LOCUS       62056_13605        9863 bp DNA     circular SYN 12-JUN-2021
DEFINITION  Cloning vector pKW20_CDFtet_pAraRedCas9_TracrRNA_rec, complete 
            sequence.
ACCESSION   MW879721
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9863)
  AUTHORS   Robertson WE, Funke LFH., de la Torre D, Fredens J, Elliott TS, 
            Spinck M, Christova Y, Cervettini D, Boge FL, Liu KC, Buse S, Maslen
            S, Salmond GPC., Chin JW.
  TITLE     Sense Codon Reassignment Enables Viral Resistance and Encoded 
            Polymer Synthesis
  JOURNAL   Science (2021) In press
REFERENCE   2  (bases 1 to 9863)
  AUTHORS   Robertson WE, Funke LFH., de la Torre D, Fredens J, Elliott TS, 
            Spinck M, Christova Y, Cervettini D, Boge FL, Liu KC, Buse S, Maslen
            S, Salmond GPC., Chin JW.
  TITLE     Direct Submission
  JOURNAL   Submitted (06-APR-2021) Protein and Nucleic Acid Chemistry, MRC 
            Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, 
            Cambridgeshire CB2 0QH, United Kingdom
REFERENCE   3  (bases 1 to 9863)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science 
            (2021) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (06-APR-2021) Protein and Nucleic Acid Chemistry, MRC Laboratory of 
            Molecular Biology, Francis Crick Avenue, Cambridge, Cambridgeshire 
            CB2 0QH, United Kingdom"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..9863
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        complement(106..184)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     misc_feature    complement(238..265)
                     /label=tracrRNA promoter predicted
                     /note="tracrRNA promoter predicted"
     misc_feature    297..331
                     /label=Cas9 promoter predicted
                     /note="Cas9 promoter predicted"
     terminator      470..497
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     CDS             complement(670..1545)
                     /label=araC
                     /note="L-arabinose regulatory protein"
     promoter        1572..1856
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     CDS             1891..2304
                     /label=Gam
                     /note="inhibitor of the host RecBCD nuclease in the lambda 
                     Red system"
     CDS             2313..3095
                     /label=Beta
                     /note="single-stranded DNA binding recombinase in the 
                     lambda Red system"
     CDS             3095..3772
                     /label=Exo
                     /note="5' to 3' double-stranded DNA exonuclease in the 
                     lambda
                     Red system"
     misc_feature    3773..3775
                     /label=TAG to TAA
                     /note="TAG to TAA"
     CDS             3799..7902
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     terminator      7942..7989
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(8163..8963)
                     /label=ApmR
                     /note="aminoglycoside 3-N-acetyltransferase type IV"
     promoter        complement(8964..9055)
