LOCUS       .                       9019 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             5131..5163
                     /label="T7 tag (gene 10 leader)"
                     /note="leader peptide from bacteriophage T7 gene 10"
     CDS             5173..8856
                     /label="LbCpf1"
                     /note="CRISPR-associated protein from Lachnospiraceae
                     bacterium ND2006 (Zetsche et al., 2015)"
     CDS             8863..8880
                     /label="6xHis"
                     /note="6xHis affinity tag"
     terminator      8947..8994
                     /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 atggctagca tgactggtgg acagcaaatg
     5161 ggtcgcggat ccatgagcaa actggagaag ttcacgaact gttattctct gtcgaaaacc
     5221 ttacgtttta aggccattcc ggtaggtaaa acgcaagaaa atatcgacaa taaacgtctc
     5281 ctggttgaag atgaaaagcg tgccgaagat tataaaggtg ttaaaaagct gcttgatcgc
     5341 tattacctgt cttttatcaa cgatgttctg cattccatca aattgaaaaa cttgaataat
     5401 tacatctcgt tatttcgcaa aaaaacccgt accgaaaaag aaaacaagga gctggaaaac
     5461 cttgaaatta atctgcgtaa agaaatcgcg aaagcattta aaggtaatga aggatataaa
     5521 tctctgttta aaaaagacat tattgaaacc atcctgcctg agttcttgga tgataaagat
     5581 gaaattgccc tggttaatag ctttaatggc tttacgactg catttaccgg cttttttgat
     5641 aaccgtgaaa acatgttcag cgaagaagcg aaatcgactt ccattgcctt ccgttgcatt
     5701 aatgaaaacc tgacccgcta catttctaat atggacatct ttgaaaaagt tgatgccatt
     5761 ttcgacaaac atgaagttca agaaatcaaa gaaaagattt taaactccga ttatgatgtt
     5821 gaagatttct ttgagggaga atttttcaat tttgttctca cccaagaggg tattgatgtc
     5881 tataacgcca ttatcggagg atttgttacg gaatcaggcg aaaagattaa aggactgaat
     5941 gaatacatta atttatacaa tcagaaaacc aaacagaaat tgccaaaatt caaaccactg
     6001 tataaacagg tattaagcga ccgtgaatcc ttgtcctttt atggcgaagg ctatacaagc
     6061 gatgaagagg tgctcgaagt attccgtaat actttgaaca aaaactcgga aatcttctcc
     6121 agcattaaaa aactggaaaa attgtttaaa aatttcgatg aatatagtag cgcaggcatc
     6181 ttcgtcaaaa atggccctgc gatttcaacc atttctaaag acattttcgg tgaatggaac
     6241 gtgatccgcg ataaatggaa cgcggagtat gacgacattc atctgaaaaa aaaagccgtt
     6301 gttaccgaaa agtatgaaga tgaccgccgc aagagcttta aaaaaattgg tagtttctca
     6361 ctggaacagt tacaagaata tgcagacgcc gatctgtcgg ttgtggaaaa gttaaaagag
     6421 attattatcc agaaagtcga tgaaatttat aaagtgtacg gctccagcga aaagctcttt
     6481 gatgccgact ttgttttaga aaaatcgctg aaaaaaaacg atgccgtggt ggccatcatg
     6541 aaagatctgt tggattcggt caaatcgttc gaaaactata tcaaggcgtt ctttggcgaa
     6601 gggaaagaga ccaatcgtga cgaatcgttc tatggcgatt ttgtgttggc gtatgatatt
     6661 cttctgaaag tagatcacat ctatgatgct atccgcaact acgtgaccca gaaaccgtat
     6721 tcgaaggata aatttaagct gtattttcaa aaccctcagt ttatgggtgg ctgggataaa
