LOCUS       .                      10099 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             5122..9222
                     /label="eSpCas9(1.1)"
                     /note="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting
                     specificity (Slaymaker et al., 2016)"
     CDS             9223..9936
                     /label="EGFP"
                     /note="the original enhanced GFP (Yang et al., 1996)"
     CDS             9943..9960
                     /label="6xHis"
                     /note="6xHis affinity tag"
     terminator      10027..10074
                     /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 atggccccaa agaagaagcg gaaggtcggt
     5101 atccacggag tcccagcagc cgacaagaag tacagcatcg gcctggacat cggcaccaac
     5161 tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg
     5221 ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac
     5281 agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga
     5341 cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac
     5401 gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag
     5461 cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc
     5521 atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc
     5581 tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg
     5641 aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag
     5701 ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc
     5761 agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag
     5821 aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc
     5881 aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac
     5941 ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag
     6001 aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag
     6061 gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg
     6121 ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag
     6181 agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag
     6241 ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac
     6301 agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc
     6361 cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag
     6421 gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct
     6481 ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc
     6541 ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg
     6601 atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg
     6661 tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg
     6721 agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag
     6781 accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc
     6841 ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac
     6901 cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac
     6961 attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa
     7021 cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg
     7081 agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag
     7141 tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg
     7201 cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc
     7261 ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag
     7321 aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac
     7381 aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag
     7441 aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag
     7501 atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac
     7561 tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc
     7621 gactacgatg tggaccatat cgtgcctcag agctttctgg ccgacgactc catcgacaac
     7681 aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag
     7741 gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag
     7801 agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc
     7861 ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc
     7921 ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa
     7981 gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa
     8041 gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga
     8101 accgccctga tcaaaaagta ccctgcgctg gaaagcgagt tcgtgtacgg cgactacaag
     8161 gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc
     8221 aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac
     8281 ggcgagatcc ggaaggcgcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg
     8341 gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc
     8401 gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg
     8461 aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc
     8521 gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc
     8581 aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc
     8641 gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg
     8701 atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg
     8761 gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac
     8821 ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag
     8881 aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc
     8941 gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac
     9001 aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc
     9061 ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag
     9121 aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc
     9181 ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acgtgagcaa gggcgaggag
     9241 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     9301 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     9361 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     9421 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     9481 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     9541 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     9601 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     9661 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     9721 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     9781 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     9841 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     9901 gccgggatca ctctcggcat ggacgagctg tacaagctcg agcaccacca ccaccaccac
     9961 tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
    10021 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
    10081 ggaggaacta tatccggat
//
