LOCUS       PX854.        7087 bp DNA     circular SYN 11-SEP-2021
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    PX854.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7087)
  AUTHORS   caoheibi
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7087)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7087
                     /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             1257..1277
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     CDS             1302..1622
                     /label=FKBP
                     /note="human FK506-binding protein FKBP12"
     CDS             1668..4052
                     /label=Cas9(C)
                     /note="C-terminal portion of Streptococcus pyogenes Cas9 
                     (Zetsche et al., 2015)"
     CDS             4053..4100
                     /label=nucleoplasmin NLS
                     /note="bipartite nuclear localization signal from
                     nucleoplasmin"
     polyA_signal    4134..4341
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4350..4490
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4565..5020
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        5302..5406
                     /label=AmpR promoter
     CDS             5407..6264
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      6438..7026
                     /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 atggccccaa
     1261 agaagaagcg gaaggtcggt atccacggag tcccagcagc cggcgtgcag gtggagacca
     1321 tcagccccgg cgacggcaga accttcccca agagaggcca gacctgcgtg gtgcactaca
     1381 ccggcatgct ggaggacggc aagaagttcg acagcagcag agacagaaac aagcccttca
     1441 agttcatgct gggcaagcag gaggtgatca gaggctggga ggagggcgtg gcccagatga
     1501 gcgtgggcca gagagccaag ctgaccatca gccccgacta cgcctacggc gccaccggcc
     1561 accccggcat catccctcct cacgccaccc tggtgttcga cgtggagctg ctgaagctgg
     1621 agggtggcgg tggctcgggt ggcggtggct cgggtggcgg tggctcgtgc ttcgactccg
     1681 tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc
     1741 tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag
     1801 atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa
     1861 cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg
     1921 gctggggcag gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga
     1981 caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc
     2041 acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg
     2101 atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc
     2161 tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga
     2221 acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc
     2281 gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag
     2341 aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga
     2401 atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg
     2461 tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga
     2521 ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga
     2581 agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg
     2641 acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca
     2701 agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc
     2761 ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc
     2821 tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga
     2881 tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga
     2941 tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg
     3001 tgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct
     3061 tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc
     3121 ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc
     3181 gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga
     3241 ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata
     3301 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca
     3361 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga
     3421 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc
     3481 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc
     3541 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg
     3601 gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc
     3661 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt
     3721 ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca
     3781 agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc
     3841 gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc
     3901 tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca
     3961 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga
     4021 cacggatcga cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg
     4081 gccaggcaaa aaagaaaaag taagaattcc tagagctcgc tgatcagcct cgactgtgcc
     4141 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     4201 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     4261 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     4321 gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc
     4381 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     4441 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
     4501 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     4561 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4621 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     4681 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     4741 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     4801 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     4861 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat
     4921 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     4981 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     5041 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     5101 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     5161 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     5221 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     5281 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5341 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5401 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     5461 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     5521 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     5581 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     5641 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     5701 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     5761 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     5821 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     5881 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     5941 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     6001 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     6061 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     6121 ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     6181 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6241 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     6301 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     6361 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6421 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     6481 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6541 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     6601 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6661 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6721 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6781 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6841 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     6901 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     6961 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     7021 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     7081 cacatgt
//
