LOCUS       sequence-261485-        5029 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Self-complementary AAV vector expressing Nme2Cas9 sgRNA targeting 
            Rosa26 and AcrIIC3Nme.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5029)
  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 5029)
  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: 
            p1192-scAAV-U6-sgRNA_Nme2Cas9_Rosa26-CB-PI-AcrIIC3Nme_FLAG_NLS
FEATURES             Location/Qualifiers
     source          1..5029
                     /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             1733..1756
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1763..1783
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1838..1855)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1844..1955
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2079..2208
                     /label=AAV2 ITR
     primer_bind     complement(2227..2244)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2227..2243)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2236..2258)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2385..2840
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2472..2491)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2682..2703
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2866..2970
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2971..3039,3040..3831)
                     /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(3189..3208)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4002..4590
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4491..4510
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4878..4899
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4914..4919,4920..4937,4938..4944)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4952..4968
                     /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     4957..4979
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4976..4992
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4976..4992
                     /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 crggsggggc ggggcggggc gaggggcggg
     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 gttcaaacgg gccattattt tcaccagctt caacggcttc gagaaggtca gtcgcacgga
     1441 aaaacggcgc cttgccaaga taattaacgc cagagttagt ataatcgacg agtatcttcg
     1501 cgccaaagac accaacgcct ccttggacgg acagtataga gcattccttt tcaatgatga
     1561 gtcaccagcg atgaccgaat tcctcgctaa gctcaaggcg ttcgcagaaa gctgtactgg
     1621 tataagcatt gatgcgtggg agattgaaga gagcgagtat gttcgattgc ccgtcgaacg
     1681 gagagatttc cttgcggcgg ccaatggcaa agaaattttc aaaattggcg gggattataa
     1741 ggatgatgat gataagggct cccctaagaa gaagagaaag gtgtgataaa gcggccatca
     1801 agcttatcga taccgtcgac tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
     1861 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     1921 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     1981 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagccta
     2041 ggtagataag tagcatggcg ggttaatcat taactacaag gaacccctag tgatggagtt
     2101 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     2161 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagcc ttaattaacc
     2221 taattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     2281 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     2341 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg
     2401 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     2461 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     2521 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     2581 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     2641 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     2701 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     2761 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     2821 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     2881 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     2941 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3001 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     3061 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     3121 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     3181 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     3241 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     3301 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     3361 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     3421 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     3481 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     3541 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     3601 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     3661 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     3721 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     3781 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     3841 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     3901 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     3961 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4021 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4081 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4141 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4201 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4261 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4321 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4381 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4441 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4501 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4561 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4621 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     4681 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     4741 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     4801 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     4861 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     4921 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     4981 aacagctatg accatgatta cgccagattt aattaaggcc ttaattagg
//
