttggccttag cgcatatgat
     5101 taagtttcgt ggtcattttt tgattgaggg agatttaaat cctgataata gtgatgtgga
     5161 caaactattt atccagttgg tacaaaccta caatcaatta tttgaagaaa accctattaa
     5221 cgcaagtgga gtagatgcta aagcgattct ttctgcacga ttgagtaaat caagacgatt
     5281 agaaaatctc attgctcagc tccccggtga gaagaaaaat ggcttatttg ggaatctcat
     5341 tgctttgtca ttgggtttga cccctaattt taaatcaaat tttgatttgg cagaagatgc
     5401 taaattacag ctttcaaaag atacttacga tgatgattta gataatttat tggcgcaaat
     5461 tggagatcaa tatgctgatt tgtttttggc agctaagaat ttatcagatg ctattttact
     5521 ttcagatatc ctaagagtaa atactgaaat aactaaggct cccctatcag cttcaatgat
     5581 taaacgctac gatgaacatc atcaagactt gactctttta aaagctttag ttcgacaaca
     5641 acttccagaa aagtataaag aaatcttttt tgatcaatca aaaaacggat atgcaggtta
     5701 tattgatggg ggagctagcc aagaagaatt ttataaattt atcaaaccaa ttttagaaaa
     5761 aatggatggt actgaggaat tattggtgaa actaaatcgt gaagatttgc tgcgcaagca
     5821 acggaccttt gacaacggct ctattcccca tcaaattcac ttgggtgagc tgcatgctat
     5881 tttgagaaga caagaagact tttatccatt tttaaaagac aatcgtgaga agattgaaaa
     5941 aatcttgact tttcgaattc cttattatgt tggtccattg gcgcgtggca atagtcgttt
     6001 tgcatggatg actcggaagt ctgaagaaac aattacccca tggaattttg aagaagttgt
     6061 cgataaaggt gcttcagctc aatcatttat tgaacgcatg acaaactttg ataaaaatct
     6121 tccaaatgaa aaagtactac caaaacatag tttgctttat gagtatttta cggtttataa
     6181 cgaattgaca aaggtcaaat atgttactga aggaatgcga aaaccagcat ttctttcagg
     6241 tgaacagaag aaagccattg ttgatttact cttcaaaaca aatcgaaaag taaccgttaa
     6301 gcaattaaaa gaagattatt tcaaaaaaat agaatgtttt gatagtgttg aaatttcagg
     6361 agttgaagat agatttaatg cttcattagg tacctaccat gatttgctaa aaattattaa
     6421 agataaagat tttttggata atgaagaaaa tgaagatatc ttagaggata ttgttttaac
     6481 attgacctta tttgaagata gggagatgat tgaggaaaga cttaaaacat atgctcacct
     6541 ctttgatgat aaggtgatga aacagcttaa acgtcgccgt tatactggtt ggggacgttt
     6601 gtctcgaaaa ttgattaatg gtattaggga taagcaatct ggcaaaacaa tattagattt
     6661 tttgaaatca gatggttttg ccaatcgcaa ttttatgcag ctgatccatg atgatagttt
     6721 gacatttaaa gaagacattc aaaaagcaca agtgtctgga caaggcgata gtttacatga
     6781 acatattgca aatttagctg gtagccctgc tattaaaaaa ggtattttac agactgtaaa
     6841 agttgttgat gaattggtca aagtaatggg gcggcataag ccagaaaata tcgttattga
     6901 aatggcacgt gaaaatcaga caactcaaaa gggccagaaa aattcgcgag agcgtatgaa
     6961 acgaatcgaa gaaggtatca aagaattagg aagtcagatt cttaaagagc atcctgttga
     7021 aaatactcaa ttgcaaaatg aaaagctcta tctctattat ctccaaaatg gaagagacat
     7081 gtatgtggac caagaattag atattaatcg tttaagtgat tatgatgtcg atcacattgt
     7141 tccacaaagt ttccttaaag acgattcaat agacaataag gtcttaacgc gttctgataa
     7201 aaatcgtggt aaatcggata acgttccaag tgaagaagta gtcaaaaaga tgaaaaacta
     7261 ttggagacaa cttctaaacg ccaagttaat cactcaacgt aagtttgata atttaacgaa
     7321 agctgaacgt ggaggtttga gtgaacttga taaagctggt tttatcaaac gccaattggt
     7381 tgaaactcgc caaatcacta agcatgtggc acaaattttg gatagtcgca tgaatactaa
     7441 atacgatgaa aatgataaac ttattcgaga ggttaaagtg attaccttaa aatctaaatt
     7501 agtttctgac ttccgaaaag atttccaatt ctataaagta cgtgagatta acaattacca
     7561 tcatgcccat gatgcgtatc taaatgccgt cgttggaact gctttgatta agaaatatcc
     7621 aaaacttgaa tcggagtttg tctatggtga ttataaagtt tatgatgttc gtaaaatgat
     7681 tgctaagtct gagcaagaaa taggcaaagc aaccgcaaaa tatttctttt actctaatat
     7741 catgaacttc ttcaaaacag aaattacact tgcaaatgga gagattcgca aacgccctct
     7801 aatcgaaact aatggggaaa ctggagaaat tgtctgggat aaagggcgag attttgccac
     7861 agtgcgcaaa gtattgtcca tgccccaagt caatattgtc aagaaaacag aagtacagac
     7921 aggcggattc tccaaggagt caattttacc aaaaagaaat tcggacaagc ttattgctcg
     7981 taaaaaagac tgggatccaa aaaaatatgg tggttttgat agtccaacgg tagcttattc
     8041 agtcctagtg gttgctaagg tggaaaaagg gaaatcgaag aagttaaaat ccgttaaaga
     8101 gttactaggg atcacaatta tggaaagaag ttcctttgaa aaaaatccga ttgacttttt
     8161 agaagctaaa ggatataagg aagttaaaaa agacttaatc attaaactac ctaaatatag
     8221 tctttttgag ttagaaaacg gtcgtaaacg gatgctggct agtgccggag aattacaaaa
     8281 aggaaatgag ctggctctgc caagcaaata tgtgaatttt ttatatttag ctagtcatta
     8341 tgaaaagttg aagggtagtc cagaagataa cgaacaaaaa caattgtttg tggagcagca
     8401 taagcattat ttagatgaga ttattgagca aatcagtgaa ttttctaagc gtgttatttt
     8461 agcagatgcc aatttagata aagttcttag tgcatataac aaacatagag acaaaccaat
     8521 acgtgaacaa gcagaaaata ttattcattt atttacgttg acgaatcttg gagctcccgc
     8581 tgcttttaaa tattttgata caacaattga tcgtaaacga tatacgtcta caaaagaagt
     8641 tttagatgcc actcttatcc atcaatccat cactggtctt tatgaaacac gcattgattt
     8701 gagtcagcta ggaggtgact ga
//
