LOCUS       sequence-255632-        5087 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Self-complementary AAV vector expressing Nme2Cas9 sgRNA targeting 
            Rosa26 and AcrIIC3Nme with 3x miR-122 binding sites in 3' UTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5087)
  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 5087)
  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: 
            p1193-scAAV-U6-sgRNA_Nme2Cas9_Rosa26-CB-PI-AcrIIC3Nme_FLAG_NLS-3XmiR
            122BS
FEATURES             Location/Qualifiers
     source          1..5087
                     /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(1896..1913)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1902..2013
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2137..2266
                     /label=AAV2 ITR
     primer_bind     complement(2285..2302)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2285..2301)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2294..2316)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2443..2898
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2530..2549)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2740..2761
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2924..3028
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3029..3097,3098..3889)
                     /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(3247..3266)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4060..4648
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4549..4568
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4936..4957
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4972..4977,4978..4995,4996..5002)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5010..5026
                     /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     5015..5037
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5034..5050
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5034..5050
                     /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 gggcgcgcgs 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 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 agcttgacaa acaccattgt cacactccaa caaacaccat tgtcacactc caacaaacac
     1861 cattgtcaca ctccaccata gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     1921 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     1981 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     2041 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcctagg
     2101 tagataagta gcatggcggg ttaatcatta actacaagga acccctagtg atggagttgg
     2161 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     2221 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagcctt aattaaccta
     2281 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     2341 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     2401 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
     2461 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     2521 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     2581 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     2641 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     2701 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     2761 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     2821 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     2881 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     2941 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3001 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     3061 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     3121 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     3181 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     3241 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     3301 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     3361 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     3421 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     3481 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     3541 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     3601 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     3661 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     3721 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     3781 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     3841 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     3901 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     3961 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     4021 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4081 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4141 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4201 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4261 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4321 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4381 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4441 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4501 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4561 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4621 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4681 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     4741 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4801 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     4861 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     4921 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     4981 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     5041 cagctatgac catgattacg ccagatttaa ttaaggcctt aattagg
//