     6781 gataaagaaa ccgactaccg cgcgacgatc ttgcgctatg gctctaagta ctatttagcc
     6841 atcatggata aaaagtatgc aaaatgcctc cagaaaatcg ataaggatga tgtcaacggg
     6901 aactacgaaa aaatcaacta caaactcttg cctggcccga ataaaatgct gccgaaagtc
     6961 tttttttcca aaaaatggat ggcctattat aacccttccg aagatattca gaagatttac
     7021 aagaacggta cctttaaaaa aggtgacatg tttaatctga atgactgtca caagcttatt
     7081 gatttcttca aagatagtat ttctcgctac cctaaatggt caaacgcgta cgattttaat
     7141 ttctcagaaa cagagaaata caaagatatt gcggggtttt atcgcgaagt cgaagaacaa
     7201 gggtataagg tgtcgttcga atcggcttcc aaaaaggaag tggataaact tgttgaagaa
     7261 ggcaaactct atatgttcca gatctacaac aaagatttct ccgacaagag ccatggcacg
     7321 ccgaacttac ataccatgta ctttaaatta ctgttcgatg aaaataatca tggtcagatt
     7381 cgcttgagcg gtggcgccga actgtttatg cgccgcgcat cattgaaaaa ggaagaactt
     7441 gttgtgcacc cagcaaacag tccgattgca aacaaaaatc cggataaccc gaagaagact
     7501 accaccttaa gctatgatgt atataaagat aaacgtttta gcgaagatca gtatgaactc
     7561 cacattccga tcgcgattaa caaatgtcct aagaacatct ttaaaatcaa taccgaggtt
     7621 cgtgtactgc tcaaacacga cgacaacccc tatgttattg gcattgaccg cggcgaacgc
     7681 aacctgctgt atattgtcgt tgtggatggc aaaggtaaca ttgttgaaca gtatagcctt
     7741 aatgaaatca tcaataattt caacggcatt cgtattaaaa cggactatca tagcctgctg
     7801 gacaaaaaag agaaagaacg cttcgaagcc cgtcagaatt ggacctctat cgaaaatatc
     7861 aaggaactga aagctgggta catttctcaa gtggtccaca agatttgcga actggtggaa
     7921 aagtatgacg cggtgatcgc actggaggat ctgaattcgg gctttaaaaa tagccgtgtg
     7981 aaggtggaga aacaggttta ccaaaagttc gaaaaaatgc tgattgacaa acttaattac
     8041 atggtggaca aaaaatctaa tccatgtgcg accggcgggg ctctcaaagg ctatcagatt
     8101 acgaataaat tcgaaagctt caaatccatg tctacccaga acggatttat cttttacatc
     8161 cccgcctggc tgacttccaa aattgatccg agtaccggtt ttgttaatct gctgaaaacc
     8221 aaatatacgt cgatcgccga ttcaaaaaaa ttcattagca gcttcgaccg tattatgtac
     8281 gtcccggaag aagacctgtt cgaattcgcc cttgactata aaaatttctc gcgtacggat
     8341 gccgattata tcaaaaaatg gaaactgtac agctacggca accgcatccg catttttcgc
     8401 aacccaaaga agaacaacgt atttgactgg gaggaagttt gcttgacgag cgcttacaaa
     8461 gagctcttta acaaatatgg gatcaattat cagcaagggg acatccgtgc gttgctgtgt
     8521 gagcagtcag ataaagcgtt ctacagctct ttcatggctt taatgtcgct gatgttgcag
     8581 atgcgcaatt cgatcactgg ccgcaccgat gtggatttcc tgattagccc ggtcaaaaat
     8641 agcgatggca ttttttacga ttcacgtaac tatgaagccc aggaaaacgc tattctgccg
     8701 aagaacgctg atgcgaacgg cgcgtacaat attgcccgta aggtactgtg ggcaattggt
     8761 cagttcaaga aagcggaaga tgaaaagttg gataaggtca aaattgcgat ctcgaataaa
     8821 gaatggctgg aatatgcgca gaccagcgtg aaacatctcg agcaccacca ccaccaccac
     8881 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     8941 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     9001 ggaggaacta tatccggat
//
