LOCUS       .                      12154 bp    DNA     circular UNK 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   
VERSION     
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     promoter        complement(1..105)
                     /label="AmpR promoter"
     rep_origin      complement(131..586)
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        1365..1605
                     /label="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        3495..3570
                     /label="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes
                     CRISPR/Cas9 system"
     promoter        3632..3843
                     /label="EF-1α core promoter"
                     /note="core promoter for human elongation factor EF-1α"
     CDS             3868..7971
                     /label="Cas9"
                     /note="S. pyogenes Cas9, codon optimized for C. elegans
                     with synthetic introns"
     CDS             8020..8043
                     /label="FLAG"
                     /note="FLAG® epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             8053..8109
                     /label="P2A"
                     /note="2A peptide from porcine teschovirus-1 polyprotein"
     CDS             8110..8703
                     /label="PuroR"
                     /note="puromycin N-acetyltransferase"
     polyA_signal    8725..8857
                     /label="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     promoter        complement(10181..10211)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(10226..10247)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(10535..11123)
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(11297..12154)
                     /label="AmpR"
                     /note="β-lactamase"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
      601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
      781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
      841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
      901 tacgttaaag ataatcatgc gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaaa caaaaactca
     1081 aaatttcttc tataaagtaa caaaactttt atgagggaca gccccccccc aaagccccca
     1141 gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct
     1201 ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg
     1261 gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag
     1321 acacctgggg ggatacgggg aaaaggcctc caaggcctac tagtgagggc ctatttccca
     1381 tgattccttc atatttgcat atacgataca aggctgttag agagataatt agaattaatt
     1441 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
     1501 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
     1561 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accggagacg
     1621 gttgtaaatg agcacacaaa atacacatgc taaaatatta tattctatga cctttataaa
     1681 atcaaccaaa atcttctttt taataacttt agtatcaata attagaattt ttatgttcct
     1741 ttttgcaaac ttttaataaa aatgagcaaa ataaaaaaac gctagtttta gtaactcgcg
     1801 ttgttttctt cacctttaat aatagctact ccaccacttg ttcctaagcg gtcagctcct
     1861 gcttcaatca ttttttgagc atcttcaaat gttctaactc caccagctgc tttaactaaa
     1921 gcattgtctt taacaactga cttcattagt ttaacatctt caaatgttgc acctgatttt
     1981 gaaaatcctg ttgatgtttt aacaaattct aatccagctt caacagctat ttcacaagct
     2041 ttcatgattt cttcttttgt taataaacaa ttttccataa tacatttaac aacatgtgat
     2101 ccagctgctt tttttacagc tttcatgtct tctaaaacta attcataatt tttgtctttt
     2161 aatgcaccaa tatttaatac catatcaatt tctgttgcac catctttaat tgcttcagaa
     2221 acttcgaatg cttttgtagc tgttgtgcat gcacctagag gaaaacctac aacatttgtt
     2281 attcctacat ttgtgccttt taataattct ttacaatagc ttgttcaata tgaattaaca
     2341 caaactgttg caaaatcaaa ttcaattgct tcatcacata attgtttaat ttcagctttc
     2401 gtagcatctt gttttaataa tgtgtgatct atatatttgt ttagtttcat tttttctcct
     2461 atatattcat ttttaatttt aattctttaa taatttcgtc tactttaact ttagcgtttt
     2521 gaacagattc accaacacct ataaaataaa tttttagttt aggttcagtt ccacttgggc
     2581 gaacagcaaa tcatgactta tcttctaaat aaaattttag taagtcttgt cctggcatat
     2641 tatacattcc atcgatgtag tcttcaacat taacaacttt aagtccagca atttgagtta
     2701 agggtgttgc tctcaatgat ttcattaatg gttcaatttt taatttcttt tcttctggtt
     2761 taaaattcaa gtttaaagtg aaagtgtaat atgcacccat ttctttaaat aaatcttcta
     2821 aatagtctac taatgtttta ttttgttttt tataaaatca agcagcctct gctattaata
     2881 tagaagcttg tattccatct ttatctctag ctgagtcatc aattacatat ccataacttt
     2941 cttcataagc aaaaacaaaa tttaatccgt tatcttcttc tttagcaatt tctctaccca
     3001 ttcatttaaa tccagttaaa gtttttacaa tattaactcc atatttttca tgagcgattc
     3061 tatcacccaa atcacttgtt acaaaacttg aatatagagc cggatttttt ggaatgctat
     3121 ttaagcgttt tagatttgat aattttcaat caattaaaat tggtcctgtt tgatttccat
     3181 ctaatcttac aaaatgacca tcatgtttta ttgccattcc aaatctgtca gcatctgggt
     3241 cattcataat aataatatct gcatcatgtt taataccata ttcaagcggt atttttcatg
     3301 caggatcaaa ttctggattt ggatttacaa catttttaaa tgtttcatct tcaaatgcat
     3361 gctcttcaac ctcaataacg ttatatcctg attcacgtaa tatttttggg gtaaatttag
     3421 ttcctgttcc attaactgcg ctaaaaataa tttttaaatc ttttttagct tcttgctctt
     3481 ttttgtacgt ctctgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
     3541 caacttgaaa aagtggcacc gagtcggtgc ttttttgaat tcgctagcta ggtcttgaaa
     3601 ggagtgggaa ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc
     3661 gagaagttgg ggggaggggt cggcaattga tccggtgcct agagaaggtg gcgcggggta
     3721 aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg
     3781 tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca
     3841 caggaccggt tctagagcgc tgccaccatg gacaagaagt acagcatcgg cctggacatc
     3901 ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa
     3961 ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg
     4021 ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga
     4081 tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc
     4141 aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag
     4201 aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag
     4261 taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg
     4321 cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag
     4381 ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc
     4441 tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc
     4501 ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc
     4561 gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac
     4621 ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac
     4681 gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg
     4741 gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag
     4801 atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac
     4861 ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc
     4921 ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag
     4981 ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg
     5041 aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc
     5101 caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca
     5161 ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac
     5221 gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa
     5281 accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc
     5341 atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac
     5401 agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc
     5461 gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg
     5521 ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa
