LOCUS       sequence-254647-        4942 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Self-complementary AAV vector expressing Nme2Cas9 sgRNA targeting 
            Rosa26 and AcrIIA4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4942)
  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 4942)
  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: 
            p1194-scAAV-U6-sgRNA_Nme2Cas9_Rosa26-CB-PI-AcrIIA4_FLAG_NLS
FEATURES             Location/Qualifiers
     source          1..4942
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        complement(156..248)
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     promoter        complement(301..549)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(359..378)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(529..549)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     intron          1209..1305
                     /label=SV40 intron
                     /note="modified SV40 intron with splice donor and acceptor 
                     sites"
     CDS             1646..1669
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1676..1696
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1751..1768)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1757..1868
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   1992..2121
                     /label=AAV2 ITR
     primer_bind     complement(2140..2157)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2140..2156)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2149..2171)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2298..2753
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2385..2404)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2595..2616
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2779..2883
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2884..2952,2953..3744)
                     /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(3102..3121)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3915..4503
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4404..4423
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4791..4812
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4827..4832,4833..4850,4851..4857)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4865..4881
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     4870..4892
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4889..4905
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4889..4905
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtgtagcc atgctctagg
      121 aagatcaatt cggtacaatt cacgcgtcaa aaaaataaac gatgcccctt aaagcagaag
      181 ctttaagggg cagagcgttg cggcacatct tttcagacgg ccttattgta gcaacggttc
      241 tcatttcgtt tccgaaatga gaaagggagc tacaacctcc caggcccagg gcggtcctcc
      301 ggtgtttcgt cctttccaca agatatataa agccaagaaa tcgaaatact ttcaagttac
      361 ggtaagcata tgatagtcca ttttaaaaca taattttaaa actgcaaact acccaagaaa
      421 ttattacttt ctacgtcacg tattttgtac taatatcttt gtgtttacag tcaaattaat
      481 tctaattatc tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa
      541 taggccctcc acgcgtcgac attgattatt gactagctct ggtcgttaca taacttacgg
      601 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      661 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      721 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      781 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
      841 ttcctacttg gcagtacatc tactcgaggc cacgttctgc ttcactctcc ccatctcccc
      901 cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg
      961 ggcggggggg gggggggggg gggcgcgcgc srggsggggs ggggsggggs grggggsggg
     1021 gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc
     1081 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg
     1141 gagcgggatc agccaccgcg gtggcggcct agagtcgacg aggaactgaa aaaccagaaa
     1201 gttaactggt aagtttagtc tttttgtctt ttatttcagg tcccggatcc ggtggtggtg
     1261 caaatcaaag aactgctcct cagtggatgt tgcctttact tctaggcctg tacggaagtg
     1321 ttacttctgc tctaaaagct gcggaattgt acccgcggcc gatccaccgg tcgccaccat
     1381 gaacatcaat gatctgatta gagaaataaa gaataaagac tatactgtta aattgtctgg
     1441 aactgacagt aatagcataa cccaactcat catcagggtt aataatgatg gtaacgaata
     1501 tgttataagt gagtccgaga acgaatctat cgtcgagaag ttcatcagtg ccttcaaaaa
     1561 cggatggaac caagagtacg aggatgagga ggaattttac aatgatatgc aaacaatcac
     1621 tctgaagagc gagcttaacg gcggggatta taaggatgat gatgataagg gctcccctaa
     1681 gaagaagaga aaggtgtgat aaagcggcca tcaagcttat cgataccgtc gactagagct
     1741 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     1801 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     1861 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     1921 agcaaggggg aggattggga agacaatagc ctaggtagat aagtagcatg gcgggttaat
     1981 cattaactac aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     2041 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     2101 agtgagcgag cgagcgcgca gccttaatta acctaattca ctggccgtcg ttttacaacg
     2161 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     2221 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     2281 cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2341 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2401 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2461 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2521 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2581 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2641 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2701 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     2761 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2821 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2881 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     2941 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     3001 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     3061 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     3121 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     3181 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     3241 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     3301 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     3361 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     3421 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     3481 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     3541 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     3601 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     3661 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     3721 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     3781 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     3841 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3901 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3961 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4021 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     4081 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     4141 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     4201 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     4261 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     4321 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     4381 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     4441 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     4501 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     4561 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     4621 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     4681 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     4741 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     4801 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     4861 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaga
     4921 tttaattaag gccttaatta gg
//