                     /label=AmpR promoter
     rep_origin      complement(9103..9841)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
ORIGIN
        1 taaaaggatt aagagattaa tttccctaaa aatgataaaa caagcgtttt gaaagcgctt
       61 gtttttttgg tttgcagtca gagtagaata gaagtatcaa aaaaagcacc gactcggtgc
      121 cactttttca agttgataac ggactagcct tattttaact tgctatgctg ttttgaatgg
      181 ttccaacaag attattttat aacttttata acaaataatc aaggagaaat tcaaagaaat
      241 ttatcagcca taaaacaata cttaatacta tagaatgata acaaaataaa ctacttttta
      301 aaagaatttt gtgttataat ctatttatta ttaagtattg ggtaatattt tttgaagaga
      361 tattttgaaa aagaaaaatt aaagcatatt aaactaattt cggaggtcat taaaactatt
      421 attgaaatca tcaaatcatt atggatttaa tttaaacttt ttattaagca gaaggccatc
      481 ctgacggatg gcctttttgc gtttctagat caggaggaat tggccacgat gcgtccggcg
      541 tagaggatct gctcatgttt gacagcttat catcgatgca taatgtgcct gtcaaatgga
      601 cgaagcaggg attctgcaaa ccctatgcta ctccgtcaag ccgtcaattg tctgattcgt
      661 taccaattaa ctcaacttga cggctacatc attcactttt tcttcacaac cggcacggaa
      721 ctcgctcggg ctggccccgg tgcatttttt aaatacccgg ctgaaataga gttgatcgtc
      781 aaaaccaaca ttgcgaccga cggtggcgat aggcatccgg gtggtgctca aaagcagctt
      841 cgcctggctg atacgttggt cctcgcgcca gcttaagacg ctaatcccta actgctggcg
      901 gaaaagatga ctcagacggc tcgggctcaa gcaaacatgc tgtgcgacgc tggcgatatc
      961 aaaattgctg tctgccaggt gatcgctgat gtactgacaa gcctcgcgta cccgattatc
     1021 catcggtgga tggaggctct cgttaatcgc ttccatgcgc cgcagtaaca attgctcaag
     1081 cagatttatc gccagcagct cgctatagcg cccttcccct tgcccggcgt taatgatttg
     1141 cccaaacagg tcgctgaaat gcggctggtg cgcttcatcc gggcgaaaga accccgtatt
     1201 ggcaaatata ctcggccagt taagccattc atgccagtag gcgcgcggac gaaagtaaac
     1261 ccactggtga taccattcgc gagcctccgg atgacgaccg tagtgatgaa tctctcctgg
     1321 cgggaacagc aaaatatcac ccggtcggca aacaaattct cgtccctgat ttttcaccac
     1381 cccctgaccg cgaatggtga gattgagaat ataacctttc attcccagcg gtcggtcgat
     1441 aaaaaaatcg agataaccgt tggcctcaat cggcgttaaa cccgccacca gatgggcatt
     1501 aaagctgtat cccggcagca ggggatcatt ttgcgcttca gccatacttt tcatactccc
     1561 gccattcaga gaagaaacca attgtccata ttgcatcaga cattgccgtc actgcgtctt
     1621 ttactggctc ttctcgctaa ccaaaccggt aaccccgctt attaaaagca ttctgtaaca
     1681 aagcgggacc aaagccatga caaaaacgcg taacaaaagt gtctataatc acggcagaaa
     1741 agtccacatt gattatttgc acggcgtcac actttgctat gccatagcat ttttatccat
     1801 aagattagcg gatcctacct gacgcttttt atcgcaactc tctactgttt ctccataccc
     1861 gtttttttgg gctagcagga ggaattcacc atggatatta atactgaaac tgagatcaag
     1921 caaaagcata gtctaacccc ctttcctgtt ttcctaatca gcccggcatt tcgcgggcga
     1981 tattttcaca gctatttcag gagtagtgcc atgaacgctt attacattca ggatcgtctt
     2041 gaggctcaga gctgggcgcg tcactaccag cagctcgccc gtgaagagaa agaggcagaa
     2101 ctggcagacg acatggaaaa aggcctgccc cagcacctgt ttgaaagcct atgcatcgat
     2161 catttgcaac gccacggggc cagcaaaaaa tccattaccc gtgcgtttga tgacgatgtt
     2221 gagtttcagg agcgcatggc agaacacatc cggtacatgg ttgaaaccat tgctcaccac
     2281 caggttgata ttgatagtga ggtataaaac gaatgagtac tgcactcgca acgctggctg
