LOCUS       sequence-254646-        6833 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector expressing Nme2Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6833)
  AUTHORS   Lee J, Mou H, Ibraheim R, Liang SQ, Liu P, Xue W, Sontheimer EJ
  TITLE     Tissue-restricted Genome Editing in vivo Specified by 
            MicroRNA-repressible Anti-CRISPR Proteins.
  JOURNAL   RNA. 2019 Aug 22. pii: rna.071704.119. doi: 10.1261/rna.071704.119.
  PUBMED    31439808
REFERENCE   2  (bases 1 to 6833)
  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"; journalName: "RNA. 2019 
            Aug 22. pii: rna.071704.119. doi: 10.1261/rna.071704.119."
COMMENT     Sequence Label: p1195-pssAAV.U1a.hNme2Cas9
FEATURES             Location/Qualifiers
     source          1..6833
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      405..414
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             417..437
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             444..491
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3744..3791
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3792..3881
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             3888..3914
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     primer_bind     complement(3920..3939)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3985..4040
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     repeat_region   4053..4193
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4053..4182
                     /label=AAV2 ITR
     rep_origin      4268..4723
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4355..4374)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4565..4586
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4740..4759)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4859..4881
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4919..4937)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5005..5109
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5110..5178,5179..5970)
                     /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(5328..5347)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6141..6729
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6630..6649
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6791..6833,1..87)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag
       61 tggccaactc catcactagg ggttcctgcg gcctctagaa tggaggcggt actatgtaga
      121 tgagaattca ggagcaaact gggaaaagca actgcttcca aatatttgtg atttttacag
      181 tgtagttttg gaaaaactct tagcctacca attcttctaa gtgttttaaa atgtgggagc
      241 cagtacacat gaagttatag agtgttttaa tgaggcttaa atatttaccg taactatgaa
      301 atgctacgca tatcatgctg ttcaggctcc gtggccacgc aactcatact taagcagaca
      361 gtggttcaaa gtttttttct tccatttcag gtgtcgtgaa caccgccacc atggtgccta
      421 agaagaagag aaaggtggaa gataaacgcc cagcagctac aaagaaggca ggtcaagcca
      481 agaaaaagaa aatggccgcc ttcaagccta acccaatcaa ttacatcctg ggactggaca
      541 tcggaatcgc atccgtggga tgggctatgg tggagatcga cgaggaggag aatcctatcc
      601 ggctgatcga tctgggcgtg agagtgtttg agagggccga ggtgccaaag accggcgatt
      661 ctctggctat ggcccggaga ctggcacgga gcgtgaggcg cctgacacgg agaagggcac
      721 acaggctgct gagggcacgc cggctgctga agagagaggg cgtgctgcag gcagcagact
      781 tcgatgagaa tggcctgatc aagagcctgc caaacacccc ctggcagctg agagcagccg
      841 ccctggacag gaagctgaca ccactggagt ggtctgccgt gctgctgcac ctgatcaagc
      901 accgcggcta cctgagccag cggaagaacg agggagagac agcagacaag gagctgggcg
      961 ccctgctgaa gggagtggcc aacaatgccc acgccctgca gaccggcgat ttcaggacac
     1021 ctgccgagct ggccctgaat aagtttgaga aggagtccgg ccacatcaga aaccagaggg
     1081 gcgactatag ccacaccttc tcccgcaagg atctgcaggc cgagctgatc ctgctgttcg
     1141 agaagcagaa ggagtttggc aatccacacg tgagcggagg cctgaaggag ggaatcgaga
     1201 ccctgctgat gacacagagg cctgccctgt ccggcgacgc agtgcagaag atgctgggac
     1261 actgcacctt cgagcctgca gagccaaagg ccgccaagaa cacctacaca gccgagcggt
     1321 ttatctggct gacaaagctg aacaatctga gaatcctgga gcagggatcc gagaggccac
     1381 tgaccgacac agagagggcc accctgatgg atgagcctta ccggaagtct aagctgacat
     1441 atgcccaggc cagaaagctg ctgggcctgg aggacaccgc cttctttaag ggcctgagat
     1501 acggcaagga taatgccgag gcctccacac tgatggagat gaaggcctat cacgccatct
     1561 ctcgcgccct ggagaaggag ggcctgaagg acaagaagtc ccccctgaac ctgagctccg
     1621 agctgcagga tgagatcggc accgccttct ctctgtttaa gaccgacgag gatatcacag
     1681 gccgcctgaa ggacagggtg cagcctgaga tcctggaggc cctgctgaag cacatctctt
     1741 tcgataagtt tgtgcagatc agcctgaagg ccctgagaag gatcgtgcca ctgatggagc
     1801 agggcaagcg gtacgacgag gcctgcgccg agatctacgg cgatcactat ggcaagaaga
     1861 acacagagga gaagatctat ctgcccccta tccctgccga cgagatcaga aatcctgtgg
     1921 tgctgagggc cctgtcccag gcaagaaaag tgatcaacgg agtggtgcgc cggtacggat
     1981 ctccagcccg gatccacatc gagaccgcca gagaagtggg caagagcttc aaggaccgga
     2041 aggagatcga gaagagacag gaggagaatc gcaaggatcg ggagaaggcc gccgccaagt
     2101 ttagggagta cttccctaac tttgtgggcg agccaaagtc taaggacatc ctgaagctgc
     2161 gcctgtacga gcagcagcac ggcaagtgtc tgtatagcgg caaggagatc aatctggtgc