     5581 atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg
     5641 ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa
     5701 aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg
     5761 atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg
     5821 aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg
     5881 gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga
     5941 aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc
     6001 caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc
     6061 gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg
     6121 ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag
     6181 aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg
     6241 ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg
     6301 tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac
     6361 cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa ggacgactcc
     6421 atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc
     6481 tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg
     6541 attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg
     6601 gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg
     6661 gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg
     6721 gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag
     6781 ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc
     6841 gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc
     6901 gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag
     6961 gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc
     7021 ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga aaccggggag
     7081 atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa
     7141 gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg
     7201 cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac
     7261 ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag
     7321 ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga
     7381 agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa
     7441 aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag
     7501 agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa
     7561 tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat
     7621 aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag
     7681 cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg
     7741 tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac
     7801 ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc
     7861 gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc
     7921 atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga caagcgacct
     7981 gccgccacaa agaaggctgg acaggctaag aagaagaaag attacaaaga cgatgacgat
     8041 aagggatccg gcgcaacaaa cttctctctg ctgaaacaag ccggagatgt cgaagagaat
     8101 cctggaccga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg
     8161 gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat
     8221 ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg
     8281 ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg
     8341 ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg
     8401 agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc
     8461 aaggagcccg cgtggttcct ggccaccgtc ggagtctcgc ccgaccacca gggcaagggt
     8521 ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc
     8581 ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc
     8641 accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt
     8701 gcctgaacgc gttaagtcga cgttaacttg tttattgcag cttataatgg ttacaaataa
     8761 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     8821 ttgtccaaac tcatcaatgt atcttatcat gtctggaatt gactcaaatg atgtcaatta
     8881 gtctatcaga agctatctgg tctcccttcc gggggacaag acatccctgt ttaatattta
     8941 aacagcagtg ttcccaaact gggttcttat atcccttgct ctggtcaacc aggttgcagg
     9001 gtttcctgtc ctcacaggaa cgaagtccct aaagaaacag tggcagccag gtttagcccc
     9061 ggaattgact ggattccttt tttagggccc attggtatgg ctttttcccc gtatcccccc
     9121 aggtgtctgc aggctcaaag agcagcgaga agcgttcaga ggaaagcgat cccgtgccac
     9181 cttccccgtg cccgggctgt ccccgcacgc tgccggctcg gggatgcggg gggagcgccg
     9241 gaccggagcg gagccccggg cggctcgctg ctgcccccta gcgggggagg gacgtaatta
     9301 catccctggg ggctttgggg gggggctgtc cctgatatct ataacaagaa aatatatata
     9361 taataagtta tcacgtaagt agaacatgaa ataacaatat aattatcgta tgagttaaat
     9421 cttaaaagtc acgtaaaaga taatcatgcg tcattttgac tcacgcggtc gttatagttc
     9481 aaaatcagtg acacttaccg cattgacaag cacgcctcac gggagctcca agcggcgact
     9541 gagatgtcct aaatgcacag cgacggattc gcgctattta gaaagagaga gcaatatttc
     9601 aagaatgcat gcgtcaattt tacgcagact atctttctag ggttaatcta gctgcatcag
     9661 gatcatatcg tcgggtcttt tttccggctc agtcatcgcc caagctggcg ctatctgggc
     9721 atcggggagg aagaagcccg tgccttttcc cgcgaggttg aagcggcatg gaaagagttt
     9781 gccgaggatg actgctgctg cattgacgtt gagcgaaaac gcacgtttac catgatgatt
     9841 cgggaaggtg tggccatgca cgcctttaac ggtgaactgt tcgttcaggc cacctgggat
     9901 accagttcgt cgcggctttt ccggacacag ttccggatgg tcagcccgaa gcgcatcagc
     9961 aacccgaaca ataccggcga cagccggaac tgccgtgccg gtgtgcagat taatgacagc
    10021 ggtgcggcgc tgggatatta cgtcagcgag gacgggtatc ctggctggat gccgcagaaa
    10081 tggacatgga taccccgtga gttacccggc gggcgcgctt ggcgtaatca tggtcatagc
    10141 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
    10201 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
    10261 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
    10321 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
    10381 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
    10441 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
    10501 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
    10561 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
    10621 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
    10681 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
    10741 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
    10801 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
    10861 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
    10921 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
    10981 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
    11041 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
    11101 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
    11161 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
    11221 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
    11281 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
    11341 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
    11401 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
    11461 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
    11521 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
    11581 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
    11641 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
    11701 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
    11761 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
    11821 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
    11881 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
    11941 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
    12001 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
    12061 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
    12121 gaataagggc gacacggaaa tgttgaatac tcat
//
