LOCUS       sequence-385819-        8096 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Hsp1-Hsp2Cas9, cloning backbone for sgRNA and puromycin 
            resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8096)
  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 8096)
  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-Hsp1-Hsp2Cas9-P2A-puro
FEATURES             Location/Qualifiers
     source          1..8096
                     /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        154..533
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        534..737
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     688..708
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      756..765
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             768..788
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3954..4001
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4023..4079
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4080..4676
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4080..4099)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4576..4596
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4677..4757
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4785..4802)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4791..4998
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        5005..5253
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5005..5025
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5176..5195
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        5272..5344
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     repeat_region   5359..5499
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5359..5488
                     /label=AAV2 ITR
     rep_origin      5574..6029
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5661..5680)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5871..5892
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6046..6065)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6165..6187
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6225..6243)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6311..6415
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6416..6484,6485..7276)
                     /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(6634..6653)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7447..8035
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7936..7955
                     /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 gcggcctcta gactcgaggc gttgacattg attattgact agttattaat
      181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      721 tctatataag cagagctctc tggctaacta ccggtgccac catggcccca aagaagaagc
      781 ggaaggtcgg tatccacgga gtcccagcag ccagcgacaa atccaagatc ctgggattcg
      841 acatcggcat caccagcatc ggctgggcct acgtggaagg cgaagagctg atcgactgcg
      901 gcgtccggat ctttaccaag gccgagaatc ccaagaccaa agaatccctg gccctgccta
      961 gacgggaagc tagaggcgtg cggcggagac tggcccgaag aaaaggcaga ctgaatgcca
     1021 tcaagcaact gatctgcaag gaattcggcc tgaactacca agactacatc gctcaggacg
     1081 gcgagctgcc caaagcctac gccgggaagc tgattaaccc ttatgagctg agatctaagg
     1141 cccttagaga gaagctggaa aaggaagagc tggcccgagt gattctgcac atcgccaagc
     1201 accggggcta cggaaacaag catgccaagg aaaccgacga tgccgagagc ggcaagatca
     1261 agaaagcaat tagcaacaat gagaagatcc tgaatgagaa gggatatcag agcgccggag
     1321 agtacctgta ccaggaattt ttccagaaag aaagactgca ggaggaaaag aagggcaaca
     1381 acacgaccaa agaattcaaa aatgtgagga acaaagctgg cgactacgag cactgtatcg
     1441 ctcagaccca gatgcagaac gagttgaggc ttattttcga gaagcagcga gagtttggcc
     1501 tgaacttcag cgaggaattc aagaagaaag tgctggagac ggcctttttt cagagacctc
     1561 tgaaagactt cagcgataag atcggcgatt gcaccttcat cgagggcgcc aagcgggccc
     1621 ctaaggacag cctgagcgcc atcgagttcg tggccctggg aagaatcatc aacaccctga
     1681 aaaatctgca gaacacaagc ggcgaggtgt acagcagaga taagatcaac gagatcctgc
     1741 aaatcgtgct ggataagggc gagatcagct acaagaaact gagagaaatc ctggacatcg
     1801 agagcagcgt gcgtttcaag gacagcaaac tggattacac caaagaactg aaggaagccg
     1861 agaaaaccaa gctgttcgag agcaagaacc tccagaaatt caagaaagct ctgggcagct
     1921 tcgaaggcat ctccagagag atcttggaca gtatcgccaa ggagatcatg atcacaaagg
     1981 ataaagagaa gctggccaag gagctgaaga agttccctct ggatgaaacc cagatccaga
     2041 acctttctaa cttgtccttc agccaaacaa tcaacctgtc actggaagcc ctgtctcaga
     2101 tcctgccctt catgagaggc gagagagagg acaaatgcct gagatacgac gaggctgtgg
     2161 aaaaggccgg cctgaaggag gtggccaaaa acaagtctaa ggatgacttc ctgcctcctc
     2221 tggaaaacta cgagccttac ctggccaatc ctgtggtggc ccgggccctc tctgagtata
     2281 gaaaggtgct gaacgccctg ctgagaaagt acggcaagat gcacaagatt cacatcgagt
     2341 ttaccagaga agtgggcaag agcaaagagg agcggaacaa aatcgaaaaa gaacagaaag
     2401 agaatttcgc caaaaaccag gaggcccaga agcagtgcga gatcctgggc ctgcctatca
     2461 acggcgctaa tatcctgaag atgaagctgt ggcgggagca gcaggagtcc tgcgtgtaca
     2521 gcggcaagga aatcgccatc gacgacctga agaaccccgc catcctgcag gtggatcaca
     2581 tctatcctta ctctagaagc tttgatgatt cttataacaa caaggtgctg gtgttctcca
     2641 aagaaaacca gaacaaactc aacaagacac ctttcgaggc tttcggcgaa aacaaggaca
     2701 aatgggagaa aatcctgtcc ctggcttcta agcttcctaa gcctaagcgg cagaggatct
     2761 acaacatgca gttcaaggac aaagaacaag gctttctgac ccggaatatc aacgacaccg
     2821 gctacatcgc tagactggtc agccagtgga caaagaactg cctgagcttc gagaagctga
     2881 gcgaggatga ggtttccatc gccggcgaga aaggaagcaa agtgcacgtg gaagtgatca
     2941 gcggctccct gacagccatg ctgcggcact actggggcct gggcgacaag aaccgggaca
     3001 accacctgca ccacgcggtg gacgccatca tcatcgctta tagcaacgaa aaggtgatca
