LOCUS       sequence-388184-        6808 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CcuCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6808)
  AUTHORS   Gao S, Wang Y, Qi T, Wei J, Hu Z, Liu J, Sun S, Liu H, Wang Y
  TITLE     Genome editing with natural and engineered CjCas9 orthologs.
  JOURNAL   Mol Ther. 2023 Apr 5;31(4):1177-1187. doi: 
            10.1016/j.ymthe.2023.01.029. Epub 2023 Feb 1.
  PUBMED    36733251
REFERENCE   2  (bases 1 to 6808)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.ymthe.2023.01.029"; journalName: "Mol Ther"; date: 
            "2023-04-5- 5"; volume: "31"; issue: "4"; pages: "1177-1187"
COMMENT     Sequence Label: pAAV-CMV-CcuCas9
FEATURES             Location/Qualifiers
     source          1..6808
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        206..509
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        510..713
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     664..684
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      757..766
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             769..789
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3895..3915
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3934..3953)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    3963..4044
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3988..4007
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4071..4211
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4071..4200
                     /label=AAV2 ITR
     rep_origin      4286..4741
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4373..4392)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4583..4604
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4758..4777)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4877..4899
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4937..4955)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5023..5127
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5128..5196,5197..5988)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5346..5365)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6159..6747
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6648..6667
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtctagtt attaatagta atcaattacg gggtcattag
      181 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      241 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      301 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      361 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      421 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      481 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      541 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      601 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      661 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag
      721 tgaaccgtca gatccgctag agatccgcgg ccgcgggcca ccatggcccc gaagaaaaag
      781 cgcaaggtcg aagcgtccaa gatcttgggc tttgatatcg gaatctccag catcggatgg
      841 gccttcgtgg aaaatgagga actgcgggac tgcggcgtcc ggatcttcac caaggccgag
      901 aaccccgaga acggcgagag ccttgccctg cctagacggg aagctagagg cgtgagaaga
      961 aggctggcta gaagaaaggc ccggctgaac ggcgtgaaga aactgctgtg ccaggagttc
     1021 ggcctcaagc tgaacgacta cttcagcaac gacggcgagc tgcctaaggc ttatcagatg
     1081 agcaagaata ccaagagcgt gtacgagctg agaaccctgg ccctgagctg caagctggag
     1141 ttcaaggacc tggctagagt gtttctgcac atcgccaaac atagaggcta cgggaacaag
     1201 cacgccaaga tcagccaaga cagcaaggat atcggccaaa tcaagaaggc cctgaaacat
     1261 aatgaagagg agctgaagaa ttaccggagt gtgggcgagt acctgtacaa gaacttctac
     1321 cagaaggtgc ggaatttcga agaaatcaag acaaagaaca agaacaacca gagcaccttc
     1381 gagttcaaga acgtgcgaaa caagtctggc aactacaagc aatgtgtgag ccaggagtgg
     1441 ctgaaaaacg agctgaagct gatcttcgag aagcagagat ctttcggcgt gaaactgtcc
     1501 aaggatttcg agaacgagct gctggacaag atctttttcc agcgggagct aaaggatttt
     1561 agcgacaaaa tcggcttttg catatacttt gaaggcaaga aaagagccag caaggaaagc
     1621 ctcagcgcca aggaattcat cgcgttgaca agaatgatca acacattcaa gaaccttgaa
     1681 cagcagaacc ggaagaacca gtctggggag atctactccc aggagatcat caaggagatt
     1741 ttgaacatca tcctggacaa gggcatgatg aagtacaaag aactgaggac cctgctgaag
     1801 cttgataaat ccatccagtt caaggatagc aagcttagtt acgagaaggg gatcgagaag
     1861 gccgaggaaa ccaaattcat tgatttcaag gaactgcgca acttcaagaa ggctcttggc
     1921 ggatctttcg aaggcctgga gagaaagcat attgacgaaa ttgcccacaa gataacactg
     1981 atcaagtcca aggagaagct gaaaattgct ctggagaact acatcaagga acacaagctg
     2041 gaacagagac ttaacgagtg cctgggaaac ctgtctgagc tggatttcag cggccacatc
     2101 gacctgtctc tggaggccct ggagcagatc ctgcctttta tgagagaggg cttcagatac
     2161 gacgaggccg tgaagaaggc cggcctgaga gagatccgga aagattacct gaagaaggac
     2221 ttcctgccac ctctgaagga ctttgagaac ctggctaatc ctgtggtggt gagagccctc
     2281 gctgaataca gaaaagtgct gaacgccctg ctgaagaagt acggaaaaat gcacaagatt
