LOCUS       sequence-317319-        7728 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutagenesis of Kcnd3.
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  JOURNAL   Unpublished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            "Unpublished"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-sgKcnd3
FEATURES             Location/Qualifiers
     source          1..7728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        154..533
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        534..737
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     688..708
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    complement(774..807)
                     /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(858..891)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(903..983)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(990..1037)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(1038..4193)
                     /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(4218..4238)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    4270..4303
                     /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    4354..4387
                     /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(4412..4429)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4418..4625
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4632..4880
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4632..4652
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4803..4822
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4902..4977
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4991..5131
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4991..5120
                     /label=AAV2 ITR
     rep_origin      5206..5661
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5293..5312)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5503..5524
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5678..5697)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5797..5819
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5857..5875)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5943..6047
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6048..6116,6117..6908)
                     /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(6266..6285)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7079..7667
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7568..7587
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat
      181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      721 tctatataag cagagctctc tggctaacta ccggtgctag cgtcgactcc ggaataactt
      781 cgtataggat actttatacg aagttatgca gaatggtagc tggattgtag ctgctattag
      841 caatatgaaa cctcttaata acttcgtata gcatacatta tacgaagtta tggcgcgcct
      901 taagcgtaat ctggaacatc gtatgggtaa gcgtaatctg gaacatcgta tgggtaagcg
      961 taatctggaa catcgtatgg gtaggatccc tttttctttt ttgcctggcc ggcctttttc
     1021 gtggccgccg gccttttgcc ctttttgatg atctgagggt gcttcttaga tttcacttca
     1081 tacaggttgc ccagaatgtc tgtgctgtac ttcttaatgc tctgggtctt ggaggcgatt
     1141 gtcttaatga tcctgggggg cctcttgtcg ttcatgtttt ccaggtactc gcggtaggtg
     1201 atgtcgatca tgttcacttc gatccggttc agcaggtcgt tgttcacgcc gatcactcta
     1261 tacagctcgc cgttgatctt gatcagatcg ttgttgtaga aggaggcgat aaactcggcc
     1321 tggttgctga tcttcttcag cttcttagct tcctcatagc acttgctatt cacttcgtag
     1381 tagttttctt ttttgatcac atccagattc ttcacggtca cgaacttgta cacgccattg
     1441 tccaggtaca cgtcgaatct gtagggcttc agggacagct tcacgacctt gtttctgctg
     1501 ttggggtagt cgtcggtgat gtccagatgg gcgttcagtt tgttgccgta atacttaatc
     1561 ttcttgatca cggggccgtt gtcctttttg gagtacttgg tcaggtagtt cccggtttcc
     1621 tcgtagtact tgtacagggg attcttctcg tcgccgtact gttccataat cagcttcagt
     1681 ttctggtagg tctgggggtc gtggtggtac atcagcagct tttcggggct cttgttgatc
     1741 agctttttca gcttgtcatt gtccttgtcg tacaggccgt tcagattgtt cacgatcagg
     1801 gtgttgccct tgtcgtcctt ccgggtggag tacagggtgt cgttaatcag ctctctatta
     1861 ggcttcttgt ccacccggtg gctgtacttg tagtccttga agtccttaat gtgcttgatc
     1921 tggtgggggg tgatgaagat ctctttgtac tcctgctcgg tttcgatctc gggcatgctc
     1981 tcggcctgct tttcctcgaa catctggttt tccatcactt ttttggcctt gtccagtttc
     2041 ttccactctt tgaagatgaa atcggcgttg gcaatgatca gggcgtcctc ggcgtggtgc
     2101 ttgtacccct tgttccgctc tttcttaaac ttccacttcc gccgcagaaa gctggtgaag
     2161 ccgccattga tggacttcac tttcacgtcc aggttgttca ctctgaagta gctccgcagc
     2221 aggttcatca ggcctctggt ggcgtatctg gtatccacca ggttccggtt gatgaagtct
     2281 ttctgcacgg agaacctgtt gatgtcccgt tcttccagca gatactcttt cttggtcttg
     2341 ctgattctgc ccttgccctt ggccagattc aggatgtgct tcttgaaggt ttcgtagctg
     2401 atcttgctgt cgctgctgct caggtactgg aatggggtcc ggttgccctt cttgctgttt
     2461 tcttcctgct tcacgagcac cttgttgttg aagctgttgt cgaaggacac gcttctgggg
     2521 atgatgtggt ccacctcata gttgaagggg ttgttcagca gatcttccag agggatggct
     2581 tccaggctgt acaggcactt gccttcctgc atgtcgtgca gcttgatctt ctcgatcagg
     2641 tacttggcgt tctctttgcc ggtggtccgg atgatttcct cgatccgctc gttggtctgc
     2701 cggttccgct tctgcatctc gttgatcatt ttctgggcgt ccttggagtt cttctcgcgg
     2761 gccagctcga taatgatgtc gttgggcagg ccgtacttct tgatgatggc gttgatcact
     2821 ttgatgctct ggatgaagct tctcttcacg acggggctca ggatgaagtc gtccaccagg
     2881 gtggtgggga tctctttctg ctgggacagg tccaccttct tgggcaccag cttcagccgg
