LOCUS       sequence-317315-        7728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutagenesis of Nts.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7728)
  TITLE     Zweifel CRISPR AAV Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7728)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-CMV-FLEX-SaCas9-U6-sgNts
FEATURES             Location/Qualifiers
     source          1..7728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    complement(621..654)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(705..738)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(750..830)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(837..884)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(885..4040)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             complement(4065..4085)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    4117..4150
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    4201..4234
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4259..4276)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4265..4472
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4479..4727
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4479..4499
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4650..4669
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4749..4824
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4838..4978
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4838..4967
                     /label=AAV2 ITR
     rep_origin      5053..5508
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5140..5159)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5350..5371
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5525..5544)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5644..5666
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5704..5722)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5790..5894
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5895..5963,5964..6755)
                     /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(6113..6132)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6926..7514
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7415..7434
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7576..7705
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactaccg
      601 gtgctagcgt cgactccgga ataacttcgt ataggatact ttatacgaag ttatgcagaa
      661 tggtagctgg attgtagctg ctattagcaa tatgaaacct cttaataact tcgtatagca
      721 tacattatac gaagttatgg cgcgccttaa gcgtaatctg gaacatcgta tgggtaagcg
      781 taatctggaa catcgtatgg gtaagcgtaa tctggaacat cgtatgggta ggatcccttt
      841 ttcttttttg cctggccggc ctttttcgtg gccgccggcc ttttgccctt tttgatgatc
      901 tgagggtgct tcttagattt cacttcatac aggttgccca gaatgtctgt gctgtacttc
      961 ttaatgctct gggtcttgga ggcgattgtc ttaatgatcc tggggggcct cttgtcgttc
     1021 atgttttcca ggtactcgcg gtaggtgatg tcgatcatgt tcacttcgat ccggttcagc
     1081 aggtcgttgt tcacgccgat cactctatac agctcgccgt tgatcttgat cagatcgttg
     1141 ttgtagaagg aggcgataaa ctcggcctgg ttgctgatct tcttcagctt cttagcttcc
     1201 tcatagcact tgctattcac ttcgtagtag ttttcttttt tgatcacatc cagattcttc
     1261 acggtcacga acttgtacac gccattgtcc aggtacacgt cgaatctgta gggcttcagg
     1321 gacagcttca cgaccttgtt tctgctgttg gggtagtcgt cggtgatgtc cagatgggcg
     1381 ttcagtttgt tgccgtaata cttaatcttc ttgatcacgg ggccgttgtc ctttttggag
     1441 tacttggtca ggtagttccc ggtttcctcg tagtacttgt acaggggatt cttctcgtcg
     1501 ccgtactgtt ccataatcag cttcagtttc tggtaggtct gggggtcgtg gtggtacatc
     1561 agcagctttt cggggctctt gttgatcagc tttttcagct tgtcattgtc cttgtcgtac
     1621 aggccgttca gattgttcac gatcagggtg ttgcccttgt cgtccttccg ggtggagtac
     1681 agggtgtcgt taatcagctc tctattaggc ttcttgtcca cccggtggct gtacttgtag
     1741 tccttgaagt ccttaatgtg cttgatctgg tggggggtga tgaagatctc tttgtactcc
     1801 tgctcggttt cgatctcggg catgctctcg gcctgctttt cctcgaacat ctggttttcc
     1861 atcacttttt tggccttgtc cagtttcttc cactctttga agatgaaatc ggcgttggca
     1921 atgatcaggg cgtcctcggc gtggtgcttg taccccttgt tccgctcttt cttaaacttc
     1981 cacttccgcc gcagaaagct ggtgaagccg ccattgatgg acttcacttt cacgtccagg
     2041 ttgttcactc tgaagtagct ccgcagcagg ttcatcaggc ctctggtggc gtatctggta
     2101 tccaccaggt tccggttgat gaagtctttc tgcacggaga acctgttgat gtcccgttct
     2161 tccagcagat actctttctt ggtcttgctg attctgccct tgcccttggc cagattcagg
     2221 atgtgcttct tgaaggtttc gtagctgatc ttgctgtcgc tgctgctcag gtactggaat
     2281 ggggtccggt tgcccttctt gctgttttct tcctgcttca cgagcacctt gttgttgaag
     2341 ctgttgtcga aggacacgct tctggggatg atgtggtcca cctcatagtt gaaggggttg
     2401 ttcagcagat cttccagagg gatggcttcc aggctgtaca ggcacttgcc ttcctgcatg
     2461 tcgtgcagct tgatcttctc gatcaggtac ttggcgttct ctttgccggt ggtccggatg
     2521 atttcctcga tccgctcgtt ggtctgccgg ttccgcttct gcatctcgtt gatcattttc
     2581 tgggcgtcct tggagttctt ctcgcgggcc agctcgataa tgatgtcgtt gggcaggccg
     2641 tacttcttga tgatggcgtt gatcactttg atgctctgga tgaagcttct cttcacgacg
     2701 gggctcagga tgaagtcgtc caccagggtg gtggggatct ctttctgctg ggacaggtcc
     2761 accttcttgg gcaccagctt cagccggttg aagatagcga tctggttgtc gttggtgtgc
     2821 cacagctcgt ccaggatcag gttgatggcc ttcaggctca ggttgtgggt gccggtatag
     2881 cccttcagat tagagatctg ctcgatctct tcctgggtca gctcggagtt cagattggtc
