LOCUS       sequence-385815-        7401 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Hsp2Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7401)
  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 7401)
  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-Hsp2Cas9
FEATURES             Location/Qualifiers
     source          1..7401
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        156..468
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     387..407
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        488..560
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     enhancer        672..975
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        976..1179
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1130..1150
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1204..1213
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1216..1236
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4462..4482
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4483..4509
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             4483..4509
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(4527..4546)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    4556..4637
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4581..4600
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4664..4804
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4664..4793
                     /label=AAV2 ITR
     rep_origin      4879..5334
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4966..4985)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5176..5197
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5351..5370)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5470..5492
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5530..5548)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5616..5720
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5721..5789,5790..6581)
                     /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(5939..5958)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6752..7340
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7241..7260
                     /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 gcggcccgct ctagagatcc gacgcgccat ctctaggccc
      181 gcgccggccc cctcgcacag acttgtggga gaagctcggc tactcccctg ccccggttaa
      241 tttgcatata atatttccta gtaactatag aggcttaatg tgcgataaaa gacagataat
      301 ctgttctttt taatactagc tacattttac atgataggct tggatttcta taagagatac
      361 aaatactaaa ttattatttt aaaaaacagc acaaaaggaa actcacccta actgtaaagt
      421 aattgtgtgt tttgagacta taaatatccc ttggagaaaa gccttgtttg cagtcttctc
      481 agaagacgtt ttagtccctg aaaagggact aaaataaaga gtttgcggga ctctgcgggg
      541 ttacaatccc ctaaaaccgc ttttttacta gtctcgaggg ggggcccggt acccaattcg
      601 ccctatagtt ctagttatta atagtaatca attacggggt cattagttca tagcccatat
      661 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
      721 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
      781 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
      841 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
      901 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
      961 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
     1021 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
     1081 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
     1141 ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc
     1201 cgcgccacca tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccaaagatc
     1261 ctgggcttcg atatcggaat cgccagcgtg ggatgggcct acgtggagga aaacgagctg
     1321 agagattgcg gcgtgcggat cttcacaaag gccgagaata gcacaggtga gtccctggcc
     1381 ctgcctcggc gggaagccag aggcacacgg agacgcctga agcggagaaa acagagactg
     1441 aacgaactta agagactgct gtgtaaacaa ttcaacttca acctgaacga ctactgcagc
     1501 aacgacggcg agctggccaa agcctatcag acaaccaagg acaccaaaag cccctatgag
     1561 ctgcgcgcac tggccctgga gcagaagctg gacagcaagg atctggtgcg cgtcatcctg
     1621 cacattgcca agcacagagg ctacggtaac aagcatgcca aaagagacct ggaagccgaa
     1681 gaaaaaatca aggccaagat cgagaaggag gaccaggaga tcacccagaa agccacccag
     1741 gagaagcaaa gcaccaccac aaagggcgac agcaagaaag agcaggagca ggtgctgaaa
     1801 gctctgtaca gaaatgaaac agccctgaaa cactaccaga ccgtgggcca gtacttctac
     1861 aaggagttca aagagaaagg ccaaagaatc cggaatacca ccaacaacta ccagcacacc
     1921 atgcggcagg actggctgaa gaaggaactg gagctgatct tcgagaagca gaaagagttt
     1981 ggcgctaaat tctctgaagg attcgagaac aagatcatcg agatcgcctt cttccagagg
     2041 gacctgaagt cttttgagga caaggtgggc caatgtacct tctacccaaa cgaacctcgg
     2101 gcccctaagg acagcctgag cgccatcgag ttcgtgacac tgacaagaat catcaacacc
     2161 ctgaagaatc tggaggaaaa gagcaagaac ctgggcatag gcgaagtgta cggaaaggat
     2221 aagatcaagg agattctgaa catcgtgctg gacaaaggcg agatcagcta caagaagctg
     2281 agggaaatcc tgggcctgaa cgagcagatc acattcaagg acaacaagct cgattacgcc
     2341 aaagacatga aggaggaaca aaaagtgaaa ctgatcgagt tcaagaatct gaaagacttc
     2401 aaaaaggcca tcggcggaag cttccaggct tttggctcct ttgagacatt cgacagaaag
     2461 gaactggatc tgatcgcaac cgacatcgcc gtgatcaaat ccagagaaaa cctggctaag
     2521 aagctggaaa attatcccag cctgacacag gagcaaagag aatctctctc tgagctcaat
     2581 ttcagcaagc acatcaacct gtctctgcgg gcccttgaga agatcctgcc gttcatgaga
