LOCUS       sequence-408513-        7727 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutagenesis of Ntsr1, second guide.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7727)
  TITLE     Zweifel CRISPR AAV Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7727)
  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            "Unpublished"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-FLEXfrt-SaCas9-U6-sgNtsr1(guide_2)
FEATURES             Location/Qualifiers
     source          1..7727
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        24..403
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        404..607
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     558..578
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    650..683
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(755..835)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(842..889)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATRKAGQAKKKK"
     CDS             complement(890..4045)
                     /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(4070..4090)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4280..4297)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4286..4493
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4500..4748
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4500..4520
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4671..4690
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4771..4846
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4860..5000
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4860..4989
                     /label=AAV2 ITR
     rep_origin      5075..5530
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5162..5181)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5372..5393
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5547..5566)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5666..5688
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5726..5744)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5812..5916
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5917..5985,5986..6777)
                     /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(6135..6154)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6948..7536
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7437..7456
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7598..7727
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gcggcctcta gactcgaggc gttgacattg attattgact agttattaat agtaatcaat
       61 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
      121 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
      181 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
      241 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      301 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
      361 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
      421 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
      481 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
      541 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
      601 cagagctctc tggctaacta ccggtgctag cgtcgacaga attggatccg aagttcctat
      661 tctctagaaa gtataggaac ttccaaggcc tgaagttcct attcttcaaa aggtatagga
      721 acttcgcggc cgcttgatat cttggcgcgc cttaagcgta atctggaaca tcgtatgggt
      781 aagcgtaatc tggaacatcg tatgggtaag cgtaatctgg aacatcgtat gggtaggatc
      841 cctttttctt ttttgcctgg ccggcctttc tcgtggccgc cggccttttg ccctttttga
      901 tgatctgagg gtgcttctta gatttcactt catacaggtt gcccagaatg tctgtgctgt
      961 acttcttaat gctctgggtc ttggaggcga ttgtcttaat gatcctgggg ggcctcttgt
     1021 cgttcatgtt ttccaggtac tcgcggtagg tgatgtcgat catgttcact tcgatccggt
     1081 tcagcaggtc gttgttcacg ccgatcactc tatacagctc gccgttgatc ttgatcagat
     1141 cgttgttgta gaaggaggcg ataaactcgg cctggttgct gatcttcttc agcttcttag
     1201 cttcctcata gcacttgcta ttcacttcgt agtagttttc ttttttgatc acatccagat
     1261 tcttcacggt cacgaacttg tacacgccat tgtccaggta cacgtcgaat ctgtagggct
     1321 tcagggacag cttcacgacc ttgtttctgc tgttggggta gtcgtcggtg atgtccagat
     1381 gggcgttcag tttgttgccg taatacttaa tcttcttgat cacggggccg ttgtcctttt
     1441 tggagtactt ggtcaggtag ttcccggttt cctcgtagta cttgtacagg ggattcttct
     1501 cgtcgccgta ctgttccata atcagcttca gtttctggta ggtctggggg tcgtggtggt
     1561 acatcagcag cttttcgggg ctcttgttga tcagcttttt cagcttgtca ttgtccttgt
     1621 cgtacaggcc gttcagattg ttcacgatca gggtgttgcc cttgtcgtcc ttccgggtgg
     1681 agtacagggt gtcgttaatc agctctctat taggcttctt gtccacccgg tggctgtact
     1741 tgtagtcctt gaagtcctta atgtgcttga tctggtgggg ggtgatgaag atctctttgt
     1801 actcctgctc ggtttcgatc tcgggcatgc tctcggcctg cttttcctcg aacatctggt
     1861 tttccatcac ttttttggcc ttgtccagtt tcttccactc tttgaagatg aaatcggcgt
     1921 tggcaatgat cagggcgtcc tcggcgtggt gcttgtaccc cttgttccgc tctttcttaa
     1981 acttccactt ccgccgcaga aagctggtga agccgccatt gatggacttc actttcacgt
     2041 ccaggttgtt cactctgaag tagctccgca gcaggttcat caggcctctg gtggcgtatc
     2101 tggtatccac caggttccgg ttgatgaagt ctttctgcac ggagaacctg ttgatgtccc
     2161 gttcttccag cagatactct ttcttggtct tgctgattct gcccttgccc ttggccagat
     2221 tcaggatgtg cttcttgaag gtttcgtagc tgatcttgct gtcgctgctg ctcaggtact
     2281 ggaatggggt ccggttgccc ttcttgctgt tttcttcctg cttcacgagc accttgttgt
     2341 tgaagctgtt gtcgaaggac acgcttctgg ggatgatgtg gtccacctca tagttgaagg
     2401 ggttgttcag cagatcttcc agagggatgg cttccaggct gtacaggcac ttgccttcct
     2461 gcatgtcgtg cagcttgatc ttctcgatca ggtacttggc gttctctttg ccggtggtcc
     2521 ggatgatttc ctcgatccgc tcgttggtct gccggttccg cttctgcatc tcgttgatca
     2581 ttttctgggc gtccttggag ttcttctcgc gggccagctc gataatgatg tcgttgggca
