LOCUS       pX335.        8434 bp DNA     circular SYN 11-SEP-2021
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    pX335
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8434)
  AUTHORS   caoheibi
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8434)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..241
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer;
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..1004
                     /label=chicken beta-actin promoter
     intron          1005..1233
                     /note="hybrid intron"
                     /note="hybrid between chicken beta-actin (CBA) and minute
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1254..1280
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="HA"
                     /translation="YPYDVPDYA"
     CDS             1284..1304
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /label=nuclear localization signal of SV40 large T
                     ant
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             1314..5414
                     /label=Cas9(D10A)
                     /note="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             5418..5438
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T
                     antigen"
                     /label=nuclear localization signal of SV40 large T
                     ant
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     polyA_signal    5481..5688
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5697..5837
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      5912..6367
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        6649..6753
                     /label=AmpR promoter
     CDS             6754..7611
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      7785..8373
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
      901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc
     1021 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
     1081 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
     1141 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac
     1201 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atgtacccat
     1261 acgatgttcc agattacgct tcgccgaaga aaaagcgcaa ggtcgaagcg tccgacaaga
     1321 agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg atcaccgacg
     1381 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca
     1441 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc
     1501 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag
     1561 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt
     1621 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg
     1681 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg
     1741 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt
     1801 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc
     1861 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca
     1921 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa
     1981 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggcaac ctgattgccc
     2041 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac
     2101 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg
     2161 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg
     2221 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga
     2281 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc
     2341 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg
     2401 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg
     2461 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga
     2521 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc
     2581 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc
     2641 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct
     2701 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca
     2761 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca
     2821 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc
     2881 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc
     2941 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc
     3001 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg
     3061 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca
     3121 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga
     3181 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg
     3241 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc
     3301 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga
     3361 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct
     3421 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca
     3481 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg
     3541 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg
     3601 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga
     3661 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca
     3721 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg
     3781 tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc
     3841 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc
     3901 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc
     3961 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg
     4021 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa
     4081 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg
     4141 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt
     4201 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg
     4261 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc
     4321 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca
     4381 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga
     4441 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg
     4501 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc
     4561 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg
     4621 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga
     4681 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc
     4741 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc
     4801 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag
     4861 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt
     4921 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa
     4981 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga
     5041 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc
     5101 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg
     5161 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag
     5221 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct
     5281 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg
     5341 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc
     5401 agctgggagg cgacagcccc aagaagaaga gaaaggtgga ggccagctaa gaattcctag
     5461 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     5521 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     5581 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     5641 ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcagga
     5701 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     5761 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     5821 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     5881 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     5941 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     6001 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     6061 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     6121 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     6181 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     6241 aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     6301 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     6361 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     6421 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     6481 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     6541 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     6601 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     6661 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     6721 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     6781 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     6841 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     6901 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     6961 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     7021 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     7081 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     7141 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     7201 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     7261 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     7321 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     7381 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     7441 gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca
     7501 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     7561 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     7621 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     7681 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     7741 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     7801 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     7861 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     7921 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     7981 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     8041 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     8101 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     8161 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     8221 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     8281 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     8341 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     8401 ttcctggcct tttgctggcc ttttgctcac atgt
//
