LOCUS       .                       9286 bp    DNA     circular UNK 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   
VERSION     
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     rep_origin      12..467
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(563..1375)
                     /label="KanR"
                     /note="aminoglycoside phosphotransferase"
     rep_origin      1497..2085
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     misc_feature    complement(2271..2413)
                     /label="bom"
                     /note="basis of mobility region from pBR322"
     CDS             complement(2518..2706)
                     /label="rop"
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     protein_bind    complement(3481..3502)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3518..4597)
                     /label="lacI"
                     /note="lac repressor"
     promoter        complement(4598..4675)
                     /label="lacI promoter"
     promoter        4984..5002
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5003..5027
                     /label="lac operator"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-β-D-thiogalactopyranoside (IPTG)."
     RBS             5042..5064
                     /label="RBS"
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5083..5100
                     /label="6xHis"
                     /note="6xHis affinity tag"
     CDS             5110..5127
                     /label="thrombin site"
                     /note="thrombin recognition and cleavage site"
     CDS             5134..5154
                     /label="TEV site"
                     /note="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
     CDS             5155..9075
                     /label="AsCpf1"
                     /note="CRISPR-associated protein from Acidaminococcus sp.
                     BV3L6 (Zetsche et al., 2015)"
     CDS             9130..9147
                     /label="6xHis"
                     /note="6xHis affinity tag"
     terminator      9214..9261
                     /label="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt
      481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      541 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
      601 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
      661 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
      721 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
      781 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
      841 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
      901 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
      961 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     1021 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     1081 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     1141 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     1201 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     1261 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     1321 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     1381 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     1441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     1501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     1561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     1621 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     1681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     1741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     1801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     1861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     1921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     1981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2101 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     2161 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     2221 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     2281 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta
     2341 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     2401 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     2461 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     2521 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     2581 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     2641 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     2701 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     2761 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     2821 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     2881 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     2941 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     3001 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     3061 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     3121 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     3181 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     3241 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     3301 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     3361 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     3421 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     3481 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3541 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3601 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     3661 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     3721 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     3781 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     3841 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     3901 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     3961 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     4021 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     4081 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     4141 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     4201 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     4261 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     4321 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     4381 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     4441 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     4501 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     4561 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     4621 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     4681 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     4741 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     4801 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     4861 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     4921 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