     2341 ggaagctggc tgaacgtgtc ggcatggatt ctgtcgaccc acaggaactg atcaccactc
     2401 ttcgccagac ggcatttaaa ggtgatgcca gcgatgcgca gttcatcgca ttactgatcg
     2461 ttgccaacca gtacggcctt aatccgtgga cgaaagaaat ttacgccttt cctgataagc
     2521 agaatggcat cgttccggtg gtgggcgttg atggctggtc ccgcatcatc aatgaaaacc
     2581 agcagtttga tggcatggac tttgagcagg acaatgaatc ctgtacatgc cggatttacc
     2641 gcaaggaccg taatcatccg atctgcgtta ccgaatggat ggatgaatgc cgccgcgaac
     2701 cattcaaaac tcgcgaaggc agagaaatca cggggccgtg gcagagccat cccaaacgga
     2761 tgttacgtca taaagccatg attcagtgtg cccgtctggc cttcggattt gctggtatct
     2821 atgacaagga tgaagccgag cgcattgtcg aaaatactgc atacactgca gaacgtcagc
     2881 cggaacgcga catcactccg gttaacgatg aaaccatgca ggagattaac actctgctga
     2941 tcgccctgga taaaacatgg gatgacgact tattgccgct ctgttcccag atatttcgcc
     3001 gcgacattcg tgcaagcagt gaactgacac aggccgaagc agtaaaagct cttggattcc
     3061 tgaaacagaa agccgcagag cagaaggtgg cagcatgaca ccggacatta tcctgcagcg
     3121 taccgggatc gatgtgagag ctgtcgaaca gggggatgat gcgtggcaca aattacggct
     3181 cggcgtcatc accgctagtg aagttcacaa cgtgatagca aaaccccgct ccggaaagaa
     3241 gtggcctgac atgaaaatgt cctactttca caccctgctt gctgaggttt gcaccggtgt
     3301 ggctccggaa gttaacgcta aagcactggc ctggggaaaa cagtacgaga acgacgccag
     3361 aaccctgttt gaattcactt ccggcgtgaa tgttactgaa tccccgatca tctatcgcga
     3421 cgaaagtatg cgtaccgcct gctctcccga tggtttatgc agtgacggca acggccttga
     3481 actgaaatgc ccgtttacct cccgggattt catgaagttc cggctcggtg gtttcgaggc
     3541 cataaagagt gcttacatgg cccaggtgca gtacagcatg tgggtgacgc gaaaaaatgc
     3601 ctggtacttt gccaactatg acccgcgtat gaagcgtgaa ggcctgcatt atgtcgtgat
     3661 tgagcgggat gaaaagtaca tggcgagttt tgacgagatc gtgccggagt tcatcgaaaa
     3721 aatggacgag gcactggctg aaattggttt tgtatttggg gagcaatggc gataaatcct
     3781 agcaaggagg aaatcatcat ggataagaaa tacagtatag gcttagatat cggcacaaat
     3841 agcgtcggat gggcggtgat cactgatgaa tataaggttc cgtctaaaaa gttcaaggtt
     3901 ctgggaaata cagaccgcca cagtatcaaa aaaaatctta taggggctct tttatttgac
     3961 agtggagaga cagcggaagc gactcgtctc aaacggacag ctcgtagaag gtatacacgt
     4021 cggaagaatc gtatttgtta tctacaggag atttttagta atgagatggc gaaagtagat
     4081 gatagtttct ttcatcgact tgaagagtct tttttggtgg aagaagacaa gaagcatgaa
     4141 cgtcatccta tttttggaaa tatagtagat gaagttgctt atcatgagaa atatccaact
     4201 atctatcatc tgcgaaaaaa attggtagat tctactgata aagcggattt gcgcttaatc
     4261 tatttggcct tagcgcatat gattaagttt cgtggtcatt ttttgattga gggagattta
     4321 aatcctgata atagtgatgt ggacaaacta tttatccagt tggtacaaac ctacaatcaa
     4381 ttatttgaag aaaaccctat taacgcaagt ggagtagatg ctaaagcgat tctttctgca
     4441 cgattgagta aaagtagacg attagaaaat ctcattgctc agctccccgg tgagaagaaa
     4501 aatggcttat ttgggaatct cattgctttg agtttgggtt tgacccctaa ttttaaaagt
     4561 aattttgatt tggcagaaga tgctaaatta cagcttagta aagatactta cgatgatgat
     4621 ttagataatt tattggcgca aattggagat caatatgctg atttgttttt ggcagctaag
     4681 aatttaagtg atgctatttt acttagtgat atcctaagag taaatactga aataactaag
     4741 gctcccctaa gtgctagtat gattaaacgc tacgatgaac atcatcaaga cttgactctt
     4801 ttaaaagctt tagttcgaca acaacttcca gaaaagtata aagaaatctt ttttgatcaa
