LOCUS       62056_8305        9458 bp DNA     circular SYN 24-NOV-2020
DEFINITION  Expression vector pdCas9d.e.f, complete sequence.
ACCESSION   MW221490
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9458)
  AUTHORS   Alvarez B, Mencia M, de Lorenzo V, Fernandez LA.
  TITLE     In vivo diversification of target genomic sites using processive 
            base deaminase fusions blocked by dCas9
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9458)
  AUTHORS   Alvarez B, Fernandez LA.
  TITLE     Direct Submission
  JOURNAL   Submitted (05-NOV-2020) Microbial Biotechnology, CNB-CSIC (Centro 
            Nacional de Biotecnologia-CSIC), Darwin 3, Cantoblanco, Madrid 
            28049, Spain
REFERENCE   3  (bases 1 to 9458)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (05-NOV-2020) Microbial Biotechnology, CNB-CSIC (Centro Nacional de 
            Biotecnologia-CSIC), Darwin 3, Cantoblanco, Madrid 28049, Spain"
FEATURES             Location/Qualifiers
     source          1..9458
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(4..660)
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     promoter        complement(661..763)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      complement(1289..1834)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     promoter        1946..1974
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     misc_RNA        complement(2292..2370)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             2666..6769
                     /label=dCas9
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     misc_feature    6793..6924
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   6925..6960
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes
                     CRISPR/Cas system"
     misc_feature    6965..6990
                     /label=d spacer sequence
                     /note="d spacer sequence"
     repeat_region   6991..7026
     misc_feature    7031..7056
                     /label=e spacer sequence
                     /note="e spacer sequence"
     repeat_region   7057..7092
     misc_feature    7097..7122
                     /label=f spacer sequence
                     /note="f spacer sequence"
     repeat_region   7123..7158
ORIGIN
        1 ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac
       61 atggaagcca tcacagacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc
      121 gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc
      181 cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga aaaacatatt
      241 ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga
      301 atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt
      361 ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagctc
      421 accgtctttc attgccatac ggaattccgg atgagcattc atcaggcggg caagaatgtg
      481 aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat
      541 atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg
      601 ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat
      661 tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg gtagtgatct
      721 tatttcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtctc attttcgcca
      781 aaagttggcc cagggcttcc cggtatcaac agggacacca ggatttattt attctgcgaa
      841 gtgatcttcc gtcacaggta tttattcggc gcaaagtgcg tcgggtgatg ctgccaactt
      901 actgatttag tgtatgatgg tgtttttgag gtgctccagt ggcttctgtt tctatcagct
      961 gtccctcctg ttcagctact gacggggtgg tgcgtaacgg caaaagcacc gccggacatc