     2941 agttcttcct ggatgtcctc gctgctctgg tagatggtca ggatcttggc aatctgatcc
     3001 agcagctcgg cgttctcaat aatctctttc cgggcggtaa tgtccttgat gtcgtggtac
     3061 accttcaggt tggtgaactc gggcttgccg gtgctggtca ctctgtagcc cttaatatcc
     3121 tcttcgttca cgaggatttc tttggcgatc tgcttcaggg tgggcttctt cttctgcttg
     3181 aacacgttct cgatgatctg gaacttctcg taatattcca gcttctcgtt ctcgtccctg
     3241 gtgatcacga gattgttcag gtcgttcagg gcgttgtaca ggtcggcgtt gtaggcgtac
     3301 ttcacgctcc gcagttcctc ggggaagtag gtgcagtggc ccatcagcat ctcgtaccat
     3361 tctttgatgt ccttccagcc gaaggggctg ccctcgccag gtccctcata gtaggtccgc
     3421 cgggtttcca gcaggtcgat gtaggtgtcg atgaagctct ggtccagctg gtggtaggcc
     3481 ttctgcacct tcagcagctg tttggcttct ttcacgtagt cgctggtctt gaatctgttg
     3541 atgctgcccc gcacttcgcc gtctttcttc agccgttcca gctgcagttc ggccacgtat
     3601 ttctcttcca gggccttgct gttccggctg atctgctctt tggtggacag ctcgttgccg
     3661 gtgtcctctt ccacctcgtt cacgttgtgc acgcctcttc tcttggccag gtgcagcagg
     3721 gcggcagaga actcttcctc gctcagcttc tggctcaggc ccttcactct ggcctcgtag
     3781 gggttgatgc cgctcagctc gctgtggtcg gtcagcaggt tgtagtcgaa cagcagcttc
     3841 ttcactctct ggattctatg ccgcctccgc cgcttcagcc ttctggcgcc tctcttgctc
     3901 cgcctgccct cgttgttttc cacgttggcc tctttgaaca gccgcacgcc ggcatcgatc
     3961 acgtcccgtg tctcgtagtc gatgatgccg tagcccacgc tggtgatgcc gatgtccagg
     4021 cccaggatgt agttccgctt ggctgctggg actccgtgga taccgacctt ccgcttcttc
     4081 tttggggcca tgcggccgct gcagaccggt gctagcataa cttcgtataa agtatcctat
     4141 acgaagttat ttgccttaac ccagaaatta tcactgttat tctttagaat ggtgcaaaga
     4201 ataacttcgt ataatgtatg ctatacgaag ttatgaattc ctagagctcg ctgatcagcc
     4261 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     4321 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     4381 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     4441 gattgggaag agaatagcag gcatgctggg gaggtaccga gggcctattt cccatgattc
     4501 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
     4561 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     4621 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     4681 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgac gttatcaagg
     4741 atatcttcgt tttagtactc tggaaacaga atctactaaa acaaggcaaa atgccgtgtt
     4801 tatctcgtca acttgttggc gagatttttg cggccgcagg aacccctagt gatggagttg
     4861 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     4921 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     4981 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     5041 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     5101 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
     5161 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     5221 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     5281 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     5341 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
     5401 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
     5461 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     5521 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     5581 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     5641 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     5701 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     5761 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5821 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5881 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5941 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     6001 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     6061 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     6121 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     6181 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     6241 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     6301 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     6361 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     6421 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     6481 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6541 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6601 gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6661 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6721 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6781 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6841 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6901 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6961 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     7021 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     7081 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     7141 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     7201 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     7261 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     7321 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     7381 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     7441 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     7501 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     7561 cttttgctca catgtcctgc aggcagctgc gcgctcgctc gctcactgag gccgcccggg
     7621 cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg
     7681 gccaactcca tcactagggg ttcctgcggc ctctagactc gaggcgtt
//