     4861 agtaaaaacg gatatgcagg ttatattgat gggggagcta gccaagaaga attttataaa
     4921 tttatcaaac caattttaga aaaaatggat ggtactgagg aattattggt gaaactaaat
     4981 cgtgaagatt tgctgcgcaa gcaacggacc tttgacaacg gctctattcc ccatcaaatt
     5041 cacttgggtg agctgcatgc tattttgaga agacaagaag acttttatcc atttttaaaa
     5101 gacaatcgtg agaagattga aaaaatcttg acttttcgaa ttccttatta tgttggtcca
     5161 ttggcgcgtg gcaatagtcg ttttgcatgg atgactcgga agtctgaaga aacaattacc
     5221 ccatggaatt ttgaagaagt tgtcgataaa ggtgctagtg ctcaaagttt tattgaacgc
     5281 atgacaaact ttgataaaaa tcttccaaat gaaaaagtac taccaaaaca tagtttgctt
     5341 tatgagtatt ttacggttta taacgaattg acaaaggtca aatatgttac tgaaggaatg
     5401 cgaaaaccag catttcttag tggtgaacag aagaaagcca ttgttgattt actcttcaaa
     5461 acaaatcgaa aagtaaccgt taagcaatta aaagaagatt atttcaaaaa aatagaatgt
     5521 tttgatagtg ttgaaattag tggagttgaa gatagattta atgctagttt aggtacctac
     5581 catgatttgc taaaaattat taaagataaa gattttttgg ataatgaaga aaatgaagat
     5641 atcttagagg atattgtttt aacattgacc ttatttgaag atagggagat gattgaggaa
     5701 agacttaaaa catatgctca cctctttgat gataaggtga tgaaacagct taaacgtcgc
     5761 cgttatactg gttggggacg tttgtctcga aaattgatta atggtattag ggataagcaa
     5821 tctggcaaaa caatattaga ttttttgaaa agtgatggtt ttgccaatcg caattttatg
     5881 cagctgatcc atgatgatag tttgacattt aaagaagaca ttcaaaaagc acaagtgtct
     5941 ggacaaggcg atagtttaca tgaacatatt gcaaatttag ctggtagccc tgctattaaa
     6001 aaaggtattt tacagactgt aaaagttgtt gatgaattgg tcaaagtaat ggggcggcat
     6061 aagccagaaa atatcgttat tgaaatggca cgtgaaaatc agacaactca aaagggccag
     6121 aaaaatagcc gagagcgtat gaaacgaatc gaagaaggta tcaaagaatt aggaagtcag
     6181 attcttaaag agcatcctgt tgaaaatact caattgcaaa atgaaaagct ctatctctat
     6241 tatctccaaa atggaagaga catgtatgtg gaccaagaat tagatattaa tcgtttaagt
     6301 gattatgatg tcgatcacat tgttccacaa agtttcctta aagacgatag tatagacaat
     6361 aaggtcttaa cgcgttctga taaaaatcgt ggtaaaagcg ataacgttcc aagtgaagaa
     6421 gtagtcaaaa agatgaaaaa ctattggaga caacttctaa acgccaagtt aatcactcaa
     6481 cgtaagtttg ataatttaac gaaagctgaa cgtggaggtt tgagtgaact tgataaagct
     6541 ggttttatca aacgccaatt ggttgaaact cgccaaatca ctaagcatgt ggcacaaatt
     6601 ttggatagtc gcatgaatac taaatacgat gaaaatgata aacttattcg agaggttaaa
     6661 gtgattacct taaaatctaa attagtttct gacttccgaa aagatttcca attctataaa
     6721 gtacgtgaga ttaacaatta ccatcatgcc catgatgcgt atctaaatgc cgtcgttgga
     6781 actgctttga ttaagaaata tccaaaactt gaaagcgagt ttgtctatgg tgattataaa
     6841 gtttatgatg ttcgtaaaat gattgctaag tctgagcaag aaataggcaa agcaaccgca
     6901 aaatatttct tttactctaa tatcatgaac ttcttcaaaa cagaaattac acttgcaaat
     6961 ggagagattc gcaaacgccc tctaatcgaa actaatgggg aaactggaga aattgtctgg
     7021 gataaagggc gagattttgc cacagtgcgc aaagtattgt ccatgcccca agtcaatatt
     7081 gtcaagaaaa cagaagtaca gacaggcgga ttctccaagg agagtatttt accaaaaaga
     7141 aatagcgaca agcttattgc tcgtaaaaaa gactgggatc caaaaaaata tggtggtttt
     7201 gatagtccaa cggtagctta tagtgtccta gtggttgcta aggtggaaaa agggaaaagc
     7261 aagaagttaa aatccgttaa agagttacta gggatcacaa ttatggaaag aagttccttt
     7321 gaaaaaaatc cgattgactt tttagaagct aaaggatata aggaagttaa aaaagactta