     3061 aggccttctc tgattacaaa aaacatagag agcagaacaa ggccaagttc tacgccaagg
     3121 agatcgccga gagcgagtac aagaagcaga aggttttctt cgaacctacc aaccacttca
     3181 gacaaaaagt gctggaaaag attgacagca tcttcgtgag caaacctcca aggaagagag
     3241 cccggggagc cctgcacgag gaaacattct acagcttcga gagactgtgc aacgacaagg
     3301 agtacgccaa gaaagagaag atggactacg gcggaaaggc tggcatccag aaggctatcg
     3361 ccctgggtaa aattagacag gttggaacaa agatcgtgtc taacggcacc atggtgcggg
     3421 tggacatctt caaagataag aagggcaagt tctacggcgt ccccatctac accatggact
     3481 tcgccctggg catcctgcct aataaggccg tgatcagcgg aaagaaagac ggcgtgatca
     3541 aggactggct ggaaatggat agcaactacg agttctgctt cagcctgttt aaggatgatc
     3601 tgatcctcgt gcagaagtct gagatggaag agcctgagct gtgttactac gtgggcttcg
     3661 acagctgcag ccctagaatc atcgtggaca agcacaacaa gtgctataag aacatgagcg
     3721 acaacgagaa gaagctgttt accctgaagg agcgggaacg gaacaacaag aaggaagagc
     3781 agaaacagga cgacgagaac aaggaacaga agatcatcgg cggcagactg ggaattcaga
     3841 atctgaaaac cttcaagaaa taccaggtgt ccccactggg cgaggtgaag gaagtgggca
     3901 aagagacaag acaggctatc gccctgaaat ctacaaagca catcaagcaa gacaaaaggc
     3961 cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gggatccgct agcggcagcg
     4021 gcgccaccaa cttcagcctg ctgaagcagg ccggcgacgt ggaggagaac cccggcccca
     4081 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac
     4141 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc
     4201 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
     4261 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga
     4321 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
     4381 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
     4441 ccgcgtggtt cctggccacc gtcggagtct cgcccgacca ccagggcaag ggtctgggca
     4501 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     4561 aaacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     4621 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctacc
     4681 catacgatgt tccagattac gcttacccat acgatgttcc agattacgct tacccatacg
     4741 atgttccaga ttacgcttaa gaattcctag agctcgctga tcagcctcga ctgtgccttc
     4801 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     4861 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     4921 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa
     4981 tagcaggcat gctggggagg taccgagggc ctatttccca tgattccttc atatttgcat
     5041 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
     5101 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     5161 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     5221 gctttatata tcttgtggaa aggacgaaac accgggtctt cgagaagacc tgttttagtc
     5281 cctgaaaagg gactaaaata aagagtttgc gggactctgc ggggttacaa tcccctaaaa
     5341 ccgctttttt gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     5401 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     5461 cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc
     5521 tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc
     5581 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     5641 tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     5701 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     5761 acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc
     5821 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     5881 gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat
     5941 tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     6001 ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga
     6061 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     6121 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     6181 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     6241 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     6301 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     6361 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     6421 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     6481 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     6541 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     6601 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     6661 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     6721 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     6781 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     6841 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     6901 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6961 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     7021 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     7081 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg
     7141 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     7201 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     7261 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     7321 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     7381 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     7441 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     7501 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     7561 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     7621 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     7681 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     7741 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     7801 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     7861 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     7921 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     7981 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     8041 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
