LOCUS       sequence-385768-        6883 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Hsp1Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6883)
  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 6883)
  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-Hsp1Cas9
FEATURES             Location/Qualifiers
     source          1..6883
                     /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             3970..3990
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4009..4028)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    4038..4119
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4063..4082
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4146..4286
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4146..4275
                     /label=AAV2 ITR
     rep_origin      4361..4816
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4448..4467)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4658..4679
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4833..4852)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4952..4974
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5012..5030)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5098..5202
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5203..5271,5272..6063)
                     /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(5421..5440)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6234..6822
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6723..6742
                     /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 aagcgtccag cgacaaatcc aagatcctgg gattcgacat cggcatcacc
      841 agcatcggct gggcctacgt ggaaggcgaa gagctgatcg actgcggcgt ccggatcttt
      901 accaaggccg agaatcccaa gaccaaagaa tccctggccc tgcctagacg ggaagctaga
      961 ggcgtgcggc ggagactggc ccgaagaaaa ggcagactga atgccatcaa gcaactgatc
     1021 tgcaaggaat tcggcctgaa ctaccaagac tacatcgctc aggacggcga gctgcccaaa
     1081 gcctacgccg ggaagctgat taacccttat gagctgagat ctaaggccct tagagagaag
     1141 ctggaaaagg aagagctggc ccgagtgatt ctgcacatcg ccaagcaccg gggctacgga
     1201 aacaagcatg ccaaggaaac cgacgatgcc gagagcggca agatcaagaa agcaattagc
     1261 aacaatgaga agatcctgaa tgagaaggga tatcagagcg ccggagagta cctgtaccag
     1321 gaatttttcc agaaagaaag actgcaggag gaaaagaagg gcaacaacac gaccaaagaa
     1381 ttcaaaaatg tgaggaacaa agctggcgac tacgagcact gtatcgctca gacccagatg
     1441 cagaacgagt tgaggcttat tttcgagaag cagcgagagt ttggcctgaa cttcagcgag
     1501 gaattcaaga agaaagtgct ggagacggcc ttttttcaga gacctctgaa agacttcagc
     1561 gataagatcg gcgattgcac cttcatcgag ggcgccaagc gggcccctaa ggacagcctg
     1621 agcgccatcg agttcgtggc cctgggaaga atcatcaaca ccctgaaaaa tctgcagaac
     1681 acaagcggcg aggtgtacag cagagataag atcaacgaga tcctgcaaat cgtgctggat
     1741 aagggcgaga tcagctacaa gaaactgaga gaaatcctgg acatcgagag cagcgtgcgt
     1801 ttcaaggaca gcaaactgga ttacaccaaa gaactgaagg aagccgagaa aaccaagctg
     1861 ttcgagagca agaacctcca gaaattcaag aaagctctgg gcagcttcga aggcatctcc
     1921 agagagatct tggacagtat cgccaaggag atcatgatca caaaggataa agagaagctg
     1981 gccaaggagc tgaagaagtt ccctctggat gaaacccaga tccagaacct ttctaacttg
     2041 tccttcagcc aaacaatcaa cctgtcactg gaagccctgt ctcagatcct gcccttcatg
     2101 agaggcgaga gagaggacaa atgcctgaga tacgacgagg ctgtggaaaa ggccggcctg
     2161 aaggaggtgg ccaaaaacaa gtctaaggat gacttcctgc ctcctctgga aaactacgag
     2221 ccttacctgg ccaatcctgt ggtggcccgg gccctctctg agtatagaaa ggtgctgaac
     2281 gccctgctga gaaagtacgg caagatgcac aagattcaca tcgagtttac cagagaagtg
     2341 ggcaagagca aagaggagcg gaacaaaatc gaaaaagaac agaaagagaa tttcgccaaa
     2401 aaccaggagg cccagaagca gtgcgagatc ctgggcctgc ctatcaacgg cgctaatatc
     2461 ctgaagatga agctgtggcg ggagcagcag gagtcctgcg tgtacagcgg caaggaaatc
     2521 gccatcgacg acctgaagaa ccccgccatc ctgcaggtgg atcacatcta tccttactct
     2581 agaagctttg atgattctta taacaacaag gtgctggtgt tctccaaaga aaaccagaac
     2641 aaactcaaca agacaccttt cgaggctttc ggcgaaaaca aggacaaatg ggagaaaatc
     2701 ctgtccctgg cttctaagct tcctaagcct aagcggcaga ggatctacaa catgcagttc
     2761 aaggacaaag aacaaggctt tctgacccgg aatatcaacg acaccggcta catcgctaga
     2821 ctggtcagcc agtggacaaa gaactgcctg agcttcgaga agctgagcga ggatgaggtt
     2881 tccatcgccg gcgagaaagg aagcaaagtg cacgtggaag tgatcagcgg ctccctgaca
     2941 gccatgctgc ggcactactg gggcctgggc gacaagaacc gggacaacca cctgcaccac
     3001 gcggtggacg ccatcatcat cgcttatagc aacgaaaagg tgatcaaggc cttctctgat
     3061 tacaaaaaac atagagagca gaacaaggcc aagttctacg ccaaggagat cgccgagagc
     3121 gagtacaaga agcagaaggt tttcttcgaa cctaccaacc acttcagaca aaaagtgctg
     3181 gaaaagattg acagcatctt cgtgagcaaa cctccaagga agagagcccg gggagccctg
     3241 cacgaggaaa cattctacag cttcgagaga ctgtgcaacg acaaggagta cgccaagaaa
     3301 gagaagatgg actacggcgg aaaggctggc atccagaagg ctatcgccct gggtaaaatt
     3361 agacaggttg gaacaaagat cgtgtctaac ggcaccatgg tgcgggtgga catcttccag
     3421 cacaagagca acggcaagtt ttacggcgtc cccatctaca caatggattt tgccctcggc
     3481 gtgctgccta acaaggctgt ggtgtccgga aaagataagc agggagtgat caaggactgg
     3541 ctggaaatgg acagcaacta cgagttttgc ttcagtctct tcaaggacga cctgatcctg
     3601 gtgcagaaaa aggagatgga aaaagcagaa ctgtgctact acaccgattt taacagcaac
     3661 acggccggca tcaaattcga gaagcacaac aacctgtacg tgaacctgaa tgagaatgaa
     3721 agaaagctgt tcaccagcaa ggaagttgtt aagggcggca agaagaacgt gtggaaggac
     3781 aaaaccccta aagaaatcga gcagctggag aagaacagaa agatcatcgg aggcttcgtg
     3841 accatcgagg atctgaaaac cttcgagaaa taccaggtgt ctcctctggg agaggtgcag
     3901 aaggccgact tcgcccctag acaggccatc accctgaaaa gcagccctaa gcacagaatc
     3961 aacaagagcc ccaagaagaa gagaaaggtg tagctcgagc tcgagctcga tgagtttgga
     4021 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     4081 gctttatttg taaccattat aagctgcaat aaacaagtta acccacgtgc ggaccgagcg
     4141 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     4201 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     4261 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     4321 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     4381 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     4441 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     4501 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4561 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     4621 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     4681 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     4741 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     4801 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     4861 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     4921 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     4981 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     5041 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     5101 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5161 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     5221 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     5281 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     5341 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     5401 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     5461 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     5521 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     5581 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     5641 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     5701 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     5761 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     5821 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     5881 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     5941 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6001 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6061 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6121 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6181 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6241 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6301 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6361 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     6421 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6481 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6541 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6601 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     6661 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6721 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     6781 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     6841 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