     7381 atcattaaac tacctaaata tagtcttttt gagttagaaa acggtcgtaa acggatgctg
     7441 gctagtgccg gagaattaca aaaaggaaat gagctggctc tgccaagcaa atatgtgaat
     7501 tttttatatt tagctagtca ttatgaaaag ttgaagggta gtccagaaga taacgaacaa
     7561 aaacaattgt ttgtggagca gcataagcat tatttagatg agattattga gcaaatcagt
     7621 gaattttcta agcgtgttat tttagcagat gccaatttag ataaagttct tagtgcatat
     7681 aacaaacata gagacaaacc aatacgtgaa caagcagaaa atattattca tttatttacg
     7741 ttgacgaatc ttggagctcc cgctgctttt aaatattttg atacaacaat tgatcgtaaa
     7801 cgatatacgt ctacaaaaga agttttagat gccactctta tccatcaatc catcactggt
     7861 ctttatgaaa cacgcattga tttgagtcag ctaggaggtg actgaagaga cccctaggct
     7921 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
     7981 ggttttttgc tgaaacctca ggcatttgag aagcacacgg tcacactgct tccggtagtc
     8041 aataaaccgg taaaccagca atagacataa gcggctattt aacgaccctg ccctgaaccg
     8101 acgaccgggt catcgtggcc ggatcttgcg gcccctcggc ttgaacgaat tgttagacat
     8161 cagccaatgc tctggcgagc ggcatcgcat tcttcgcatc ccgcctctgg cggatgcagg
     8221 aagatcaacg gatctcggcc cagttgaccc agggctgtcg ccacaatgtc gcgggagcgg
     8281 atcaaccgag caaaggcatg accgactgga ccttccttct gaaggctctt ctccttgagc
     8341 cacctgtccg ccaaggcaaa gcgctcacag cagtggtcat tctcgagata atcgacgcgt
     8401 accaacttgc catcctgaag aatggtgcag tgtctcggca ccccataggg aacctttgcc
     8461 atcaactcgg caagatgcag cgtcgtgttg gcatcgtgtc ccacgccgag gagaagtacc
     8521 tgcccatcga gttcatggac acgggcgacc gggcttgcag ggctgtgagg tggcaggggc
     8581 aatggatcag agatgatctg ctctgcctgt ggccccgctg ccgcaaaggc aaatggatgg
     8641 gcgctgcgct ttacatttgg caggcgccag aatgtgtcag agacaactcc aaggtccggt
     8701 gtaacggggc tcgtggcagg atcgaacggc tcgtcgtcca gaccactcca gctgggcatg
     8761 acgagcgtcc ctcccggacc cagcgcagca cgcagggcct cgatcagtcc aagtggccca
     8821 tcttcgaggg gccggacgct acggaaggag ctgtggacca gcagcacacc gccgggggta
     8881 accccaaggt tgagaagctg accgatgagc tcggcttttc gccattcgta ttgcacgaca
     8941 ttgcactcca ccgcactact catactcttc ctttttcaat attattgaag catttatcag
     9001 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaatagct
     9061 agctcactcg gtcgctacgc tccgggcgtg agactgcggc gggcgctgcg gacacataca
     9121 aagttaccca cagattccgt ggataagcag gggactaaca tgtgaggcaa aacagcaggg
     9181 ccgcgccggt ggcgtttttc cataggctcc gccctcctgc cagagttcac ataaacagac
     9241 gcttttccgg tgcatctgtg ggagccgtga ggctcaacca tgaatctgac agtacgggcg
     9301 aaacccgaca ggacttaaag atccccaccg tttccggcgg gtcgctccct cttgcgctct
     9361 cctgttccga ccctgccgtt taccggatac ctgttccgcc tttctccctt acgggaagtg
     9421 tggcgctttc tcatagctca cacactggta tctcggctcg gtgtaggtcg ttcgctccaa
     9481 gctgggctgt aagcaagaac tccccgttca gcccgactgc tgcgccttat ccggtaactg
     9541 ttcacttgag tccaacccgg aaaagcacgg taaaacgcca ctggcagcag ccattggtaa
     9601 ctgggagttc gcagaggatt tgtttagcta aacacgcggt tgctcttgaa gtgtgcgcca
     9661 aagtccggct acactggaag gacagatttg gttgctgtgc tctgcgaaag ccagttacca
     9721 cggttaagca gttccccaac tgacttaacc ttcgatcaaa ccacctcccc aggtggtttt
     9781 ttcgtttaca gggcaaaaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     9841 cttttctact gaaccgctct aga
//
