LOCUS       sequence-248743-        4934 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 4934)
  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 4934)
  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..4934
                     /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..548)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(358..377)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(528..548)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     intron          1205..1301
                     /label=SV40 intron
                     /note="modified SV40 intron with splice donor and acceptor 
                     sites"
     CDS             1642..1665
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1672..1692
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1747..1764)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1753..1864
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   1984..2113
                     /label=AAV2 ITR
     primer_bind     complement(2132..2149)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2132..2148)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2141..2163)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2290..2745
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2377..2396)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2587..2608
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2771..2875
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2876..2944,2945..3736)
                     /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(3094..3113)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3907..4495
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4396..4415
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4783..4804
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4819..4824,4825..4842,4843..4849)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4857..4873
                     /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     4862..4884
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4881..4897
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4881..4897
                     /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 ccttccacaa gatatataaa gccaagaaat cgaaatactt tcaagttacg
      361 gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta cccaagaaat
      421 tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt caaattaatt
      481 ctaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat catgggaaat
      541 aggccctcca cgcgtcgaca ttgattattg actctggtcg ttacataact tacggtaaat
      601 ggcccgcctg gctgaccgcc caacgacccc gcccattgac gtcaataatg acgtatgttc
      661 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      721 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      781 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      841 cttggcagta catctactcg aggccacgtt ctgcttcact ctccccatct cccccccctc
      901 cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg
      961 gggggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg
     1021 cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta
     1081 tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagcgg
     1141 gatcagccac cgcggtggcg gccctagagt cgatcgagga actgaaaaac cagaaagtta
     1201 actggtaagt ttagtctttt tgtcttttat ttcaggtccc ggatccggtg gtggtgcaaa
     1261 tcaaagaact gctcctcagt ggatgttgcc tttacttcta ggcctgtacg gaagtgttac
     1321 ttctgctcta aaagctgcgg aattgtaccc gcggccgatc caccggtcgc caccatgaac
     1381 atcaatgatc tgattagaga aataaagaat aaagactata ctgttaaatt gtctggaact
     1441 gacagtaata gcataaccca actcatcatc agggttaata atgatggtaa cgaatatgtt
     1501 ataagtgagt ccgagaacga atctatcgtc gagaagttca tcagtgcctt caaaaacgga
     1561 tggaaccaag agtacgagga tgaggaggaa ttttacaatg atatgcaaac aatcactctg
     1621 aagagcgagc ttaacggcgg ggattataag gatgatgatg ataagggctc ccctaagaag
     1681 aagagaaagg tgtgataaag cggccatcaa gcttatcgat accgtcgact agagctcgct
     1741 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     1801 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     1861 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     1921 agggggagga ttgggaagac aattaggtag ataagtagca tggcgggtta atcattaact
     1981 acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     2041 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     2101 agcgagcgcg cagccttaat taacctaatt cactggccgt cgttttacaa cgtcgtgact
     2161 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     2221 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     2281 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2341 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2401 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2461 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2521 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2581 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     2641 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     2701 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     2761 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2821 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     2881 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     2941 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     3001 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     3061 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     3121 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     3181 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     3241 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     3301 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     3361 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     3421 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     3481 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     3541 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     3601 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     3661 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     3721 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     3781 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3841 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3901 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3961 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4021 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     4081 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4141 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4201 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4261 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4321 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4381 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4441 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4501 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4561 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4621 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4681 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     4741 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     4801 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     4861 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca gatttaatta
     4921 aggccttaat tagg
//