     1021 agcgctagcg gagtgtatac tggcttacta tgttggcact gatgagggtg tcagtgaagt
     1081 gcttcatgtg gcaggagaaa aaaggctgca ccggtgcgtc agcagaatat gtgatacagg
     1141 atatattccg cttcctcgct cactgactcg ctacgctcgg tcgttcgact gcggcgagcg
     1201 gaaatggctt acgaacgggg cggagatttc ctggaagatg ccaggaagat acttaacagg
     1261 gaagtgagag ggccgcggca aagccgtttt tccataggct ccgcccccct gacaagcatc
     1321 acgaaatctg acgctcaaat cagtggtggc gaaacccgac aggactataa agataccagg
     1381 cgtttccccc tggcggctcc ctcgtgcgct ctcctgttcc tgcctttcgg tttaccggtg
     1441 tcattccgct gttatggccg cgtttgtctc attccacgcc tgacactcag ttccgggtag
     1501 gcagttcgct ccaagctgga ctgtatgcac gaaccccccg ttcagtccga ccgctgcgcc
     1561 ttatccggta actatcgtct tgagtccaac ccggaaagac atgcaaaagc accactggca
     1621 gcagccactg gtaattgatt tagaggagtt agtcttgaag tcatgcgccg gttaaggcta
     1681 aactgaaagg acaagttttg gtgactgcgc tcctccaagc cagttacctc ggttcaaaga
     1741 gttggtagct cagagaacct tcgaaaaacc gccctgcaag gcggtttttt cgttttcaga
     1801 gcaagagatt acgcgcagac caaaacgatc tcaagaagat catcttatta atcagataaa
     1861 atatttctag atttcagtgc aatttatctc ttcaaatgta gcacctgaag tcagccccat
     1921 acgatataag ttgtaattct catgtttgac agcttatcat cgataagctt taatgcggta
     1981 gtttatcaca gttaaattgc taacgcagtc aggcaccgtg tatgaaatct aacaatgcgc
     2041 tcatcgtcat cctcggcacc gtcaccctgg atgctgtagg cataggcttg gttatgccgg
     2101 tactgccggg cctcttgcgg gattacgaaa tcatcctgtg gagcttagta ggtttagcaa
     2161 gatggcagcg cctaaatgta gaatgataaa aggattaaga gattaatttc cctaaaaatg
     2221 ataaaacaag cgttttgaaa gcgcttgttt ttttggtttg cagtcagagt agaatagaag
     2281 tatcaaaaaa agcaccgact cggtgccact ttttcaagtt gataacggac tagccttatt
     2341 ttaacttgct atgctgtttt gaatggttcc aacaagatta ttttataact tttataacaa
     2401 ataatcaagg agaaattcaa agaaatttat cagccataaa acaatactta atactataga
     2461 atgataacaa aataaactac tttttaaaag aattttgtgt tataatctat ttattattaa
     2521 gtattgggta atattttttg aagagatatt ttgaaaaaga aaaattaaag catattaaac
     2581 taatttcgga ggtcattaaa actattattg aaatcatcaa actcattatg gatttaattt
     2641 aaacttttta ttttaggagg caaaaatgga taagaaatac tcaataggct tagctatcgg
     2701 cacaaatagc gtcggatggg cggtgatcac tgatgaatat aaggttccgt ctaaaaagtt
     2761 caaggttctg ggaaatacag accgccacag tatcaaaaaa aatcttatag gggctctttt
     2821 atttgacagt ggagagacag cggaagcgac tcgtctcaaa cggacagctc gtagaaggta
     2881 tacacgtcgg aagaatcgta tttgttatct acaggagatt ttttcaaatg agatggcgaa
     2941 agtagatgat agtttctttc atcgacttga agagtctttt ttggtggaag aagacaagaa
     3001 gcatgaacgt catcctattt ttggaaatat agtagatgaa gttgcttatc atgagaaata
     3061 tccaactatc tatcatctgc gaaaaaaatt ggtagattct actgataaag cggatttgcg
     3121 cttaatctat ttggccttag cgcatatgat taagtttcgt ggtcattttt tgattgaggg
     3181 agatttaaat cctgataata gtgatgtgga caaactattt atccagttgg tacaaaccta
     3241 caatcaatta tttgaagaaa accctattaa cgcaagtgga gtagatgcta aagcgattct
     3301 ttctgcacga ttgagtaaat caagacgatt agaaaatctc attgctcagc tccccggtga
     3361 gaagaaaaat ggcttatttg ggaatctcat tgctttgtca ttgggtttga cccctaattt
     3421 taaatcaaat tttgatttgg cagaagatgc taaattacag ctttcaaaag atacttacga
     3481 tgatgattta gataatttat tggcgcaaat tggagatcaa tatgctgatt tgtttttggc
     3541 agctaagaat ttatcagatg ctattttact ttcagatatc ctaagagtaa atactgaaat
     3601 aactaaggct cccctatcag cttcaatgat taaacgctac gatgaacatc atcaagactt
     3661 gactctttta aaagctttag ttcgacaaca acttccagaa aagtataaag aaatcttttt
     3721 tgatcaatca aaaaacggat atgcaggtta tattgatggg ggagctagcc aagaagaatt
     3781 ttataaattt atcaaaccaa ttttagaaaa aatggatggt actgaggaat tattggtgaa