     2641 gagggcctga gatacaacga ggcagtcgag aaggccggac tgcaggagca cagaaagcac
     2701 aagcaaaagt ccgacttcct gcctcctctg caggactacg agccatacct cgccaacccc
     2761 gtggtggaaa gagccctgag cgaataccgg aaagttctga acgccctgct gagaaagtac
     2821 ggccaggtgc acaagatcca cctggagttc accagagagg ctaagctgag cagcaaggaa
     2881 cggcagaagt acgagaagga gcaacgggaa aacttcgcca gaaaccagga ggccgagaag
     2941 cagtgcgagg ccatcggcct gcctctgaat gatgccaaaa acatcctgaa gatgaagctg
     3001 tggattgagc agagacagct gtgcgcctac agcggcgaaa agatcaccat tgagcacctg
     3061 aaggacagca caatgctgga aatcgaccac atctaccctt acagcagatc ctacgatgac
     3121 tcttacaaca acaaggtgct gtgtctggcc agagaaaatc agaataaagg caaccacaca
     3181 ccttttgagg ccttcggcaa ggacacggaa aagtgggacc ggatcctgag cctcgctagc
     3241 aagctcccta aacctaagta cagaagaatc agcaacaagc agttcaagga caaggaggcc
     3301 ggcttcctgg ctagaaacat cgtggacacc agctatatcg cgagactggc cagtgaatac
     3361 acaaagcatt ctctgaactt cctgccactg tctgaggacg aggtgatgat caccggcgaa
     3421 aaggattcta aaaagcacgt cgaggtggtg agcggcagcc tcaccgccac cctgcggcac
     3481 tactggggcc tgggcggaaa gaacggggac aaggacagag ataaccacct gcaccacgtg
     3541 gtggacgcta tcatcatcgc ttacaccaac gccaaggtta tcaaggcctt ctctgatttc
     3601 aggaagctgc aggagcagaa cagagccaag ttctacgcta aggagatcgc ccagtctaag
     3661 gacaagacca aaaaaggctt tgaacctcca tgtgaaaatt tccggaacaa gatccaggag
     3721 aaggtcgaca gtatcttcgt gagcaagccc ccccggaaga gagccagagg cgccctgcac
     3781 gaggaaacct tttatagcct gaatgatgcc agccttctta agtcttacgg cggccaggag
     3841 ggcgtgcaga gggcactgga gctgggcaag atcagacgga tcggaacaaa agtcgtgagc
     3901 aacggcgaga tgaagagagt ggatatcttc aaggacaaga agggcaagtt ctacggagtg
     3961 cccatctaca ccatggactt cgcgctggga atcctgccta acaaggccgt gatcagcggc
     4021 aagaaagacg gcgtgatcaa ggactggctg gaaatggaca gcaactacga attctgcttc
     4081 tctctgttta aggatgacct gatcctggtg cagaagtccg agatggaaga gcccgagctg
     4141 tgctactacg tgggctttga tagctgcagc cctcggatca tcgtggataa acacaataag
     4201 tgctacaaga acatgagcga taacgagaag aaactgttca ccctgaagga aagagagcgg
     4261 aacaacaaga aggaggaaca gaaacaggat gatgagaata aggaacagaa gatcatcggc
     4321 ggcagactcg gaattcagaa cctgaaaaca tttaagaagt accaagtgtc ccctctgggc
     4381 gaggtgaagg aggtgggaaa agagaccaga caggccatcg ccctgaagag caccaagcat
     4441 atcaagcagg actccggacc tccaaagaaa aagagaaaag tataccccta cgacgtgccc
     4501 gactacgcct aataactctc gagctcgatg agtttggaca aaccacaact agaatgcagt
     4561 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
     4621 gctgcaataa acaagttaac gcacgtgcgg accgagcggc cgcaggaacc cctagtgatg
     4681 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     4741 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     4801 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     4861 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4921 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     4981 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     5041 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     5101 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5161 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     5221 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     5281 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     5341 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     5401 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5461 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5521 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     5581 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5641 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5701 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5761 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5821 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5881 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5941 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     6001 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     6061 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     6121 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6181 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6241 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6301 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6361 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6421 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6481 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6541 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6601 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6661 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6721 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6781 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6841 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6901 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6961 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     7021 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     7081 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     7141 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     7201 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     7261 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     7321 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     7381 gctggccttt tgctcacatg t
//