     2941 ttgaagatag cgatctggtt gtcgttggtg tgccacagct cgtccaggat caggttgatg
     3001 gccttcaggc tcaggttgtg ggtgccggta tagcccttca gattagagat ctgctcgatc
     3061 tcttcctggg tcagctcgga gttcagattg gtcagttctt cctggatgtc ctcgctgctc
     3121 tggtagatgg tcaggatctt ggcaatctga tccagcagct cggcgttctc aataatctct
     3181 ttccgggcgg taatgtcctt gatgtcgtgg tacaccttca ggttggtgaa ctcgggcttg
     3241 ccggtgctgg tcactctgta gcccttaata tcctcttcgt tcacgaggat ttctttggcg
     3301 atctgcttca gggtgggctt cttcttctgc ttgaacacgt tctcgatgat ctggaacttc
     3361 tcgtaatatt ccagcttctc gttctcgtcc ctggtgatca cgagattgtt caggtcgttc
     3421 agggcgttgt acaggtcggc gttgtaggcg tacttcacgc tccgcagttc ctcggggaag
     3481 taggtgcagt ggcccatcag catctcgtac cattctttga tgtccttcca gccgaagggg
     3541 ctgccctcgc caggtccctc atagtaggtc cgccgggttt ccagcaggtc gatgtaggtg
     3601 tcgatgaagc tctggtccag ctggtggtag gccttctgca ccttcagcag ctgtttggct
     3661 tctttcacgt agtcgctggt cttgaatctg ttgatgctgc cccgcacttc gccgtctttc
     3721 ttcagccgtt ccagctgcag ttcggccacg tatttctctt ccagggcctt gctgttccgg
     3781 ctgatctgct ctttggtgga cagctcgttg ccggtgtcct cttccacctc gttcacgttg
     3841 tgcacgcctc ttctcttggc caggtgcagc agggcggcag agaactcttc ctcgctcagc
     3901 ttctggctca ggcccttcac tctggcctcg taggggttga tgccgctcag ctcgctgtgg
     3961 tcggtcagca ggttgtagtc gaacagcagc ttcttcactc tctggattct atgccgcctc
     4021 cgccgcttca gccttctggc gcctctcttg ctccgcctgc cctcgttgtt ttccacgttg
     4081 gcctctttga acagccgcac gccggcatcg atcacgtccc gtgtctcgta gtcgatgatg
     4141 ccgtagccca cgctggtgat gccgatgtcc aggcccagga tgtagttccg cttggctgct
     4201 gggactccgt ggataccgac cttccgcttc ttctttgggg ccatgcggcc gctgcagacc
     4261 ggtgctagca taacttcgta taaagtatcc tatacgaagt tatttgcctt aacccagaaa
     4321 ttatcactgt tattctttag aatggtgcaa agaataactt cgtataatgt atgctatacg
     4381 aagttatgaa ttcctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca
     4441 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     4501 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     4561 gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct
     4621 ggggaggtac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     4681 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
     4741 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     4801 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     4861 tgtggaaagg acgaaacacc gagcgctatc ccgacacctt ggttttagta ctctggaaac
     4921 agaatctact aaaacaaggc aaaatgccgt gtttatctcg tcaacttgtt ggcgagattt
     4981 ttgcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     5041 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     5101 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     5161 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     5221 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     5281 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     5341 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     5401 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     5461 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     5521 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
     5581 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     5641 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     5701 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     5761 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     5821 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     5881 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     5941 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     6001 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     6061 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     6121 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     6181 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     6241 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     6301 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     6361 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     6421 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     6481 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     6541 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     6601 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     6661 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     6721 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg
     6781 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6841 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6901 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6961 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     7021 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     7081 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     7141 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     7201 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     7261 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     7321 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     7381 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     7441 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     7501 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     7561 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     7621 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     7681 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