     3841 actaaatcgt gaagatttgc tgcgcaagca acggaccttt gacaacggct ctattcccca
     3901 tcaaattcac ttgggtgagc tgcatgctat tttgagaaga caagaagact tttatccatt
     3961 tttaaaagac aatcgtgaga agattgaaaa aatcttgact tttcgaattc cttattatgt
     4021 tggtccattg gcgcgtggca atagtcgttt tgcatggatg actcggaagt ctgaagaaac
     4081 aattacccca tggaattttg aagaagttgt cgataaaggt gcttcagctc aatcatttat
     4141 tgaacgcatg acaaactttg ataaaaatct tccaaatgaa aaagtactac caaaacatag
     4201 tttgctttat gagtatttta cggtttataa cgaattgaca aaggtcaaat atgttactga
     4261 aggaatgcga aaaccagcat ttctttcagg tgaacagaag aaagccattg ttgatttact
     4321 cttcaaaaca aatcgaaaag taaccgttaa gcaattaaaa gaagattatt tcaaaaaaat
     4381 agaatgtttt gatagtgttg aaatttcagg agttgaagat agatttaatg cttcattagg
     4441 tacctaccat gatttgctaa aaattattaa agataaagat tttttggata atgaagaaaa
     4501 tgaagatatc ttagaggata ttgttttaac attgacctta tttgaagata gggagatgat
     4561 tgaggaaaga cttaaaacat atgctcacct ctttgatgat aaggtgatga aacagcttaa
     4621 acgtcgccgt tatactggtt ggggacgttt gtctcgaaaa ttgattaatg gtattaggga
     4681 taagcaatct ggcaaaacaa tattagattt tttgaaatca gatggttttg ccaatcgcaa
     4741 ttttatgcag ctgatccatg atgatagttt gacatttaaa gaagacattc aaaaagcaca
     4801 agtgtctgga caaggcgata gtttacatga acatattgca aatttagctg gtagccctgc
     4861 tattaaaaaa ggtattttac agactgtaaa agttgttgat gaattggtca aagtaatggg
     4921 gcggcataag ccagaaaata tcgttattga aatggcacgt gaaaatcaga caactcaaaa
     4981 gggccagaaa aattcgcgag agcgtatgaa acgaatcgaa gaaggtatca aagaattagg
     5041 aagtcagatt cttaaagagc atcctgttga aaatactcaa ttgcaaaatg aaaagctcta
     5101 tctctattat ctccaaaatg gaagagacat gtatgtggac caagaattag atattaatcg
     5161 tttaagtgat tatgatgtcg atgccattgt tccacaaagt ttccttaaag acgattcaat
     5221 agacaataag gtcttaacgc gttctgataa aaatcgtggt aaatcggata acgttccaag
     5281 tgaagaagta gtcaaaaaga tgaaaaacta ttggagacaa cttctaaacg ccaagttaat
     5341 cactcaacgt aagtttgata atttaacgaa agctgaacgt ggaggtttga gtgaacttga
     5401 taaagctggt tttatcaaac gccaattggt tgaaactcgc caaatcacta agcatgtggc
     5461 acaaattttg gatagtcgca tgaatactaa atacgatgaa aatgataaac ttattcgaga
     5521 ggttaaagtg attaccttaa aatctaaatt agtttctgac ttccgaaaag atttccaatt
     5581 ctataaagta cgtgagatta acaattacca tcatgcccat gatgcgtatc taaatgccgt
     5641 cgttggaact gctttgatta agaaatatcc aaaacttgaa tcggagtttg tctatggtga
     5701 ttataaagtt tatgatgttc gtaaaatgat tgctaagtct gagcaagaaa taggcaaagc
     5761 aaccgcaaaa tatttctttt actctaatat catgaacttc ttcaaaacag aaattacact
     5821 tgcaaatgga gagattcgca aacgccctct aatcgaaact aatggggaaa ctggagaaat
     5881 tgtctgggat aaagggcgag attttgccac agtgcgcaaa gtattgtcca tgccccaagt
     5941 caatattgtc aagaaaacag aagtacagac aggcggattc tccaaggagt caattttacc
     6001 aaaaagaaat tcggacaagc ttattgctcg taaaaaagac tgggatccaa aaaaatatgg
     6061 tggttttgat agtccaacgg tagcttattc agtcctagtg gttgctaagg tggaaaaagg
     6121 gaaatcgaag aagttaaaat ccgttaaaga gttactaggg atcacaatta tggaaagaag
     6181 ttcctttgaa aaaaatccga ttgacttttt agaagctaaa ggatataagg aagttaaaaa
     6241 agacttaatc attaaactac ctaaatatag tctttttgag ttagaaaacg gtcgtaaacg
     6301 gatgctggct agtgccggag aattacaaaa aggaaatgag ctggctctgc caagcaaata
     6361 tgtgaatttt ttatatttag ctagtcatta tgaaaagttg aagggtagtc cagaagataa
     6421 cgaacaaaaa caattgtttg tggagcagca taagcattat ttagatgaga ttattgagca
     6481 aatcagtgaa ttttctaagc gtgttatttt agcagatgcc aatttagata aagttcttag
     6541 tgcatataac aaacatagag acaaaccaat acgtgaacaa gcagaaaata ttattcattt