     2341 cacattgagc tgacaagaga gatcgggctg aaccacaaag aaagacagaa ctatatcgag
     2401 gaacaaaaga acaactttaa ctttaacgag gaagccagaa agaagtgcga ggagatcggc
     2461 ttggatgccg agaagtacct gctgaagatg aaactgttcc tggagcagga cgagttctgt
     2521 atctacagcg gagagaagat caagatcgag cacctgagag atgccaagat gctggaaatc
     2581 gaccacatct acccctacag cagaagcttc gatgacagct acctgaacaa ggtgctggtg
     2641 ttctccaagg aaaaccagaa caaaggaaac aagacccctt tcgaggcttt tggcaatgac
     2701 aaagagaagt gggacaagat caaaggcctg acccagggcc tgaagaagct gcccagcaag
     2761 aaaaagaaaa gaatcctgaa cctctacttc aatgacaagg aagctggctt catcagcaga
     2821 aacctgaacg acaccgccta catcgccaga ttctccgcca actacacaaa atacttcctg
     2881 gacttcctgc ccctgagcga gaacgaagtg tccatatccg gagagaaagg ctctaaggtg
     2941 cacgtggaag ccatcagcgg cagactgaca gccatgctga gacactactg gggcctgggc
     3001 cagaaggaca gaaacaacca cctgcaccac gccgtggacg cagtgatcgt ggcctacacc
     3061 aacgccaaga tcatcaagaa cttcgccgac ttcaagaagt tccgggagca aaacaaggcc
     3121 aagatctacg ccaaggaatt cgcccagacc accgacaaga aaatctttaa accctgtgaa
     3181 aatttcagaa acaagatcct catgaagatt gacgagatct ttgtgtccaa acctccaaga
     3241 aagcgggccc ggggcgccct gcacaaggag agcttcatga gcttcgacga caagaagctg
     3301 ctcaaaagct acggaggaag agagggcgtc aagaaggccc tgtccctggg caagatcaga
     3361 aagatcggta caaagatagt tgagaacaag aatatggtgc gggtcgacat cttcaaggac
     3421 aagaagggca agttctacgg cgtccccatc tatacaatgg atttcgctct gggaatcctg
     3481 cctaacaaag ccgtggtgag tggagaagat aagaaaaccc gggaaatcaa ggactggaag
     3541 gaaatggacg agaagtacga attctgcttc agcctgttca aagacgatct gattaaaatt
     3601 cagaaaaagg atatgaagga acctaagttc tgctactaca agaatttcga cgtgtccaac
     3661 gcttctatct gcatcgcaaa acacgacaac aagttcgaga atagagacga ggacgaaaaa
     3721 atcctgtaca gcaaagccac ctacaagaag gtggaagcca gaggcatcgg catccagaat
     3781 ctgaagatcc tggagaagtg gcaggtgagt cctctgggcg aaaagcggga attcgagttc
     3841 gagcctcgga agaatatctc actgaaaacc accaacagaa agaatgtgtt cagccccaag
     3901 aagaagagaa aggtgtagct cgagctcgag ctcgatgagt ttggacaaac cacaactaga
     3961 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     4021 attataagct gcaataaaca agttaaccca cgtgcggacc gagcggccgc aggaacccct
     4081 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     4141 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     4201 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4261 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4321 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     4381 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4441 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4501 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4561 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     4621 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     4681 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     4741 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     4801 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4861 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4921 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     4981 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5041 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5101 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5161 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5221 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5281 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5341 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5401 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5461 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5521 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5581 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5641 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     5701 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     5761 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     5821 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     5881 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     5941 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6001 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6061 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6121 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6181 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6241 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6301 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6361 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6421 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6481 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6541 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6601 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6661 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6721 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6781 gccttttgct ggccttttgc tcacatgt
//