     4981 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
     5041 ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac
     5101 agcagcggcc tggtgccgcg cggcagccat atggaaaacc tgtattttca gggcatgaca
     5161 cagttcgagg gctttaccaa cctgtatcag gtgagcaaga cactgcggtt tgagctgatc
     5221 ccacagggca agaccctgaa gcacatccag gagcagggct tcatcgagga ggacaaggcc
     5281 cgcaatgatc actacaagga gctgaagccc atcatcgatc ggatctacaa gacctatgcc
     5341 gaccagtgcc tgcagctggt gcagctggat tgggagaacc tgagcgccgc catcgactcc
     5401 tatagaaagg agaaaaccga ggagacaagg aacgccctga tcgaggagca ggccacatat
     5461 cgcaatgcca tccacgacta cttcatcggc cggacagaca acctgaccga tgccatcaat
     5521 aagagacacg ccgagatcta caagggcctg ttcaaggccg agctgtttaa tggcaaggtg
     5581 ctgaagcagc tgggcaccgt gaccacaacc gagcacgaga acgccctgct gcggagcttc
     5641 gacaagttta caacctactt ctccggcttt tatgagaaca ggaagaacgt gttcagcgcc
     5701 gaggatatca gcacagccat cccacaccgc atcgtgcagg acaacttccc caagtttaag
     5761 gagaattgtc acatcttcac acgcctgatc accgccgtgc ccagcctgcg ggagcacttt
     5821 gagaacgtga agaaggccat cggcatcttc gtgagcacct ccatcgagga ggtgttttcc
     5881 ttcccttttt ataaccagct gctgacacag acccagatcg acctgtataa ccagctgctg
     5941 ggaggaatct ctcgggaggc aggcaccgag aagatcaagg gcctgaacga ggtgctgaat
     6001 ctggccatcc agaagaatga tgagacagcc cacatcatcg cctccctgcc acacagattc
     6061 atccccctgt ttaagcagat cctgtccgat aggaacaccc tgtctttcat cctggaggag
     6121 tttaagagcg acgaggaagt gatccagtcc ttctgcaagt acaagacact gctgagaaac
     6181 gagaacgtgc tggagacagc cgaggccctg tttaacgagc tgaacagcat cgacctgaca
     6241 cacatcttca tcagccacaa gaagctggag acaatcagca gcgccctgtg cgaccactgg
     6301 gatacactga ggaatgccct gtatgagcgg agaatctccg agctgacagg caagatcacc
     6361 aagtctgcca aggagaaggt gcagcgcagc ctgaagcacg aggatatcaa cctgcaggag
     6421 atcatctctg ccgcaggcaa ggagctgagc gaggccttca agcagaaaac cagcgagatc
     6481 ctgtcccacg cacacgccgc cctggatcag ccactgccta caaccctgaa gaagcaggag
     6541 gagaaggaga tcctgaagtc tcagctggac agcctgctgg gcctgtacca cctgctggac
     6601 tggtttgccg tggatgagtc caacgaggtg gaccccgagt tctctgcccg gctgaccggc
     6661 atcaagctgg agatggagcc ttctctgagc ttctacaaca aggccagaaa ttatgccacc
     6721 aagaagccct actccgtgga gaagttcaag ctgaactttc agatgcctac actggcctct
     6781 ggctgggacg tgaataagga gaagaacaat ggcgccatcc tgtttgtgaa gaacggcctg
     6841 tactatctgg gcatcatgcc aaagcagaag ggcaggtata aggccctgag cttcgagccc
     6901 acagagaaaa ccagcgaggg ctttgataag atgtactatg actacttccc tgatgccgcc
     6961 aagatgatcc caaagtgcag cacccagctg aaggccgtga cagcccactt tcagacccac
     7021 acaaccccca tcctgctgtc caacaatttc atcgagcctc tggagatcac aaaggagatc
     7081 tacgacctga acaatcctga gaaggagcca aagaagtttc agacagccta cgccaagaaa
     7141 accggcgacc agaagggcta cagagaggcc ctgtgcaagt ggatcgactt cacaagggat
     7201 tttctgtcca agtataccaa gacaacctct atcgatctgt ctagcctgcg gccatcctct
     7261 cagtataagg acctgggcga gtactatgcc gagctgaatc ccctgctgta ccacatcagc
     7321 ttccagagaa tcgccgagaa ggagatcatg gatgccgtgg agacaggcaa gctgtacctg
     7381 ttccagatct ataacaagga ctttgccaag ggccaccacg gcaagcctaa tctgcacaca
     7441 ctgtattgga ccggcctgtt ttctccagag aacctggcca agacaagcat caagctgaat
     7501 ggccaggccg agctgttcta ccgccctaag tccaggatga agaggatggc acaccggctg
     7561 ggagagaaga tgctgaacaa gaagctgaag gatcagaaaa ccccaatccc cgacaccctg
     7621 taccaggagc tgtacgacta tgtgaatcac agactgtccc acgacctgtc tgatgaggcc
     7681 agggccctgc tgcccaacgt gatcaccaag gaggtgtctc acgagatcat caaggatagg
     7741 cgctttacca gcgacaagtt ctttttccac gtgcctatca cactgaacta tcaggccgcc
     7801 aattccccat ctaagttcaa ccagagggtg aatgcctacc tgaaggagca ccccgagaca
     7861 cctatcatcg gcatcgatcg gggcgagaga aacctgatct atatcacagt gatcgactcc
     7921 accggcaaga tcctggagca gcggagcctg aacaccatcc agcagtttga ttaccagaag
     7981 aagctggaca acagggagaa ggagagggtg gcagcaaggc aggcctggtc tgtggtgggc
     8041 acaatcaagg atctgaagca gggctatctg agccaggtca tccacgagat cgtggacctg
     8101 atgatccact accaggccgt ggtggtgctg gagaacctga atttcggctt taagagcaag
     8161 aggaccggca tcgccgagaa ggccgtgtac cagcagttcg agaagatgct gatcgataag
     8221 ctgaattgcc tggtgctgaa ggactatcca gcagagaaag tgggaggcgt gctgaaccca
     8281 taccagctga cagaccagtt cacctccttt gccaagatgg gcacccagtc tggcttcctg
     8341 ttttacgtgc ctgccccata tacatctaag atcgatcccc tgaccggctt cgtggacccc
     8401 ttcgtgtgga aaaccatcaa gaatcacgag agccgcaagc acttcctgga gggcttcgac
     8461 tttctgcact acgacgtgaa aaccggcgac ttcatcctgc actttaagat gaacagaaat
     8521 ctgtccttcc agaggggcct gcccggcttt atgcctgcat gggatatcgt gttcgagaag
     8581 aacgagacac agtttgacgc caagggcacc cctttcatcg ccggcaagag aatcgtgcca
     8641 gtgatcgaga atcacagatt caccggcaga taccgggacc tgtatcctgc caacgagctg
     8701 atcgccctgc tggaggagaa gggcatcgtg ttcagggatg gctccaacat cctgccaaag
     8761 ctgctggaga atgacgattc tcacgccatc gacaccatgg tggccctgat ccgcagcgtg
     8821 ctgcagatgc ggaactccaa tgccgccaca ggcgaggact atatcaacag ccccgtgcgc
     8881 gatctgaatg gcgtgtgctt cgactcccgg tttcagaacc cagagtggcc catggacgcc
     8941 gatgccaatg gcgcctacca catcgccctg aagggccagc tgctgctgaa tcacctgaag
     9001 gagagcaagg atctgaagct gcagaacggc atctccaatc aggactggct ggcctacatc
     9061 caggagctgc gcaacaagcg acctgccgcc acaaagaagg ctggacaggc taagaagaag
     9121 aaactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
     9181 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     9241 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggat
//