     2221 ggctgaacga gaagggctat gtggagatcg atcacgccct gcctttctcc agaacctggg
     2281 acgattcttt taacaataag gtgctggtgc tgggcagcga gaaccagaat aagggcaatc
     2341 agacaccata cgagtatttc aatggcaagg acaactccag ggagtggcag gagttcaagg
     2401 cccgcgtgga gacctctaga tttcccagga gcaagaagca gcggatcctg ctgcagaagt
     2461 tcgacgagga tggctttaag gagtgcaacc tgaatgacac cagatacgtg aaccggttcc
     2521 tgtgccagtt tgtggccgat cacatcctgc tgaccggcaa gggcaagaga agggtgttcg
     2581 cctctaatgg ccagatcaca aacctgctga ggggattttg gggactgagg aaggtgcggg
     2641 cagagaatga cagacaccac gcactggatg cagtggtggt ggcatgcagc accgtggcaa
     2701 tgcagcagaa gatcacaaga ttcgtgaggt ataaggagat gaacgccttt gacggcaaga
     2761 ccatcgataa ggagacaggc aaggtgctgc accagaagac ccacttcccc cagccttggg
     2821 agttctttgc ccaggaagtg atgatccggg tgttcggcaa gccagacggc aagcctgagt
     2881 ttgaggaggc cgatacccca gagaagctga ggacactgct ggcagagaag ctgtctagca
     2941 ggccagaggc agtgcacgag tacgtgaccc cactgttcgt gtccagggca cccaatcgga
     3001 agatgtctgg cgcccacaag gacacactga gaagcgccaa gaggtttgtg aagcacaacg
     3061 agaagatctc cgtgaagaga gtgtggctga ccgagatcaa gctggccgat ctggagaaca
     3121 tggtgaatta caagaacggc agggagatcg agctgtatga ggccctgaag gcaaggctgg
     3181 aggcctacgg aggaaatgcc aagcaggcct tcgacccaaa ggataacccc ttttataaga
     3241 agggaggaca gctggtgaag gccgtgcggg tggagaagac ccaggagagc ggcgtgctgc
     3301 tgaataagaa gaacgcctac acaatcgccg acaatggcga tatggtgaga gtggacgtgt
     3361 tctgtaaggt ggataagaag ggcaagaatc agtactttat cgtgcctatc tatgcctggc
     3421 aggtggccga gaacatcctg ccagacatcg attgcaaggg ctacagaatc gacgatagct
     3481 atacattctg tttttccctg cacaagtatg acctgatcgc cttccagaag gatgagaagt
     3541 ccaaggtgga gtttgcctac tatatcaatt gcgactcctc taacggcagg ttctacctgg
     3601 cctggcacga taagggcagc aaggagcagc agtttcgcat ctccacccag aatctggtgc
     3661 tgatccagaa gtatcaggtg aacgagctgg gcaaggagat caggccatgt cggctgaaga
     3721 agcgcccacc cgtgcgggag gataagcggc ccgcagcaac caagaaggca ggacaggcca
     3781 agaagaagaa gtacccctat gacgtgcctg actacgccgg gtatccctac gacgtgcctg
     3841 attacgccgg gtcctatccc tacgacgtgc cagattacgc tgcagctcca gcagcgaaga
     3901 aaaagaagct ggattaagat ctttttccct ctgccaaaaa ttatggggac atcatgaagc
     3961 cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt
     4021 ggaatttttt gtgtctctca ctcggcggcc gcaggaaccc ctagtgatgg agttggccac
     4081 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     4141 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     4201 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     4261 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4321 gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt
     4381 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4441 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     4501 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     4561 aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt
     4621 gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa
     4681 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     4741 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     4801 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4861 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     4921 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     4981 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5041 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5101 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5161 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5221 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5281 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5341 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     5401 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5461 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     5521 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     5581 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     5641 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     5701 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     5761 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     5821 cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     5881 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     5941 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6001 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6061 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6121 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6181 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6241 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     6301 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6361 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6421 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6481 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6541 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6601 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6661 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6721 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     6781 gctcacatgt cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccg
//