     2641 ggccgtactt cttgatgatg gcgttgatca ctttgatgct ctggatgaag cttctcttca
     2701 cgacggggct caggatgaag tcgtccacca gggtggtggg gatctctttc tgctgggaca
     2761 ggtccacctt cttgggcacc agcttcagcc ggttgaagat agcgatctgg ttgtcgttgg
     2821 tgtgccacag ctcgtccagg atcaggttga tggccttcag gctcaggttg tgggtgccgg
     2881 tatagccctt cagattagag atctgctcga tctcttcctg ggtcagctcg gagttcagat
     2941 tggtcagttc ttcctggatg tcctcgctgc tctggtagat ggtcaggatc ttggcaatct
     3001 gatccagcag ctcggcgttc tcaataatct ctttccgggc ggtaatgtcc ttgatgtcgt
     3061 ggtacacctt caggttggtg aactcgggct tgccggtgct ggtcactctg tagcccttaa
     3121 tatcctcttc gttcacgagg atttctttgg cgatctgctt cagggtgggc ttcttcttct
     3181 gcttgaacac gttctcgatg atctggaact tctcgtaata ttccagcttc tcgttctcgt
     3241 ccctggtgat cacgagattg ttcaggtcgt tcagggcgtt gtacaggtcg gcgttgtagg
     3301 cgtacttcac gctccgcagt tcctcgggga agtaggtgca gtggcccatc agcatctcgt
     3361 accattcttt gatgtccttc cagccgaagg ggctgccctc gccaggtccc tcatagtagg
     3421 tccgccgggt ttccagcagg tcgatgtagg tgtcgatgaa gctctggtcc agctggtggt
     3481 aggccttctg caccttcagc agctgtttgg cttctttcac gtagtcgctg gtcttgaatc
     3541 tgttgatgct gccccgcact tcgccgtctt tcttcagccg ttccagctgc agttcggcca
     3601 cgtatttctc ttccagggcc ttgctgttcc ggctgatctg ctctttggtg gacagctcgt
     3661 tgccggtgtc ctcttccacc tcgttcacgt tgtgcacgcc tcttctcttg gccaggtgca
     3721 gcagggcggc agagaactct tcctcgctca gcttctggct caggcccttc actctggcct
     3781 cgtaggggtt gatgccgctc agctcgctgt ggtcggtcag caggttgtag tcgaacagca
     3841 gcttcttcac tctctggatt ctatgccgcc tccgccgctt cagccttctg gcgcctctct
     3901 tgctccgcct gccctcgttg ttttccacgt tggcctcttt gaacagccgc acgccggcat
     3961 cgatcacgtc ccgtgtctcg tagtcgatga tgccgtagcc cacgctggtg atgccgatgt
     4021 ccaggcccag gatgtagttc cgcttggctg ctgggactcc gtggataccg accttccgct
     4081 tcttctttgg ggccatgcgg ccgctgcaga ccggtttaga tctttactag tttaagcttc
     4141 aattggaagt tcctattctt tctagagtat aggaacttcc tgcagtaggc tagcgaagtt
     4201 cctattcctt ttgaagtata ggaacttcgt cgaggggggg cccgagctta tcgatgaatt
     4261 cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     4321 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     4381 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     4441 gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggaggtaccg
     4501 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
     4561 taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
     4621 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
     4681 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
     4741 gaaacaccgc ttggtgcggc tgcgggacat gttttagtac tctggaaaca gaatctacta
     4801 aaacaaggca aaatgccgtg tttatctcgt caacttgttg gcgagatttt tgcggccgca
     4861 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     4921 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     4981 agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     5041 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     5101 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc
     5161 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     5221 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     5281 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     5341 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     5401 ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat
     5461 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     5521 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     5581 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     5641 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     5701 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     5761 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     5821 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5881 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5941 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     6001 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     6061 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     6121 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     6181 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     6241 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     6301 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     6361 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     6421 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     6481 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     6541 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     6601 attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg
     6661 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     6721 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     6781 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6841 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6901 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6961 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     7021 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     7081 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     7141 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     7201 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     7261 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     7321 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     7381 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     7441 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     7501 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     7561 cggttcctgg ccttttgctg gccttttgct cacatgtcct gcaggcagct gcgcgctcgc
     7621 tcgctcactg aggccgcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga
     7681 gcgagcgcgc agagagggag tggccaactc catcactagg ggttcct
//