     6601 atttacgttg acgaatcttg gagctcccgc tgcttttaaa tattttgata caacaattga
     6661 tcgtaaacga tatacgtcta caaaagaagt tttagatgcc actcttatcc atcaatccat
     6721 cactggtctt tatgaaacac gcattgattt gagtcagcta ggaggtgact gaagtatatt
     6781 ttagatgaag attatttctt aataactaaa aatatggtat aatactctta ataaatgcag
     6841 taatacaggg gcttttcaag actgaagtct agctgagaca aatagtgcga ttacgaaatt
     6901 ttttagacaa aaatagtcta cgaggtttta gagctatgct gttttgaatg gtcccaaaac
     6961 cggtaacacc gtgaaactgc agtacagcgc gttttagagc tatgctgttt tgaatggtcc
     7021 caaaacgctg ggtgtggtgg gtccaggtat cgtttcgttt tagagctatg ctgttttgaa
     7081 tggtcccaaa acgagtgcga aatccgacaa agacttcgtt atgttttaga gctatgctgt
     7141 tttgaatggt cccaaaactt cagcacactg agacttgttg agttccatgt tttagagcta
     7201 tgctgttttg aatggactcc attcaacatt gccgatgata acttgagaaa gagggttaat
     7261 accagcagtc ggataccttc ctattctttc tgttaaagcg ttttcatgtt ataataggca
     7321 aaagaagagt agtgtgatcg tccattccga cagcatcgcc agtcactatg gcgtgctgct
     7381 agcgctatat gcgttgatgc aatttctatg cgcacccgtt ctcggagcac tgtccgaccg
     7441 ctttggccgc cgcccagtcc tgctcgcttc gctacttgga gccactatcg actacgcgat
     7501 catggcgacc acacccgtcc tgtggatcct ctacgccgga cgcatcgtgg ccggcatcac
     7561 cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg
     7621 ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt
     7681 ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct
     7741 caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg
     7801 tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat
     7861 gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc
     7921 ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg
     7981 cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc
     8041 cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccgacgcgct
     8101 gggctacgtc ttgctggcgt tcgcgacgcg aggctggatg gccttcccca ttatgattct
     8161 tctcgcttcc ggcggcatcg ggatgcccgc gttgcaggcc atgctgtcca ggcaggtaga
     8221 tgacgaccat cagggacagc ttcaaggatc gctcgcggct cttaccagcc taacttcgat
     8281 cattggaccg ctgatcgtca cggcgattta tgccgcctcg gcgagcacat ggaacgggtt
     8341 ggcatggatt gtaggcgccg ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc
     8401 atggagccgg gccacctcga cctgaatgga agccggcggc acctcgctaa cggattcacc
     8461 actccaagaa ttggagccaa tcaattcttg cggagaactg tgaatgcgca aaccaaccct
     8521 tggcagaaca tatccatcgc gtccgccatc tccagcagcc gcacgcggcg catctcgggc
     8581 agcgttgggt cctggccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta
     8641 ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga
     8701 agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc
     8761 cgtgtttcgt aaagtctgga aacgcggaag tcccctacgt gctgctgaag ttgcccgcaa
     8821 cagagagtgg aaccaaccgg tgataccacg atactatgac tgagagtcaa cgccatgagc
     8881 ggcctcattt cttattctga gttacaacag tccgcaccgc tgtccggtag ctccttccgg
     8941 tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc
     9001 gtaggacagg tgccggcagc gcccaacagt cccccggcca cggggcctgc caccataccc
     9061 acgccgaaac aagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg
     9121 ctaccctgtg gaacacctac atctgtatta acgaagcgct aaccgttttt atcaggctct
     9181 gggaggcaga ataaatgatc atatcgtcaa ttattacctc cacggggaga gcctgagcaa
     9241 actggcctca ggcatttgag aagcacacgg tcacactgct tccggtagtc aataaaccgg
     9301 taaaccagca atagacataa gcggctattt aacgaccctg ccctgaaccg acgaccgggt
     9361 cgaatttgct ttcgaatttc tgccattcat ccgcttatta tcacttattc aggcgtagca
     9421 ccaggcgttt aagggcacca ataactgcct taaaaaaa
//
